Chloroflexi (class)

The Chloroflexia are one of six classes of bacteria in the phylum Chloroflexi, known as filamentous green non-sulfur bacteria. They use light for energy and are named for their green pigment, usually found in photosynthetic bodies called chlorosomes.
Chloroflexia are typically filamentous, and can move about through bacterial gliding. They are facultatively aerobic, but do not produce oxygen in the process of producing energy from light, or phototrophy. Additionally, Chloroflexia have a different method of phototrophy (photoheterotrophy) than true photosynthetic bacteria.
Etymology.
The name "Chloroflexi" is a Neolatin plural of "Chloroflexus", which is the name of the first genus described. The noun is a combination of the Greek "chloros" (χλωρός) meaning "greenish-yellow" and the Latin "flexus" (of "flecto") meaning "bent" to mean "a green bending". The name is not due to chlorine, an element confirmed as such in 1810 by Sir Humphry Davy and named after its pale green colour.
Taxonomy and molecular signatures.
The Chloroflexia class is a group of deep branching photosynthetic bacteria (with the exception of "Herpetosiphon" and "Kallotenue" species) that currently consist of three orders: Chloroflexales, Herpetosiphonales, and Kallotenuales. The Herpetosiphonales and Kallotenuales each consist of a single genus within its own family, Herpetosiphonaceae ("Herpetosiphon") and Kallotenuaceae ("Kallotenue"), respectively, whereas the Chloroflexales are more phylogenetically diverse.
Microscopic distinguishing characteristics.
Members of the phylum Chloroflexi are monoderms and stain mostly Gram negative, whereas most bacteria species are diderms and stain Gram negative, with the Gram positive exceptions of the Firmicutes (low GC Gram positives), Actinobacteria (high GC, Gram positives), and the "Deinococcus-Thermus" group (Gram positive, diderms with thick peptidoglycan).
Genetic distinguishing characteristics.
Comparative genomic analysis has recently refined the taxonomy of the class "Chloroflexia", dividing the "Chloroflexales" into the suborder "Chloroflexineae" consisting of the families "Oscillachloridaceae" and "Chloroflexaceae", and the suborder Roseiflexineae containing family "Roseiflexaceae". The revised taxonomy was based on the identification of a number of conserved signature indels (CSIs) which serve as highly reliable molecular markers of shared ancestry.
Physiological distinguishing characteristics.
Additional support for the division of the Chloroflexales into two suborders is the observed differences in physiological characteristics where each suborder is characterized by distinct carotenoids, quinones, and fatty acid profiles that are consistently absent in the other suborder.
In addition to demarcating taxonomic ranks, CSIs may play a role in the unique characteristics of members within the clade: In particular, a four-amino-acid insert in the protein pyruvate flavodoxin/ferredoxin oxidoreductase, a protein which plays important roles in photosynthetic organisms, has been found exclusively among all members in the genus "Chloroflexus", and is thought to play an important functional role.
Additional work has been done using CSIs to demarcate the phylogenetic position of "Chloroflexia" relative to neighbouring photosynthetic groups such as the Cyanobacteria.
"Chloroflexia" species form a distinct lineage with "Chlorobi" species, their closest phylogenetic relatives. A CSI has been found to be shared among both "Chloroflexia" and "Chlorobi" members, which has been interpreted as the result of a horizontal gene transfer event between the two relatives.
Taxonomy.
The currently accepted taxonomy is as follows:
Additionally, "Kouleothrix aurantiaca" and "Dehalobium chlorocoercia" have not been fully described.


Flestolol

Flestolol is a short-acting beta adrenergic receptor antagonist.
Synthesis.
Acylation of glycidol (2) with the acid chloride 1 produces the ester 3. Reaction of that intermediate with amine 4, obtained by reaction of 1,1-dimethylethylenediamine with urea, gives flestolol (5).


Growth-hormone-releasing hormone receptor

The growth-hormone-releasing hormone receptor (GHRHR) is a G-protein-coupled receptor that binds growth hormone-releasing hormone. The GHRHR activates a Gs protein that causes a cascade of cAMP via adenylate cyclase.
Function.
This gene, expressed in the pituitary, encodes a receptor for growth-hormone-releasing hormone. Binding of this hormone to the receptor leads to synthesis and release of growth hormone. Mutations in this gene have been associated with isolated growth-hormone deficiency (IGHD), also known as Dwarfism of Sindh, a disorder characterized by short stature. Many alternate transcriptional splice variants encoding different isoforms have been described, but only two have been characterized to date.


Bob Waterston

Robert Hugh "Bob" Waterston, (born September 17, 1943) is an American biologist. He is best known for his work on the Human Genome Project, for which he was a pioneer along with John Sulston.
Biography.
Robert Waterston was born in 1943 in Detroit, Michigan. He went to Princeton as an undergraduate where he majored in engineering while spending as much time as possible studying the humanities; he wrote his senior dissertation on the plays of Eugene O'Neill. By this time he was thinking of switching to medicine but had never studied biology.
While on what we might now call a 'gap year' visit to Germany he took courses in biology – in German – and returned to take up a place at the school of medicine of the University of Chicago.
By 1972 he had acquired both MD and PhD degrees. A chance meeting with Sydney Brenner on a physiology summer school at the Woods Hole Marine Biological Laboratory in 1969 had introduced him to the grand project then beginning at the MRC's Laboratory of Molecular Biology (LMB) in Cambridge, England: to understand life at the molecular level through studying the nematode worm Caenorhabditis elegans. At the earliest possible opportunity, Waterston left for Cambridge to become the third American post-doctoral researcher to join Brenner's group to work on the worm.
Brenner was looking for worms with mutations that affected their behavior, as part of the search for genes controlling the nervous system and muscles. Waterston took on the task of looking at the cellular and molecular level for abnormalities in the muscle mutants, and within a few years had made discoveries that led to the cloning of genes for two important muscle proteins.
He returned to the USA in 1976 as an assistant professor of anatomy and neurobiology, at Washington University in St. Louis and set up a lab dedicated to studying the molecular biology of muscle in the worm. There, he and his colleagues identified many more muscle genes and investigated their role in muscle assembly and contraction, as well as discovering and analyzing transposable elements and nonsense suppressors. A few years later he switched to the Department of Genetics, where by 1991 he became chair.
In the mid-1980s he made a sabbatical visit to the LMB, ostensibly to continue his work with Brenner. But the only space available was in the room where John Sulston and Alan Coulson were beginning to map the worm genome. Waterston joined them, and after his return to St Louis the worm map became a collaborative project between the two labs.
In 1989, one of the first Human Genome Project grants went to Waterston and Sulston to begin the sequencing of the worm genome. They were so successful that at the same time that the Wellcome Trust established the Sanger Centre with Sulston at its head, Waterston received funding from the National Human Genome Research Institute to undertake large-scale human sequencing at his Washington University lab. The partnership became the first to complete the sequence of a multicellular organism when the worm genome was published in December 1998, and both labs played key roles in the sequencing of the human genome, with Waterston’s lab contributing more of the finished sequence than any other US lab. The partnership went on have leading roles in the sequencing of both the mouse and chimpanzee genomes.
Waterston, like his colleague John Sulston, has always been committed to the free release of scientific information, and he was an influential voice in establishing the Bermuda Principles on data sharing in 1996. In the past few years Waterston and Sulston have jointly won numerous awards for their scientific work and their support for the scientific community, including the Gairdner Award, the General Motors prize, the Dan David Prize and the George W Beadle medal of the Genetics Society of America. Waterston also received the Gruber Prize in genetics.
In January 2003, Waterston moved from St Louis to become the new William Gates III Endowed Chair in Biomedical Science, Professor and Chair of the Department of Genome Sciences, at the University of Washington in Seattle. He is leading research into the genetic control of development in the nematode. He has developed novel technology to decipher automatically the dynamic expression of genes with single cell resolution and minute time resolution and applied the technology to describe the expression patterns of more than 200 transcription factors. As part of the NHGRI modENCODE project, with collaborators he has collected data that accurately defines the complement of both protein coding and many noncoding genes of the nematode. In another modENCODE project he and collaborators are defining the binding sites for those transcription factors. They are now combining these sets of data to construct increasingly complete regulatory networks directing C. elegans embryonic development. Recently, he has used next generation sequencing methods to develop a collection of mutant C. elegans strains containing more than million mutations to probe the function of the more than 20,000 C. elegans genes.
Collaborations, both inside and outside of the university, have helped the Genome Sciences Department bolster an exciting teamwork atmosphere. It has formed close ties with the School of Medicine to study human variation more effectively. The faculty also partner with clinicians interested in applying genomic technologies to medical problems. In addition, the Department has established a strong outreach program. By bringing leading-edge science and ethics teachers to K-12 classes, the Department helps address the unique social and ethical challenges implied in the new discoveries in genome sciences.
Today, the UW Department of Genome Sciences is carrying on its legacy of collaboration. It is a world pioneer of genome interpretation and technology innovation, ranking in the top three Genetics/ Genomics graduate programs in the United States It brings national prominence to the University of Washington and is at the forefront in unlocking life-changing secrets of the genome.
- Adapted from the Wellcome Trust biography written by Georgina Ferry.


AAC/AAD leader

The AAC/AAD 5' leader is a disputed genetic element that was proposed to be a conserved RNA structure that is found upstream of the bacterial aminoglycosides antibiotic-resistant genes and that functions as an aminoglycoside-specific riboswitch. The putative RNA is upstream of aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) genes.
According to the initial report, it has a short open reading frame (ORF) that encodes a leader peptide upstream of the resistance gene. A 75-nucleotide-long sequence from the first ribosome binding site up to the coding sequence appeared to have the regulatory role, and it was considered the minimal functional RNA. The proposed model is that aminoglycosides bind to specific regions of AAC/AAD 5' leader and induce change in the RNA structure, thereby inducing expression of the resistance protein encoded by the downstream gene. Results in the initial report are that the leader RNA is widely distributed among antibiotic-resistant bacterial pathogens.
A subsequent letter disputed these claims. This letter stated that the proposed riboswitch matches the "attI" site, which is a previously known part of integrons and is a signal for recombination, a frequent process in integrons. The letter further stated, among other points, that the putative riboswitch lacks additional conserved features beyond those previously established for "attI", that the riboswitch/"attI" are also associated with genes that confer resistance to antibiotics unrelated to aminoglycosides (or encode other unrelated proteins), and the letter proposed that the results of the original paper could be explained by a previously elucidated mechanism involving the ORF. The authors of the original paper published a response to this letter.


4-HO-DET

4-HO-DET, also known as 4-hydroxy-diethyl-tryptamine, CZ-74, is a hallucinogenic drug and psychedelic compound of moderate duration. 4-HO-DET is a substituted tryptamine, structurally related to psilocin, ethocybin, and 4-HO-DIPT.
Analogs.
The acetic acid ester of 4-HO-DET is known as 4-AcO-DET. The phosphoric acid ester of 4-HO-DET is known as 4-phosphoryloxy-DET, CEY-19, or ethocybin.
History.
4-HO-DET received the lab code CZ-74 in the late 1950s by the inventors of the substance, Albert Hofmann and Franz Troxler. The substance was used together with its phosphoryloxy-analog ethocybin in human clinical trials in the 1960s by the German researchers Hanscarl Leuner and G. Baer.
Dosage.
10–25 mg is the usual oral dosage for 4-HO-DET, while the acetate and phosphate esters are said to require a slightly higher dosage.
Effects.
4-HO-DET produces entheogenic effects similar to LSD and psilocybin.
Drug prohibition laws.
Sweden.
"Sveriges riksdags" health ministry classified 4-HO-DET as "health hazard" under the act (translated "Act on the Prohibition of Certain Goods Dangerous to Health") as of Nov 1, 2005, in their regulation SFS 2005:733 listed as 4-hydroxi-N,N-diethyltryptamin (4-HO-DET), making it illegal to sell or possess.


Raineyella antarctica

Raineyella antarctica is a Gram-positive, psychrotolerant and motile bacterium from the genus of Raineyella which has been isolated from the moss Leptobryum from the shore of the Lake Zub in
the Antarctica.


Pregnane X receptor

In the field of molecular biology, the pregnane X receptor (PXR), also known as the steroid and xenobiotic sensing nuclear receptor (SXR) or nuclear receptor subfamily 1, group I, member 2 (NR1I2) is a protein that in humans is encoded by the "NR1I2" (nuclear Receptor subfamily 1, group I, member 2) gene.
Function.
PXR is a nuclear receptor whose primary function is to sense the presence of foreign toxic substances and in response up regulate the expression of proteins involved in the detoxification and clearance of these substances from the body. PXR belongs to the nuclear receptor superfamily, members of which are transcription factors characterized by a ligand-binding domain and a DNA-binding domain. PXR is a transcriptional regulator of the cytochrome P450 gene CYP3A4, binding to the response element of the CYP3A4 promoter as a heterodimer with the 9-cis retinoic acid receptor RXR. It is activated by a range of compounds that induce CYP3A4, including dexamethasone and rifampicin.
Ligands.
Agonists.
PXR is activated by a large number of endogenous and exogenous chemicals including steroids (e.g., progesterone, 17α-hydroxyprogesterone, 17α-hydroxypregnenolone, 5α-dihydroprogesterone, 5β-dihydroprogesterone, allopregnanolone, corticosterone, cyproterone acetate, spironolactone, dexamethasone, mifepristone), antibiotics (e.g., rifampicin, rifaximin), antimycotics, bile acids, hyperforin (a constituent of the herbal antidepressant St. John's Wort), and many herbal and other compounds (e.g. meclizine, paclitaxel). Cafestol found in coffee is also an agonist.
Forskolin is another case example of a PXR agonist.
Antagonists.
Ketoconazole is an example of one of the relatively few-known antagonists of the PXR. SPA70 (also known as LC-1) was recently identified and characterized as a potent and selective PXR antagonist.
Mechanism.
Like other type II nuclear receptors, when activated, it forms a heterodimer with the retinoid X receptor, and binds to hormone response elements on DNA which elicits expression of gene products.
One of the primary targets of PXR activation is the induction of CYP3A4, an important phase I oxidative enzyme that is responsible for the metabolism of many drugs. In addition, PXR up regulates the expression of phase II conjugating enzymes such as glutathione S-transferase and phase III transport uptake and efflux proteins such as OATP2 and MDR1.


Guilty Gear Petit

Guilty Gear Petit is a sub-series of fighting video games, spin-offs of the "Guilty Gear" series, directed by Emiko Iwasaki and released for the Japan-only handheld WonderSwan Color.
"Guilty Gear Petit".
 is the first handheld version of the "Guilty Gear" series released. It is followed by the sequel Guilty Gear Petit 2. Much like "Super Gem Fighter Mini Mix", it depicts the "Guilty Gear" characters in a super deformed style. It is also notable for the inclusion of the character Fanny, who appears only in the two "Guilty Gear Petit" games. The game has seven playable characters, six of which are from other "Guilty Gear" series games. The game was only released in Japan.
"Guilty Gear Petit 2".
The sequel was released eight months after the first. The game has 13 regular characters, 2 secret characters (Testament, Venom), and four "Golden" characters (GGMillia, GGKy, GGSol, and GGMay). The game was only released in Japan for the WonderSwan Color.


Batoprazine

Batoprazine is a drug of the phenylpiperazine class which has been described as a serenic or antiaggressive agent. It acts as a 5-HT1A and 5-HT1B receptor agonist. It is closely related to eltoprazine, fluprazine, and naphthylpiperazine, of which possess similar actions and effects.


Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.
Hydration of acrylonitrile results in formation of acrylamide molecules () by nitrile hydratase. Acrylamide monomer is in a powder state before addition of water. Acrylamide is toxic to the human nervous system, therefore all safety measures must be followed when working with it. Acrylamide is soluble in water and upon addition of water it polymerizes resulting in formation of polyacrylamide. It is useful to make polyacrylamide gel via acrylmide hydration because pore size can be regulated. Increased concentrations of acrylamide result in decreased pore size after polymerization. Polyacrylamide gel with small pores helps to examine smaller molecules better since the small molecules can enter the pores and travel through the gel while large molecules get trapped at the pore openings.
As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules' higher-order structure. This method is called native-PAGE. Alternatively, a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured molecule whose mobility depends only on its length (because the protein-SDS complexes all have a similar mass-to-charge ratio). This procedure is called SDS-PAGE. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a method of separating molecules based on the difference of their molecular weight. At the pH at which gel electrophoresis is carried out the SDS molecules are negatively charged and bind to proteins in a set ratio, approximately one molecule of SDS for every 2 amino acids. In this way, the detergent provides all proteins with a uniform charge-to-mass ratio. By binding to the proteins the detergent destroys their secondary, tertiary and/or quaternary structure denaturing them and turning them into negatively charged linear polypeptide chains. When subjected to an electric field in PAGE, the negatively charged polypeptide chains travel toward the anode with different mobility. Their mobility, or the distance traveled by molecules, is inversely proportional to the logarithm of their molecular weight. By comparing the relative ratio of the distance traveled by each protein to the length of the gel (Rf) one can make conclusions about the relative molecular weight of the proteins, where the length of the gel is determined by the distance traveled by a small molecule like a tracking dye.
For nucleic acids, urea is the most commonly used denaturant. For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to coat proteins in order to impart two negative charges (from every SDS molecule) to every two amino acids of the denatured protein. 2-Mercaptoethanol may also be used to disrupt the disulfide bonds found between the protein complexes, which helps further denature the protein. In most proteins, the binding of SDS to the polypeptide chains impart an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. Proteins that have a greater hydrophobic content – for instance, many membrane proteins, and those that interact with surfactants in their native environment – are intrinsically harder to treat accurately using this method, due to the greater variability in the ratio of bound SDS. Procedurally, using both Native and SDS-PAGE together can be used to purify and to separate the various subunits of the protein. Native-PAGE keeps the oligomeric form intact and will show a band on the gel that is representative of the level of activity. SDS-PAGE will denature and separate the oligomeric form into its monomers, showing bands that are representative of their molecular weights. These bands can be used to identify and assess the purity of the protein.
Procedure.
Sample preparation.
Samples may be any material containing proteins or nucleic acids. These may be biologically derived, for example from prokaryotic or eukaryotic cells, tissues, viruses, environmental samples, or purified proteins. In the case of solid tissues or cells, these are often first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), by sonicator or by using cycling of high pressure, and a combination of biochemical and mechanical techniques – including various types of filtration and centrifugation – may be used to separate different cell compartments and organelles prior to electrophoresis. Synthetic biomolecules such as oligonucleotides may also be used as analytes.
The sample to analyze is optionally mixed with a chemical denaturant if so desired, usually SDS for proteins or urea for nucleic acids. SDS is an anionic detergent that denatures secondary and non–disulfide–linked tertiary structures, and additionally applies a negative charge to each protein in proportion to its mass. Urea breaks the hydrogen bonds between the base pairs of the nucleic acid, causing the constituent strands to anneal. Heating the samples to at least 60 °C further promotes denaturation.
In addition to SDS, proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol (beta-mercaptoethanol/BME), which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits). This is known as reducing SDS-PAGE.
A tracking dye may be added to the solution. This typically has a higher electrophoretic mobility than the analytes to allow the experimenter to track the progress of the solution through the gel during the electrophoretic run.
Preparing acrylamide gels.
The gels typically consist of acrylamide, bisacrylamide, the optional denaturant (SDS or urea), and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization. Alternatively, butanol may be added to the resolving gel (for proteins) after it is poured, as butanol removes bubbles and makes the surface smooth.
A source of free radicals and a stabilizer, such as ammonium persulfate and TEMED are added to initiate polymerization. The polymerization reaction creates a gel because of the added bisacrylamide, which can form cross-links between two acrylamide molecules. The ratio of bisacrylamide to acrylamide can be varied for special purposes, but is generally about 1 part in 35. The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%. Lower percentage gels are better for resolving very high molecular weight molecules, while much higher percentages of acrylamide are needed to resolve smaller proteins. The average pore diameter of polyacrylamide gels is determined by the total concentration of acrylamides (% T with T = Total concentration of acrylamide and bisacrylamide) and the concentration of the cross-linker bisacrylamide (%C with C = bisacrylamide concentration).<ref name="DOI10.1016/S0021-9673(00)95641-3"></ref> The pore size is reduced reciprocally to the %T. Concerning %C, a concentration of 5% produces the smallest pores, since the influence of bisacrylamide on the pore size has a parabola-shape with a vertex at 5%.
Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at the top to create the sample wells. After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis.
Electrophoresis.
Various buffer systems are used in PAGE depending on the nature of the sample and the experimental objective. The buffers used at the anode and cathode may be the same or different.
An electric field is applied across the gel, causing the negatively charged proteins or nucleic acids to migrate across the gel away from the negative electrode (which is the cathode being that this is an electrolytic rather than galvanic cell) and towards the positive electrode (the anode). Depending on their size, each biomolecule moves differently through the gel matrix: small molecules more easily fit through the pores in the gel, while larger ones have more difficulty. The gel is run usually for a few hours, though this depends on the voltage applied across the gel; migration occurs more quickly at higher voltages, but these results are typically less accurate than at those at lower voltages. After the set amount of time, the biomolecules have migrated different distances based on their size. Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point of origin. Biomolecules may therefore be separated roughly according to size, which depends mainly on molecular weight under denaturing conditions, but also depends on higher-order conformation under native conditions. The gel mobility is defined as the rate of migration traveled with a voltage gradient of 1V/cm and has units of cm2/sec/V. For analytical purposes, the relative mobility of biomolecules, "Rf", the ratio of the distance the molecule traveled on the gel to the total travel distance of a tracking dye is plotted versus the molecular weight of the molecule (or sometimes the log of MW, or rather the Mr, molecular radius). Such typically linear plots represent the standard markers or calibration curves that are widely used for the quantitative estimation of a variety of biomolecular sizes.
Certain glycoproteins, however, behave anomalously on SDS gels. Additionally, the analysis of larger proteins ranging from 250,000 to 600,000 Da is also reported to be problematic due to the fact that such polypeptides move improperly in the normally used gel systems.
Further processing.
Following electrophoresis, the gel may be stained (for proteins, most commonly with Coomassie Brilliant Blue R-250 or autoradiography; for nucleic acids, ethidium bromide; or for either, silver stain), allowing visualization of the separated proteins, or processed further (e.g. Western blot). After staining, different species biomolecules appear as distinct bands within the gel. It is common to run molecular weight size markers of known molecular weight in a separate lane in the gel to calibrate the gel and determine the approximate molecular mass of unknown biomolecules by comparing the distance traveled relative to the marker.
For proteins, SDS-PAGE is usually the first choice as an assay of purity due to its reliability and ease. The presence of SDS and the denaturing step make proteins separate, approximately based on size, but aberrant migration of some proteins may occur. Different proteins may also stain differently, which interferes with quantification by staining. PAGE may also be used as a preparative technique for the purification of proteins. For example, quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) is a method for separating native metalloproteins in complex biological matrices.
Chemical ingredients and their roles.
Polyacrylamide gel (PAG) had been known as a potential embedding medium for sectioning tissues as early as 1964, and two independent groups employed PAG in electrophoresis in 1959. It possesses several electrophoretically desirable features that make it a versatile medium. It is a synthetic, thermo-stable, transparent, strong, chemically relatively inert gel, and can be prepared with a wide range of average pore sizes. The pore size of a gel and the reproducibility in gel pore size are determined by three factors, the total amount of acrylamide present (%T) (T = Total concentration of acrylamide and bisacrylamide monomer), the amount of cross-linker (%C) (C = bisacrylamide concentration), and the time of polymerization of acrylamide (cf. QPNC-PAGE). Pore size decreases with increasing %T; with cross-linking, 5%C gives the smallest pore size. Any increase or decrease in %C from 5% increases the pore size, as pore size with respect to %C is a parabolic function with vertex as 5%C. This appears to be because of non-homogeneous bundling of polymer strands within the gel. This gel material can also withstand high voltage gradients, is amenable to various staining and destaining procedures, and can be digested to extract separated fractions or dried for autoradiography and permanent recording.
Components.
Polyacrylamide gels are composed of a stacking gel and separating gel. Stacking gels have a higher porosity relative to the separating gel, and allow for proteins to migrate in a concentrated area. Additionally, stacking gels usually have a pH of 6.8, since the neutral glycine molecules allow for faster protein mobility. Separating gels have a pH of 8.8, where the anionic glycine slows down the mobility of proteins. Separating gels allow for the separation of proteins and have a relatively lower porosity. Here, the proteins are separated based on size (in SDS-PAGE) and size/ charge (Native PAGE).
Chemical buffer stabilizes the pH value to the desired value within the gel itself and in the electrophoresis buffer. The choice of buffer also affects the electrophoretic mobility of the buffer counterions and thereby the resolution of the gel. The buffer should also be unreactive and not modify or react with most proteins. Different buffers may be used as cathode and anode buffers, respectively, depending on the application. Multiple pH values may be used within a single gel, for example in DISC electrophoresis. Common buffers in PAGE include Tris, Bis-Tris, or imidazole.
Counterion balance the intrinsic charge of the buffer ion and also affect the electric field strength during electrophoresis. Highly charged and mobile ions are often avoided in SDS-PAGE cathode buffers, but may be included in the gel itself, where it migrates ahead of the protein. In applications such as DISC SDS-PAGE the pH values within the gel may vary to change the average charge of the counterions during the run to improve resolution. Popular counterions are glycine and tricine. Glycine has been used as the source of trailing ion or slow ion because its pKa is 9.69 and mobility of glycinate are such that the effective mobility can be set at a value below that of the slowest known proteins of net negative charge in the pH range. The minimum pH of this range is approximately 8.0.
Acrylamide (; mW: 71.08) when dissolved in water, slow, spontaneous autopolymerization of acrylamide takes place, joining molecules together by head on tail fashion to form long single-chain polymers. The presence of a free radical-generating system greatly accelerates polymerization. This kind of reaction is known as vinyl addition polymerisation. A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over one another. Gel formation requires linking various chains together. Acrylamide is carcinogenic, a neurotoxin, and a reproductive toxin. It is also essential to store acrylamide in a cool dark and dry place to reduce autopolymerisation and hydrolysis.
Bisacrylamide ("N","N"′-Methylenebisacrylamide) (; mW: 154.17) is the most frequently used cross linking agent for polyacrylamide gels. Chemically it can be thought of as two acrylamide molecules coupled head to head at their non-reactive ends. Bisacrylamide can crosslink two polyacrylamide chains to one another, thereby resulting in a gel.
Sodium dodecyl sulfate (SDS) (; mW: 288.38) (only used in denaturing protein gels) is a strong detergent agent used to denature native proteins to individual polypeptides. This denaturation, which is referred to as reconstructive denaturation, is not accomplished by the total linearization of the protein, but instead, through a conformational change to a combination of random coil and α helix secondary structures. When a protein mixture is heated to 100 °C in presence of SDS, the detergent wraps around the polypeptide backbone. It binds to polypeptides in a constant weight ratio of 1.4 g SDS/g of polypeptide. In this process, the intrinsic charges of polypeptides become negligible when compared to the negative charges contributed by SDS. Thus polypeptides after treatment become rod-like structures possessing a uniform charge density, that is same net negative charge per unit weight. The electrophoretic mobilities of these proteins is a linear function of the logarithms of their molecular weights. Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass-charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure. This is known as native PAGE. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.
Urea (; mW: 60.06) is a chaotropic agent that increases the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds and van der Waals forces. Macromolecular structure is dependent on the net effect of these forces, therefore it follows that an increase in chaotropic solutes denatures macromolecules,
Ammonium persulfate (APS) (; mW: 228.2) is a source of free radicals and is often used as an initiator for gel formation. An alternative source of free radicals is riboflavin, which generated free radicals in a photochemical reaction.
TEMED ("N", "N", "N"′, "N"′-tetramethylethylenediamine) (; mW: 116.21) stabilizes free radicals and improves polymerization. The rate of polymerisation and the properties of the resulting gel depend on the concentrations of free radicals. Increasing the amount of free radicals results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. Decreasing the amount shows the reverse effect. The lowest catalytic concentrations that allow polymerisation in a reasonable period of time should be used. APS and TEMED are typically used at approximately equimolar concentrations in the range of 1 to 10 mM.
Chemicals for processing and visualization.
The following chemicals and procedures are used for processing of the gel and the protein samples visualized in it.
Tracking dye; as proteins and nucleic acids are mostly colorless, their progress through the gel during electrophoresis cannot be easily followed. Anionic dyes of a known electrophoretic mobility are therefore usually included in the PAGE sample buffer. A very common tracking dye is Bromophenol blue (BPB, 3',3",5',5" tetrabromophenolsulfonphthalein). This dye is coloured at alkali and neutral pH and is a small negatively charged molecule that moves towards the anode. Being a highly mobile molecule it moves ahead of most proteins. As it reaches the anodic end of the electrophoresis medium electrophoresis is stopped. It can weakly bind to some proteins and impart a blue colour. Other common tracking dyes are xylene cyanol, which has lower mobility, and Orange G, which has a higher mobility.
Loading aids; most PAGE systems are loaded from the top into wells within the gel. To ensure that the sample sinks to the bottom of the gel, sample buffer is supplemented with additives that increase the density of the sample. These additives should be non-ionic and non-reactive towards proteins to avoid interfering with electrophoresis. Common additives are glycerol and sucrose.
Coomassie Brilliant Blue R-250 (CBB)(; mW: 825.97) is the most popular protein stain. It is an anionic dye, which non-specifically binds to proteins. The structure of CBB is predominantly non-polar, and it is usually used in methanolic solution acidified with acetic acid. Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated into the gel can be removed by destaining with the same solution without the dye. The proteins are detected as blue bands on a clear background. As SDS is also anionic, it may interfere with staining process. Therefore, large volume of staining solution is recommended, at least ten times the volume of the gel.
Ethidium bromide (EtBr) is a popular nucleic acid stain. EtBr allows one to easily visualize DNA or RNA on a gel as EtBr fluoresces an orange color under UV light. Ethidium bromide binds nucleic acid chains through the process of Intercalation. While Ethidium bromide is a popular stain it is important to exercise caution when using EtBr as it is a known carcinogen. Because of this fact, many researchers opt to use stains such as SYBR Green and SYBR Safe which are safer alternatives to EtBr. EtBr is used by simply adding it to the gel mixture. Once the gel has run, the gel may be viewed through the use of a photo-documentation system.
Silver staining is used when more sensitive method for detection is needed, as classical Coomassie Brilliant Blue staining can usually detect a 50 ng protein band, Silver staining increases the sensitivity typically 10-100 fold more. This is based on the chemistry of photographic development. The proteins are fixed to the gel with a dilute methanol solution, then incubated with an acidic silver nitrate solution. Silver ions are reduced to their metallic form by formaldehyde at alkaline pH. An acidic solution, such as acetic acid stops development. Silver staining was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels. The technique has been extended to the study of other biological macromolecules that have been separated in a variety of supports. Many variables can influence the colour intensity and every protein has its own staining characteristics; clean glassware, pure reagents and water of highest purity are the key points to successful staining. Silver staining was developed in the 14th century for colouring the surface of glass. It has been used extensively for this purpose since the 16th century. The colour produced by the early silver stains ranged between light yellow and an orange-red. Camillo Golgi perfected the silver staining for the study of the nervous system. Golgi's method stains a limited number of cells at random in their entirety.
Autoradiography, also used for protein band detection post gel electrophoresis, uses radioactive isotopes to label proteins, which are then detected by using X-ray film.
Western blotting is a process by which proteins separated in the acrylamide gel are electrophoretically transferred to a stable, manipulable membrane such as a nitrocellulose, nylon, or PVDF membrane. It is then possible to apply immunochemical techniques to visualise the transferred proteins, as well as accurately identify relative increases or decreases of the protein of interest.


Brorphine

Brorphine is a piperidine-based opioid analgesic compound. Brorphine was originally discovered in a 2018 paper investigating functionally biased opioid compounds, with the intention of finding safer analgesics that produce less respiratory depression than typical opioids. Brorphine was originally reported to be highly biased, with an EC50 of 4.8nM for GTPγS binding and 182nM for β-arrestin recruitment, however a re-evaluation found no significant bias for any of the compounds tested, including brorphine. This supposed lack of bias for all tested compounds may be questionable, as it would seem to conflict with all other research on biased agonism. Its safety profile in any animal model has never been established. Despite the lack of safety information on the compound, brorphine has been sold as a designer drug since mid-2019, initially being identified in the US Midwest, though it has since been found in 2020 in Belgium. It is related in chemical structure to compounds such as benzylfentanyl and bezitramide, though it is sufficiently structurally distinct to fall outside the formal definition of a "fentanyl analogue" in jurisdictions such as the US and New Zealand which have Markush structure controls over this family of drugs.
Brorphine was first identified in the U.S. recreational drug supply in July 2020 by the Center for Forensic Science Research and Education (CFSRE) through its NPS Discovery program; however, earlier identifications by the Drug Enforcement Administration (DEA) may have come as early as late 2019. The rise of brorphine in the U.S. can be directly linked to the decline of isotonitazene due to scheduling by the DEA. Brorphine was first implicated in 20 deaths in the U.S., primarily in cases originating from midwest states. Brorphine was commonly found with fentanyl and flualprazolam, a drug combination verified by drug product testing. Brorphine has also been identified in counterfeit opioid pills and tablets. Recently data from CFSRE and NMS Labs show that brorphine has been detected in more than 100 cases as of October 2020.


Roland Winter

Roland Winter (born 1954) is a biophysical chemist who studies the structure, dynamics, energetics, and phase behavior of biological membranes and proteins. He is dean of the Department of Chemistry and Chemical Biology at Technical University of Dortmund, Germany.
Career.
Winter received his diploma in Chemistry in 1979 and his PhD in Physical Chemistry in 1982 at the Technical University of Karlsruhe. He went on to be a postdoctoral fellow at the University of Marburg and spent a year as a visiting scientist in the Department of Chemistry at the University of Illinois at Urbana-Champaign from 1987-1988. Winter then became a professor at Ruhr-University Bochum and in 1993 joined the faculty of Technical University of Dortmund as chair of Biophysical Chemistry. Winter is an executive board member of the German Center of Excellence RESOLV (Ruhr Explores Solvation), where he heads the research area "Connecting Solvation Dynamics with Biomolecular Function." He received the Dozenten-Price of the Fonds der Chemischen Industrie award in 1992. Winter is a member of the Deep Life Scientific Steering Committee for the Deep Carbon Observatory (DCO) and serves on the editorial boards of "Biophysical Chemistry", "Journal of Non-Equilibrium Thermodynamics", "Zeitschrift für Physikalische Chemie" and "Chemie in unserer Zeit".
Research Initiatives.
Winter studies membrane biophysics with an emphasis on the energetics and phase behavior of lipid membranes and the interaction between model biomembranes and proteins involved in cell signaling processes. He also investigates the effects of high pressure on lipid membranes and proteins.


Neoendorphin

Neoendorphins are a group of endogenous opioid peptides derived from the proteolytic cleavage of prodynorphin. They include α-neoendorphin and β-neoendorphin.


Nitrite reductase (NO-forming)

In enzymology, a nitrite reductase (NO-forming) () is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are nitric oxide, H2O, and ferricytochrome c, whereas its 3 products are nitrite, ferrocytochrome c, and H+.
This enzyme belongs to the family of oxidoreductases, specifically those acting on other nitrogenous compounds as donors with a cytochrome as acceptor. The systematic name of this enzyme class is nitric-oxide:ferricytochrome-c oxidoreductase. Other names in common use include cd-cytochrome nitrite reductase, [nitrite reductase (cytochrome)] [misleading, see comments.], cytochrome c-551:O2, NO2+ oxidoreductase, cytochrome cd, cytochrome cd1, hydroxylamine (acceptor) reductase, methyl viologen-nitrite reductase, nitrite reductase (cytochrome, and NO-forming). This enzyme participates in nitrogen metabolism. It has 3 cofactors: FAD, Iron, and Copper.
Structural studies.
As of late 2007, 20 structures have been solved for this class of enzymes, with PDB accession codes , , , , , , , , , , , , , , , , , , , and .


Joseph P. Curseen


Tyne Renewable Energy Plant

Tyne Renewable Energy Plant (or Tyne REP) is a proposed biomass power station, to be built on the north bank of the River Tyne at North Shields. The plant has been developed by MGT Power, along with their similar project, the Teesport Renewable Energy Plant on Teesside. It is expected to have a generating capacity of 295 megawatts, enough to power around 600,000 homes, making it one of the biggest of its kind in Europe. It is hoped the plant will be opened in 2014, costing £400 million.
The plant would be built on a industrial site at the Port of Tyne in North Shields adjacent to the proposed North Shields Bio Diesel Plant on the north bank of the River Tyne. The construction of the plant would create around 600 jobs, as well as 150 full-time jobs once the plant was completed, and 300 to 400 indirect jobs in the supply chain. There would be an annual spend of £30 million in the local economy.


Diphenidol

Diphenidol is a muscarinic antagonist employed as an antiemetic and as an antivertigo agent. It is not marketed in the United States or Canada.
Although the mechanism of action of diphenidol on the vestibular system has not yet been elucidated, it exerts an anticholinergic effect due to interactions with mACh receptors, particularly M1, M2, M3 and M4. Hence, its actions may take place at the vestibular nuclei, where a significant excitatory input is mediated by ACh receptors, and also at the vestibular periphery where mACh receptors are expressed at efferent synapses. A series of selective mACh-receptor antagonists based on the diphenidol molecule has been synthesized, but they have not yet been the subject of clinical trials.


SMCR2

In molecular biology, Smith-Magenis syndrome chromosome region, candidate 2 (non-protein coding), also known as SMCR2 is a long non-coding RNA. In humans, it is found in a region of chromosome 17 that is commonly deleted in Smith–Magenis syndrome.


Robert Wellesley Mann

Robert Wellesley Mann (1924 Brooklyn, New York–2006) was a pioneer in the field of medical prosthetics.
Mann was a professor at the Massachusetts Institute of Technology, where professor of mechanical engineering for almost 40 years and a member of the Institute of Medicine of the National Academy of Sciences.
Biography.
Mann graduated from Brooklyn Technical High School before serving in the Pacific theater in the Army during WWII. He came to MIT in 1947 on the GI Bill and received the S.B. degree in 1950, the S.M. in 1951 and the Sc.D. in 1957.
Boston Arm.
In September 1968, a team of physicians and designers, led by Mann, introduced the "Boston Digital Arm", the first prosthetic limb controlled by a brain–computer interface, wherein the wearer could control the movement of the arm by the electric signals sent by the brain to electronic instruments designed to interpret the signals.


Benzedrone

Benzedrone (4-MBC) is a designer drug which has been found since 2010 as an ingredient in a number of "bath salt" mixes sold as recreational drugs.


POLRMT

DNA-directed RNA polymerase, mitochondrial is an enzyme that in humans is encoded by the "POLRMT" gene.
Function.
This gene encodes a mitochondrial DNA-directed RNA polymerase. The gene product is responsible for mitochondrial gene expression as well as for providing RNA primers for initiation of replication of the mitochondrial genome. Although this polypeptide has the same function as the three nuclear DNA-directed RNA polymerases, it is more closely related to RNA polymerases of bacteriophage (including T7 RNA polymerase), mitochondrial polymerases of lower eukaryotes as well as chloroplastic RpoT polymerases.
Structure.
The structure of the enzyme has been solved. It exhibits an overall structure similar to that of phage RNAP, but the initiation mechanism is different in that it requires initiation factors TFAM (only in mammals) and TFB2M. Elongation requires the elongation factor TEFM. The exact termination process is less understood, but MTERF1 is thought to play a role.


5-Methoxytryptamine

5-Methoxytryptamine (5-MT), also known as mexamine, is a tryptamine derivative closely related to the neurotransmitters serotonin and melatonin. 5-MT has been shown to occur naturally in the body in low levels. It is biosynthesized via the deacetylation of melatonin in the pineal gland.
5-MT acts as a full agonist at the 5-HT1, 5-HT2, 5-HT4, 5-HT6, and 5-HT7 receptors. It has no affinity for the 5-HT3 receptor and is affinity for the 5-HT1E receptor is very weak in comparison to the other 5-HT1 receptors. Its affinity for the 5-HT5A receptor is unknown.
Measured affinity for some receptors (not a complete list):


Alternative splicing

Alternative splicing, or alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to code for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. Consequently, the proteins translated from alternatively spliced mRNAs will contain differences in their amino acid sequence and, often, in their biological functions (see Figure). Notably, alternative splicing allows the human genome to direct the synthesis of many more proteins than would be expected from its 20,000 protein-coding genes.
Alternative splicing occurs as a normal phenomenon in eukaryotes, where it greatly increases the biodiversity of proteins that can be encoded by the genome; in humans, ~95% of multi-exonic genes are alternatively spliced. There are numerous modes of alternative splicing observed, of which the most common is exon skipping. In this mode, a particular exon may be included in mRNAs under some conditions or in particular tissues, and omitted from the mRNA in others.
The production of alternatively spliced mRNAs is regulated by a system of trans-acting proteins that bind to cis-acting sites on the primary transcript itself. Such proteins include splicing activators that promote the usage of a particular splice site, and splicing repressors that reduce the usage of a particular site. Mechanisms of alternative splicing are highly variable, and new examples are constantly being found, particularly through the use of high-throughput techniques. Researchers hope to fully elucidate the regulatory systems involved in splicing, so that alternative splicing products from a given gene under particular conditions ("splicing variants") could be predicted by a "splicing code".
Abnormal variations in splicing are also implicated in disease; a large proportion of human genetic disorders result from splicing variants. Abnormal splicing variants are also thought to contribute to the development of cancer, and splicing factor genes are frequently mutated in different types of cancer.
Discovery.
Alternative splicing was first observed in 1977. The Adenovirus produces five primary transcripts early in its infectious cycle, prior to viral DNA replication, and an additional one later, after DNA replication begins. The early primary transcripts continue to be produced after DNA replication begins. The additional primary transcript produced late in infection is large and comes from 5/6 of the 32kb adenovirus genome. This is much larger than any of the individual adenovirus mRNAs present in infected cells. Researchers found that the primary RNA transcript produced by adenovirus type 2 in the late phase was spliced in many different ways, resulting in mRNAs encoding different viral proteins. In addition, the primary transcript contained multiple polyadenylation sites, giving different 3’ ends for the processed mRNAs.
In 1981, the first example of alternative splicing in a transcript from a normal, endogenous gene was characterized. The gene encoding the thyroid hormone calcitonin was found to be alternatively spliced in mammalian cells. The primary transcript from this gene contains 6 exons; the calcitonin mRNA contains exons 1–4, and terminates after a polyadenylation site in exon 4. Another mRNA is produced from this pre-mRNA by skipping exon 4, and includes exons 1–3, 5, and 6. It encodes a protein known as CGRP (calcitonin gene related peptide). Examples of alternative splicing in immunoglobin gene transcripts in mammals were also observed in the early 1980s.
Since then, alternative splicing has been found to be ubiquitous in eukaryotes. The "record-holder" for alternative splicing is a "D. melanogaster" gene called Dscam, which could potentially have 38,016 splice variants.
In 2021, it was discovered that the genome of adenovirus type 2, the adenovirus in which alternative splicing was first identified, was able to produce a much greater variety of mRNA than previously thought. By using next generation sequencing technology, researchers were able to update the human adenovirus type 2 transcriptome, and present a mind-boggling 904 unique mRNA, produced by the virus through a complex pattern of alternative splicing.
Modes.
Five basic modes of alternative splicing are generally recognized.
In addition to these primary modes of alternative splicing, there are two other main mechanisms by which different mRNAs may be generated from the same gene; multiple promoters and multiple polyadenylation sites. Use of multiple promoters is properly described as a transcriptional regulation mechanism rather than alternative splicing; by starting transcription at different points, transcripts with different 5'-most exons can be generated. At the other end, multiple polyadenylation sites provide different 3' end points for the transcript. Both of these mechanisms are found in combination with alternative splicing and provide additional variety in mRNAs derived from a gene.
<br>These modes describe basic splicing mechanisms, but may be inadequate to describe complex splicing events. For instance, the figure to the right shows 3 spliceforms from the mouse hyaluronidase 3 gene. Comparing the exonic structure shown in the first line (green) with the one in the second line (yellow) shows intron retention, whereas the comparison between the second and the third spliceform (yellow vs. blue) exhibits exon skipping. A model nomenclature to uniquely designate all possible splicing patterns has recently been proposed.
Mechanisms.
General splicing mechanism.
When the pre-mRNA has been transcribed from the DNA, it includes several introns and exons. (In nematodes, the mean is 4–5 exons and introns; in the fruit fly "Drosophila" there can be more than 100 introns and exons in one transcribed pre-mRNA.) The exons to be retained in the mRNA are determined during the splicing process. The regulation and selection of splice sites are done by trans-acting splicing activator and splicing repressor proteins as well as cis-acting elements within the pre-mRNA itself such as exonic splicing enhancers and exonic splicing silencers.
The typical eukaryotic nuclear intron has consensus sequences defining important regions. Each intron has the sequence GU at its 5' end. Near the 3' end there is a branch site. The nucleotide at the branchpoint is always an A; the consensus around this sequence varies somewhat. In humans the branch site consensus sequence is yUnAy. The branch site is followed by a series of pyrimidines – the polypyrimidine tract – then by AG at the 3' end.
Splicing of mRNA is performed by an RNA and protein complex known as the spliceosome, containing snRNPs designated U1, U2, U4, U5, and U6 (U3 is not involved in mRNA splicing). U1 binds to the 5' GU and U2, with the assistance of the U2AF protein factors, binds to the branchpoint A within the branch site. The complex at this stage is known as the spliceosome A complex. Formation of the A complex is usually the key step in determining the ends of the intron to be spliced out, and defining the ends of the exon to be retained. (The U nomenclature derives from their high uridine content).
The U4,U5,U6 complex binds, and U6 replaces the U1 position. U1 and U4 leave. The remaining complex then performs two transesterification reactions. In the first transesterification, 5' end of the intron is cleaved from the upstream exon and joined to the branch site A by a 2',5'-phosphodiester linkage. In the second transesterification, the 3' end of the intron is cleaved from the downstream exon, and the two exons are joined by a phosphodiester bond. The intron is then released in lariat form and degraded.
Regulatory elements and proteins.
Splicing is regulated by trans-acting proteins (repressors and activators) and corresponding cis-acting regulatory sites (silencers and enhancers) on the pre-mRNA. However, as part of the complexity of alternative splicing, it is noted that the effects of a splicing factor are frequently position-dependent. That is, a splicing factor that serves as a splicing activator when bound to an intronic enhancer element may serve as a repressor when bound to its splicing element in the context of an exon, and vice versa. The secondary structure of the pre-mRNA transcript also plays a role in regulating splicing, such as by bringing together splicing elements or by masking a sequence that would otherwise serve as a binding element for a splicing factor. Together, these elements form a "splicing code" that governs how splicing will occur under different cellular conditions.
There are two major types of cis-acting RNA sequence elements present in pre-mRNAs and they have corresponding trans-acting RNA-binding proteins. Splicing "silencers" are sites to which splicing repressor proteins bind, reducing the probability that a nearby site will be used as a splice junction. These can be located in the intron itself (intronic splicing silencers, ISS) or in a neighboring exon (exonic splicing silencers, ESS). They vary in sequence, as well as in the types of proteins that bind to them. The majority of splicing repressors are heterogeneous nuclear ribonucleoproteins (hnRNPs) such as hnRNPA1 and polypyrimidine tract binding protein (PTB). Splicing "enhancers" are sites to which splicing activator proteins bind, increasing the probability that a nearby site will be used as a splice junction. These also may occur in the intron (intronic splicing enhancers, ISE) or exon (exonic splicing enhancers, ESE). Most of the activator proteins that bind to ISEs and ESEs are members of the SR protein family. Such proteins contain RNA recognition motifs and arginine and serine-rich (RS) domains.
In general, the determinants of splicing work in an inter-dependent manner that depends on context, so that the rules governing how splicing is regulated from a splicing code. The presence of a particular cis-acting RNA sequence element may increase the probability that a nearby site will be spliced in some cases, but decrease the probability in other cases, depending on context. The context within which regulatory elements act includes cis-acting context that is established by the presence of other RNA sequence features, and trans-acting context that is established by cellular conditions. For example, some cis-acting RNA sequence elements influence splicing only if multiple elements are present in the same region so as to establish context. As another example, a cis-acting element can have opposite effects on splicing, depending on which proteins are expressed in the cell (e.g., neuronal versus non-neuronal PTB). The adaptive significance of splicing silencers and enhancers is attested by studies showing that there is strong selection in human genes against mutations that produce new silencers or disrupt existing enhancers.
DNA methylation and alternative splicing in social insects.
CpG DNA methylation has showed a role to regulate the alternative splicing in social insects. In honey bees ("Apis mellifera"), CpG DNA methylation seems to regulate the exon skipping based on the first few genomic studies after honey bee genome was available. CpG DNA methylation regulated alternative splicing more extensively, not only affect exon skipping, but also intron retention, and other splicing events.
Examples.
Exon skipping: "Drosophila" "dsx".
Pre-mRNAs from the "D. melanogaster" gene "dsx" contain 6 exons. In males, exons 1,2,3,5,and 6 are joined to form the mRNA, which encodes a transcriptional regulatory protein required for male development. In females, exons 1,2,3, and 4 are joined, and a polyadenylation signal in exon 4 causes cleavage of the mRNA at that point. The resulting mRNA is a transcriptional regulatory protein required for female development.
This is an example of exon skipping. The intron upstream from exon 4 has a polypyrimidine tract that doesn't match the consensus sequence well, so that U2AF proteins bind poorly to it without assistance from splicing activators. This 3' splice acceptor site is therefore not used in males. Females, however, produce the splicing activator Transformer (Tra) (see below). The SR protein Tra2 is produced in both sexes and binds to an ESE in exon 4; if Tra is present, it binds to Tra2 and, along with another SR protein, forms a complex that assists U2AF proteins in binding to the weak polypyrimidine tract. U2 is recruited to the associated branchpoint, and this leads to inclusion of exon 4 in the mRNA.
Alternative acceptor sites: "Drosophila" "".
Pre-mRNAs of the "Transformer" (Tra) gene of "Drosophila melanogaster" undergo alternative splicing via the alternative acceptor site mode. The gene Tra encodes a protein that is expressed only in females. The primary transcript of this gene contains an intron with two possible acceptor sites. In males, the upstream acceptor site is used. This causes a longer version of exon 2 to be included in the processed transcript, including an early stop codon. The resulting mRNA encodes a truncated protein product that is inactive. Females produce the master sex determination protein Sex lethal (Sxl). The Sxl protein is a splicing repressor that binds to an ISS in the RNA of the Tra transcript near the upstream acceptor site, preventing U2AF protein from binding to the polypyrimidine tract. This prevents the use of this junction, shifting the spliceosome binding to the downstream acceptor site. Splicing at this point bypasses the stop codon, which is excised as part of the intron. The resulting mRNA encodes an active Tra protein, which itself is a regulator of alternative splicing of other sex-related genes (see "dsx" above).
Exon definition: Fas receptor.
Multiple isoforms of the Fas receptor protein are produced by alternative splicing. Two normally occurring isoforms in humans are produced by an exon-skipping mechanism. An mRNA including exon 6 encodes the membrane-bound form of the Fas receptor, which promotes apoptosis, or programmed cell death. Increased expression of Fas receptor in skin cells chronically exposed to the sun, and absence of expression in skin cancer cells, suggests that this mechanism may be important in elimination of pre-cancerous cells in humans. If exon 6 is skipped, the resulting mRNA encodes a soluble Fas protein that does not promote apoptosis. The inclusion or skipping of the exon depends on two antagonistic proteins, TIA-1 and polypyrimidine tract-binding protein (PTB).
This mechanism is an example of exon definition in splicing. A spliceosome assembles on an intron, and the snRNP subunits fold the RNA so that the 5' and 3' ends of the intron are joined. However, recently studied examples such as this one show that there are also interactions between the ends of the exon. In this particular case, these exon definition interactions are necessary to allow the binding of core splicing factors prior to assembly of the spliceosomes on the two flanking introns.
Repressor-activator competition: HIV-1 "tat" exon 2.
HIV, the retrovirus that causes AIDS in humans, produces a single primary RNA transcript, which is alternatively spliced in multiple ways to produce over 40 different mRNAs. Equilibrium among differentially spliced transcripts provides multiple mRNAs encoding different products that are required for viral multiplication. One of the differentially spliced transcripts contains the "tat" gene, in which exon 2 is a cassette exon that may be skipped or included. The inclusion of tat exon 2 in the RNA is regulated by competition between the splicing repressor hnRNP A1 and the SR protein SC35. Within exon 2 an exonic splicing silencer sequence (ESS) and an exonic splicing enhancer sequence (ESE) overlap. If A1 repressor protein binds to the ESS, it initiates cooperative binding of multiple A1 molecules, extending into the 5’ donor site upstream of exon 2 and preventing the binding of the core splicing factor U2AF35 to the polypyrimidine tract. If SC35 binds to the ESE, it prevents A1 binding and maintains the 5’ donor site in an accessible state for assembly of the spliceosome. Competition between the activator and repressor ensures that both mRNA types (with and without exon 2) are produced.
Adaptive significance.
Alternative splicing is one of several exceptions to the original idea that one DNA sequence codes for one polypeptide (the One gene-one enzyme hypothesis). It might be more correct now to say "One gene – many polypeptides". External information is needed in order to decide which polypeptide is produced, given a DNA sequence and pre-mRNA. Since the methods of regulation are inherited, this provides novel ways for mutations to affect gene expression.
It has been proposed that for eukaryotes alternative splicing was a very important step towards higher efficiency, because information can be stored much more economically. Several proteins can be encoded by a single gene, rather than requiring a separate gene for each, and thus allowing a more varied proteome from a genome of limited size. It also provides evolutionary flexibility. A single point mutation may cause a given exon to be occasionally excluded or included from a transcript during splicing, allowing production of a new protein isoform without loss of the original protein. Studies have identified intrinsically disordered regions (see Intrinsically unstructured proteins) as enriched in the non-constitutive exons suggesting that protein isoforms may display functional diversity due to the alteration of functional modules within these regions. Such functional diversity achieved by isoforms is reflected by their expression patterns and can be predicted by machine learning approaches. Comparative studies indicate that alternative splicing preceded multicellularity in evolution, and suggest that this mechanism might have been co-opted to assist in the development of multicellular organisms.
Research based on the Human Genome Project and other genome sequencing has shown that humans have only about 30% more genes than the roundworm "Caenorhabditis elegans", and only about twice as many as the fly "Drosophila melanogaster". This finding led to speculation that the perceived greater complexity of humans, or vertebrates generally, might be due to higher rates of alternative splicing in humans than are found in invertebrates.
However, a study on samples of 100,000 ESTs each from human, mouse, rat, cow, fly ("D. melanogaster"), worm ("C. elegans"), and the plant "Arabidopsis thaliana" found no large differences in frequency of alternatively spliced genes among humans and any of the other animals tested. Another study, however, proposed that these results were an artifact of the different numbers of ESTs available for the various organisms. When they compared alternative splicing frequencies in random subsets of genes from each organism, the authors concluded that vertebrates do have higher rates of alternative splicing than invertebrates.
Disease.
Changes in the RNA processing machinery may lead to mis-splicing of multiple transcripts, while single-nucleotide alterations in splice sites or cis-acting splicing regulatory sites may lead to differences in splicing of a single gene, and thus in the mRNA produced from a mutant gene's transcripts. A study in 2005 involving probabilistic analyses indicated that greater than 60% of human disease-causing mutations affect splicing rather than directly affecting coding sequences. A more recent study indicates that one-third of all hereditary diseases are likely to have a splicing component. Regardless of exact percentage, a number of splicing-related diseases do exist. As described below, a prominent example of splicing-related diseases is cancer.
Abnormally spliced mRNAs are also found in a high proportion of cancerous cells. Combined RNA-Seq and proteomics analyses have revealed striking differential expression
of splice isoforms of key proteins in important cancer pathways. It is not always clear whether such aberrant patterns of splicing contribute to the cancerous growth, or are merely consequence of cellular abnormalities associated with cancer. For certain types of cancer, like in colorectal and prostate, the number of splicing errors per cancer has been shown to vary greatly between individual cancers, a phenomenon referred to as transcriptome instability. Transcriptome instability has further been shown to correlate grealty with reduced expression level of splicing factor genes. Mutation of "DNMT3A" has been demonstrated to contribute to hematologic malignancies, and that "DNMT3A"-mutated cell lines exhibit transcriptome instability as compared to their isogenic wildtype counterparts.
In fact, there is actually a reduction of alternative splicing in cancerous cells compared to normal ones, and the types of splicing differ; for instance, cancerous cells show higher levels of intron retention than normal cells, but lower levels of exon skipping. Some of the differences in splicing in cancerous cells may be due to the high frequency of somatic mutations in splicing factor genes, and some may result from changes in phosphorylation of trans-acting splicing factors. Others may be produced by changes in the relative amounts of splicing factors produced; for instance, breast cancer cells have been shown to have increased levels of the splicing factor SF2/ASF. One study found that a relatively small percentage (383 out of over 26000) of alternative splicing variants were significantly higher in frequency in tumor cells than normal cells, suggesting that there is a limited set of genes which, when mis-spliced, contribute to tumor development. It is believed however that the deleterious effects of mis-spliced transcripts are usually safeguarded and eliminated by a cellular posttranscriptional quality control mechanism termed Nonsense-mediated mRNA decay [NMD].
One example of a specific splicing variant associated with cancers is in one of the human DNMT genes. Three DNMT genes encode enzymes that add methyl groups to DNA, a modification that often has regulatory effects. Several abnormally spliced DNMT3B mRNAs are found in tumors and cancer cell lines. In two separate studies, expression of two of these abnormally spliced mRNAs in mammalian cells caused changes in the DNA methylation patterns in those cells. Cells with one of the abnormal mRNAs also grew twice as fast as control cells, indicating a direct contribution to tumor development by this product.
Another example is the "Ron" ("MST1R") proto-oncogene. An important property of cancerous cells is their ability to move and invade normal tissue. Production of an abnormally spliced transcript of "Ron" has been found to be associated with increased levels of the SF2/ASF in breast cancer cells. The abnormal isoform of the Ron protein encoded by this mRNA leads to cell motility.
Overexpression of a truncated splice variant of the FOSB gene – ΔFosB – in a specific population of neurons in the nucleus accumbens has been identified as the causal mechanism involved in the induction and maintenance of an addiction to drugs and natural rewards.
Recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation determines not only what parts of the genome are expressed but also how they are spliced.
Genome-wide analysis.
Genome-wide analysis of alternative splicing is a challenging task. Typically, alternatively spliced transcripts have been found by comparing EST sequences, but this requires sequencing of very large numbers of ESTs. Most EST libraries come from a very limited number of tissues, so tissue-specific splice variants are likely to be missed in any case. High-throughput approaches to investigate splicing have, however, been developed, such as: DNA microarray-based analyses, RNA-binding assays, and deep sequencing. These methods can be used to screen for polymorphisms or mutations in or around splicing elements that affect protein binding. When combined with splicing assays, including "in vivo" reporter gene assays, the functional effects of polymorphisms or mutations on the splicing of pre-mRNA transcripts can then be analyzed.
In microarray analysis, arrays of DNA fragments representing individual exons ("e.g." Affymetrix exon microarray) or exon/exon boundaries ("e.g." arrays from ExonHit or Jivan) have been used. The array is then probed with labeled cDNA from tissues of interest. The probe cDNAs bind to DNA from the exons that are included in mRNAs in their tissue of origin, or to DNA from the boundary where two exons have been joined. This can reveal the presence of particular alternatively spliced mRNAs.
CLIP (Cross-linking and immunoprecipitation) uses UV radiation to link proteins to RNA molecules in a tissue during splicing. A trans-acting splicing regulatory protein of interest is then precipitated using specific antibodies. When the RNA attached to that protein is isolated and cloned, it reveals the target sequences for that protein. Another method for identifying RNA-binding proteins and mapping their binding to pre-mRNA transcripts is "Microarray Evaluation of Genomic Aptamers by shift (MEGAshift)".net This method involves an adaptation of the "Systematic Evolution of Ligands by Exponential Enrichment (SELEX)" method together with a microarray-based readout. Use of the MEGAshift method has provided insights into the regulation of alternative splicing by allowing for the identification of sequences in pre-mRNA transcripts surrounding alternatively spliced exons that mediate binding to different splicing factors, such as ASF/SF2 and PTB. This approach has also been used to aid in determining the relationship between RNA secondary structure and the binding of splicing factors.
Deep sequencing technologies have been used to conduct genome-wide analyses of mRNAs – unprocessed and processed – thus providing insights into alternative splicing. For example, results from use of deep sequencing indicate that, in humans, an estimated 95% of transcripts from multiexon genes undergo alternative splicing, with a number of pre-mRNA transcripts spliced in a tissue-specific manner. Functional genomics and computational approaches based on multiple instance learning have also been developed to integrate RNA-seq data to predict functions for alternatively spliced isoforms. Deep sequencing has also aided in the "in vivo" detection of the transient lariats that are released during splicing, the determination of branch site sequences, and the large-scale mapping of branchpoints in human pre-mRNA transcripts.
Use of reporter assays makes it possible to find the splicing proteins involved in a specific alternative splicing event by constructing reporter genes that will express one of two different fluorescent proteins depending on the splicing reaction that occurs. This method has been used to isolate mutants affecting splicing and thus to identify novel splicing regulatory proteins inactivated in those mutants.
Databases.
There is a collection of alternative splicing databases. These databases are useful for finding genes having pre-mRNAs undergoing alternative splicing and alternative splicing events or to study the functional impact of alternative splicing.


Idah Sithole-Niang

Idah Sithole-Niang (born 1957) is a Zimbabwean biochemist and educator. Her main area of research has been viruses which attack the cowpea, one of the major food crops of Zimbabwe.
Biography.
Idah Sithole was born in Hwange, Zimbabwe on 2 October 1957. She attended the University of London, on scholarship, earning a BS in biochemistry in 1982. When she was awarded a USAID Fellowship in 1983, Sithole chose to continued her education, studying plant and virus genetics. She earned a PhD in 1988 from Michigan State University (MSU) of Lansing, Michigan. She completed a post-doctoral fellowship at the Plant Research Laboratory at MSU researching the genetics of photosynthesis in cyanobacteria on the first William L. Brown Fellowship, awarded by the Resources Development Foundation. Returning to Zimbabwe, she became a Lecturer at the University of Zimbabwe in 1992 on viruses which infect plants. Her chief area of research is the potyvirus, which attacks the cowpea, a legume which is a chief food crop of Zimbabwe. That same year, she married Sheikh Ibrahima Niang, a Senegalese professor of anthropology, whom she met at Michigan State University. They have a commuting marriage, as he works at the University of Cheikh Anta Diop in Dakar, Senegal. She was awarded a Rockefeller Foundation Fellowship for careers in Biotechnology between 1992 and 1995. She has continued teaching and researching, publishing numerous papers. In 2006 she was made Associate Professor at the University of Zimbabwe.
Sithole-Niang is in favor of genetic modifications to make cowpeas resistant to disease and believes that GMO versions of traditional crops are beneficial to developing nations. Working with the Network for the Genetic Improvement of Cowpea for Africa (NGICA) as a coordinator, she has consulted with other international experts, in part because of the lack of funding available from within Zimbabwe. She has served as both a member and a board member of numerous organizations including: Steering Committee Trustee of the African Women for Agriculture and Research and Development (AWARD) Programme, the American Association for the Advancement of Science, the American Society for Virologists, Oversight Committee of the Improved Maize for African Soils (IMAS), Selection Committee Chair of the Joshua Nkomo Scholarships, the New York Academy of Sciences, Vice Chair of the Research Council of Zimbabwe, Selection Committee for the Rhodes Scholarships in Zimbabwe and the Zimbabwe Academy of Sciences. Sithole-Niang is a Technical Advisor to the Program for Biosafety Systems for sub-Saharan Africa.


Dichloroisoprenaline

Dichloroisoprenaline (DCI), also known as dichloroisoproterenol, was the first beta blocker ever to be developed. It is non-selective for the β1-adrenergic and β2-adrenergic receptors. DCI has low potency and acts as a partial agonist/antagonist at these receptors.
Although DCI was of no clinical value itself, further developments from DCI eventually led to the development of the clinical candidate pronethalol (withdrawn due to carcinogenicity) and subsequently propranolol (the first clinically successful beta blocker).
Dichloroisoprenaline is a racemic mixture of enantiomers.


Poromycena hanedai

Poromycena hanedai is a species of agaric fungus in the family Mycenaceae. Found in Japan, the species was first described by Kobayasi in 1951. It is bioluminescent.


2,5-Diamino-6-hydroxy-4-(5-phosphoribosylamino)pyrimidine

2,5-diamino-6-hydroxy-4-(5-phosphoribosylamino)pyrimidine is a metabolite in the purine metabolism, formed by the hydrolysis of GTP by GTP cyclohydrolase II. Alternatively two separate enzymes can carry out this reaction, initially GTP cyclohydrolase IIa hydrolyses the 8,9 bond to form 2-Amino-5-formylamino-6-(5-phospho-D-ribosylamino)pyrimidin-4(3H)-one, followed by de-formylation by 2-amino-5-formylamino-6-ribosylaminopyrimidin-4(3H)-one 5'-monophosphate deformylase. 2,5-diamino-6-hydroxy-4-(5-phosphoribosylamino)pyrimidine is deaminated by Diaminohydroxyphosphoribosylaminopyrimidine deaminase to form 5-amino-6-(5-phosphoribosylamino)uracil.


Linoleate diol synthase

In enzymology, a linoleate diol synthase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are linoleate and O2, whereas its product is (9Z,12Z)-(7S,8S)-dihydroxyoctadeca-9,12-dienoate.
This enzyme belongs to the family of oxidoreductases, specifically those acting on single donors with O2 as oxidant and incorporation of two atoms of oxygen into the substrate (oxygenases). The oxygen incorporated need not be derived from O2. The systematic name of this enzyme class is linoleate:oxygen 7S,8S-oxidoreductase. This enzyme is also called linoleate (8R)-dioxygenase. This enzyme participates in linoleic acid metabolism.


Epoxide hydrolase

Epoxide hydrolases (EH's), also known as epoxide hydratases, are enzymes that metabolize compounds that contain an epoxide residue; they convert this residue to two hydroxyl residues through an epoxide hydrolyis reaction to form diol products. Several enzymes possess EH activity. Microsomal epoxide hydrolase (epoxide hydrolase 1, EH1, or mEH), soluble epoxide hydrolase (sEH, epoxide hydrolase 2, EH2, or cytoplasmic epoxide hydrolase), and the more recently discovered but not as yet well defined functionally, epoxide hydrolase 3 (EH3) and epoxide hydrolase 4 (EH4) are structurally closely related isozymes. Other enzymes with epoxide hydrolase activity include leukotriene A4 hydrolase, Cholesterol-5,6-oxide hydrolase, MEST (gene) (Peg1/MEST), and Hepoxilin-epoxide hydrolase. The hydrolases are distinguished from each other by their substrate preferences and, directly related to this, their functions.
Epoxide hydrolase types.
mEH (EH1), sEH (EH2), EH3, and EH4 isozymes.
Humans express four epoxide hydrolase isozymes: mEH, sEH, EH3, and EH4. These isozymes are known (mEH and sEH) or presumed (EH3 and EH4) to share a common structure that includes containing an Alpha/beta hydrolase fold and a common reaction mechanism wherein they add water to epoxides to form vicinal cis (see (cis-trans isomerism); see (epoxide#Olefin oxidation using organic peroxides and metal catalysts)) diol products. They differ, however, in subcellular location, substrate preferences, tissue expression, and/or function.
mEH.
mEH is widely expressed in virtually all mammalian cells as an endoplasmic reticulum-bound (i.e. microsomal-bound) enzyme with its C terminal catalytic domain facing the cytoplasm; in some tissues, however, mEH has been found bound to the cell surface plasma membrane with its catalytic domain facing the extracellular space. The primary function of mEH is to convert potentially toxic xenobiotics and other compounds that possess epoxide residues (which is often due to their initial metabolism by cytochrome P450 enzymes to epoxides) to diols. Epoxides are highly reactive electrophilic compounds that form adducts with DNA and proteins and also cause strand breaks in DHA; in consequence, epoxides can cause gene mutations, cancer, and the inactivation of critical proteins. The diols thereby formed are usually not toxic or far less toxic than their epoxide predecessors, are readily further metabolized, and ultimately excreted in the urine. mEH also metabolizes certain epoxides of polyunsaturated fatty acids such as the epoxyeicosatrienoic acids (EETs) but its activity in doing this is far less than that of sEH; mEH therefore may play a minor role, compared to sEH, in limiting the bioactivity of these cell signaling compounds (see microsomal epoxide hydrolase).
sEH.
sEH is widely expressed in mammalian cells as a cytosolic enzyme where it primarily serves the function of converting epoxyeicosatrienoic acids (EETs), epoxyeicosatetraenoic acids (EPAs), and epoxydocosapentaenoic acids (DPAs) to their corresponding diols, thereby limiting or ending their cell signaling actions; in this capacity, sEH appears to play a critical in vivo role in limiting the effects of these epoxides in animal models and possibly humans. However, sEH also metabolizes the epoxides of linoleic acid viz., Vernolic acid (leukotoxins) and Coronaric acids (isoleukotoxins) to their corresponding diols which are highly toxic in animal models and possibly humans (see Vernolic acid#toxicity, Coronaric acid#toxicity, and soluble epoxide hydrolase). sEH also possesses hepoxilin-epoxide hydrolase activity, converting bioactive hepoxilins to their inactive trioxilin products (see below section "Hepoxilin-epoxide hydrolase").
EH3.
Human EH3 is a recently characterized protein with epoxy hydrolase activity for metabolizing epoxyeicosatrienoic acids (EETs) and vernolic acids (leukotoxins) to their corresponding diols; in these capacities they may thereby limit the cell signaling activity of the EETs and contribute to the toxicity of the leukotoxins. mRNA for EH3 is most strongly expressed in the lung, skin, and upper gastrointestinal tract tissues of mice. The function of EH3 in humans, mice, or other mammals has not yet been determined although the gene for EH3 has been validated as being hypermethylated on CpG sites in its promoter region in human prostate cancer tissue, particularly in the tissues of more advanced or morphologically-based (i.e. Gleason score) more aggressive cancers; this suggests that the gene silencing of EH3 due to this hypermethylation may contribute to the onset and/or progression of prostate cancer. Similar CpG site hypermethylations in the promoter of for the EH3 gene have been validated for other cancers. This promoter methylation pattern, although not yet validated, was also found in human malignant melanoma.
EH4.
The gene for EH4, EPHX4, is projected to encode an epoxide hydrolase closely related in amino acid sequence and structure to mEH, sEH, and EH3. The activity and function of EH4 has not yet been defined.
Other epoxy hydrolases.
Leukotriene A4 hydrolase.
Leukotriene A4 hydrolase (LTA4H) acts primarily, if not exclusively, to hydrolyze leukotriene A4 (LTA4, i.e. 5S,6S-oxido-7"E",9"E",11"Z",14"Z"-eicosatetetraenoic acid; IUPAC name 4-{(2S,3S)-3-[(1E,3E,5Z,8Z)-1,3,5,8-Tetradecatetraen-1-yl]-2-oxiranyl}butanoic acid) to its diol metabolite, leukotriene B4 (LTB4, i.e. 5"S",12"R"-dihydroxy-6"Z",8"E",10"E",14"Z"-icosatetraenoic acid; IUPA name 5S,6Z,8E,10E,12R,14Z)-5,12-Dihydroxy-6,8,10,14-icosatetraenoic acid). LTB4 is an important recruiter and activator of leukocytes involved in mediation in inflammatory responses and diseases. The enzyme also possess aminopeptidase activity, degrading, for example, the leukocyte chemotactic factor tripeptide, Pro-Gly-Pro (PGP); the function of the aminopeptidase activity of LTA4AH is unknown but has been proposed to be involved in limiting inflammatory reactions caused by this or other aminopeptidase-susceptible peptides.
Cholesterol-5,6-oxide hydrolase.
(Cholesterol epoxide hydrolase or ChEH), is located in the endoplasmic reticulum and to a lesser extent plasma membrane of various cell types but most highly express in liver. The enzyme catalyzes the conversion of certain 3-hydroxyl-5,6-epoxides of cholesterol to their 3,5,6-trihydroxy products (see Cholesterol-5,6-oxide hydrolase). The function of ChEH is unknown.
Peg1/MEST.
The substrate(s) and physiological function of Peg1/MEST are not known; however, the protein may play a role in mammalian development and abnormalities in its expression by its gene (PEG1/MEST)by, for example, loss of Genomic imprinting, overexpression, or promoter switching, has been linked to certain types of cancer and tumors in humans such as invasive cervical cancer, uterine leiomyomas, and cancers of the breast, lung, and colon (see MEST (gene)).
Hepoxilin-epoxide hydrolase.
Hepoxilin-epoxide hydrolase or hepoxilin hydrolase is currently best defined as an enzyme activity that converts the biologically active monohydroxy-epoxide metabolites of arachidonic acid hepoxilin A3s and hepoxilin B3s to essentially inactive trihydroxy products, the trioxilins. That is, hepoxilin A3s (8-hydroxy-11,12-oxido-5"Z",9"E",14"Z"-eicosatrienoic acid) are metabolized to trioxilin A3s (8,11,12-trihydroxy-5"Z",9"E",14"Z"-eicosatrienoic acids) and hepoxilins B3s (10-hydroxy-11,12-oxido-5"Z",8"Z",14"Z"-eicosatrienoic acids) are metabolized to trioxilin B3s (10,11,12-trihydroxy-5"Z",8"Z",14"Z"-eicosatrienoic acids). However, this activity has not been characterized at the purified protein or gene level and recent work indicate that sEH readily metabolizes an hepoxilin A3 to a trioxilin A3 and that hepoxilin-epoxide hydrolase activity is due to sEH, at least as it is detected in mouse liver.
Mycobacterium tuberculosis.
This causative agent of tuberculosis expresses at least six different forms of epoxide hydrolase (forms A-F). The structure of epoxide hydrolase B reveals that the enzyme is a monomer and contains an alpha/beta hydrolase fold. In addition to providing insights into the enzyme mechanism, this hydrolase currently serves as a platform for rational drug design of potent inhibitors. In particular, urea based inhibitors have been developed. These inhibitors directly target the catalytic cavity. It is hypothesized that the structure of epoxide hydrolase B may allow for drug design to inhibit all other Mycobacterium tuberculosis hydrolases as long as they contain similar alpha/beta folds. The structure of hydrolase B contains a cap domain, which is hypothesized to regulate the active site of the hydrolase. Furthermore, Asp104, His333, and Asp302 form the catalytic triad of the protein and is critical to function of the protein. At present, other structures of Mycobacterium tuberculosis hydrolase have not been solved. Model studies on pharmacological susceptibility of these epoxide hydrolases continue.


Christine A. Hrycyna

Christine A. Hrycyna is a Professor of Biochemistry at Purdue University. She studies multi-drug resistance in human cancer, which usually occurs due to over expression of the MDR1 gene.
Early life and education.
Hrycyna studied at Middlebury College and graduated in 1988. She was a postgraduate student at the University of California, Los Angeles, and earned her doctorate in 1993. She worked in Steven Clarke's laboratory on protein modifications. Hrycyna was a postdoctoral fellow at The Jane Coffin Childs Memorial Fund for Medical Research and the National Cancer Institute.
Research and career.
Hrycyna works at Purdue University, which she joined as an Assistant Professor in 2000. Multidrug resistance cancer often arrises due to over expression of an energy-dependent multi-drug efflux pump, the multi-drug transporter P-glycoprotein. Whilst it is understood that P-glycoproteins are important in the movement of pharmalogical agents, their mechanism as a drug efflux pump is unclear. She looked at the mechanism of the efflux pump and how it functions in normal and cancerous cells. P-glycoproteins can be recognised by several different substrates, but their affinity is reduced during a catalytic transfer state. The "MD"R1 gene results in expression of a P-glycoprotein and related drug transporter MXR1, which are involved in mitoxantrone resistance. She found that amino acid 483, which is present in MXR/BCRP/ABCP gene, changes when the gene is over-expressed. Unfortunately, cancers treated with a variety of anti-cancer drugs can develop resistance to other cytotoxic agents.
Drug transporters like MXR1 and P-glycoprotein are part of the ATP-binding cassette transporter group. Hrycyna studies the cellular function of ABC-transporters and how they have impact human disease. She has worked on new drugs for the treatment of HIV, which work by blocking the action of ZMPSTE24. Hrycyna studies the post-translational modification of eukaryotic proteins, including the small G proteins. Eukaryotic proteins undergo three modifications, including isoprenylation, proteolysis and methylesterification, and are synthesised with the C-terminal sequence Cys-Xaa-Xaa-Xaa.
In 2011 Hrycyna wrote the book "Protein Prenylation".
Academic service.
Hrycyna has been awarded the Purdue University Department of Chemistry Arthur E. Kelley Undergraduate Award for Excellence in Teaching three times. She has been involved with the curriculum review for the Department of Chemistry, recreating their chemistry sequence for life sciences majors. In 2018 Hrycyna was named as one of the Purdue University 150th Anniversary Professors.
Awards and honours.
Her awards and honours include:


Pseudopeptidoglycan

Pseudopeptidoglycan (also known as pseudomurein) is a major cell wall component of some Archaea that differs from bacterial peptidoglycan in chemical structure, but resembles bacterial peptidoglycan in function and physical structure. Pseudopeptidoglycan, in general, is only present in a few methanogenic archaea. The basic components are N-acetylglucosamine and N-acetyltalosaminuronic acid (bacterial peptidoglycan containing "N"-acetylmuramic acid instead), which are linked by β-1,3-glycosidic bonds.
Lysozyme, a host defense mechanism present in human secretions (e.g. saliva and tears) breaks β-1,4-glycosidic bonds to degrade peptidoglycan. However, because pseudopeptidoglycan has β-1,3-glycosidic bonds, lysozyme is ineffective. These fundamental differences in cell wall chemistry suggest that archaeal cell walls and bacterial cell walls have not evolved from a common ancestor but are only the result of a convergent evolution.
No archaeal enzymes are known that cleave the β-1,3-glycosidic bonds in pseudopeptidoglycan, but it can be degraded by pseudomurein endoisopeptidase encoded by two prophages. The pseudomurein endoisopeptidases function by cleaving the peptide links between adjacent pseudopeptidoglycan strands.
Structure.
Pseudopeptidoglycan is composed of two sugars, N-acetylglucosamine and N-acetyltalosaminuronic acid. These sugars are made of different amino acids, and the peptide cross-links within pseudopeptidoglycan are formed with different amino acids. The peptide bond is formed between the lysine of a N-acetyltalosaminuronic acid and a glutamine of a parallel N-acetyltalosaminuronic acid. Pseudopeptidoglycan, like peptidoglycan in bacteria, forms a mesh-like layer outside of the plasma membrane of the archaea.
Function.
Only a few methanogenic archaea have cell walls composed of pseudopeptidoglycan. This component functions much like peptidoglycan in a bacterial cell. Pseudopeptidoglycan is used by the archaeal cell to determine its shape and provide structure to the cell. It is also used to protect the cell from undesired molecules or anything harmful in its environment.
Effects of Different Bacterial Medicines on Pseudopeptidoglycan.
Lysozyme.
Lysozyme is a natural defense mechanism in humans that has the ability to break down peptidoglycan in bacterial cells. It degrades the peptidoglycan by targeting the β-1,4-glycosidic bonds that connect the alternating amino sugars in which it is composed of. This degradation of the glycosidic bonds within peptidoglycan cause the sugars to separate and inhibit the structural integrity of the peptidoglycan and the bacteria.
Pseudopeptidoglycan, however, is composed of a different acidic amino sugar, which is N-acetyltalosaminuronic acid. This difference is the reason that it has β-1,3-glycosidic bonds (as opposed to the β-1,4-glycosidic bonds in bacteria). Lysozymes targets the linkage in peptidoglycan, and without that, becomes ineffective against pseudopeptidoglycan.
Penicillin.
Penicillin is a group of antibiotics that have been effective against many bacterial infections. It attacks bacteria by targeting and inhibiting the transpeptidase that catalyzes the cross-linking of the amino sugars in peptidoglycan. However, peptidoglycan contains different amino sugars, and therefore, a different catalysis enzyme is used. The different amino acids cause antibiotics, that target cell walls like penicillin, to be ineffective against pseudopeptidoglycan.


Melanocortin 3 receptor

Melanocortin receptor 3 is a protein that in humans is encoded by the "MC3R" gene.
Function.
This gene encodes MC3, a G-protein coupled receptor for melanocyte-stimulating hormone and adrenocorticotropic hormone that is expressed in tissues other than the adrenal cortex and melanocytes. This gene maps to the same region as the locus for benign neonatal epilepsy. Mice deficient for this gene have increased fat mass despite decreased food intake suggesting a role for this gene product in the regulation of energy homeostasis.
Research.
Studies performed by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), found that two specific polymorphisms in the MC3R gene may be associated with pediatric obesity and greater body mass because of greater energy intake. Children who were homozygous for C17A + G241A consumed approximately 38% more than those who did not contain aforementioned polymorphisms. The study concluded that these genetic variants did not affect energy expenditure.


Molecular Membrane Biology

Molecular Membrane Biology is a peer-reviewed scientific journal that publishes review articles of biomembranes at the molecular level. It is published by Taylor & Francis. The editor-in-chief is Vincent Postis.