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The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.
The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.
The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions. (c) 2002 Elsevier Science Ltd.
Monooxygenases are frequently involved in the pathways that mediate the pivotal role of microorganisms in recycling carbon from the environment. A structural study of a monooxygenase from Pseudomonas aeruginosa that was identified as a phenylalanine hydroxylase has been initiated. The single-domain monomeric protein harbours a non-haem iron at the active site. The sequence identity to the catalytic domains of tyrosine and tryptophan hydroxylases suggests that the enzyme is not restricted to the substrate phenylalanine alone. Here, the cloning, purification and crystallization of native and SeMet-labelled P. aeruginosa phenylalanine hydroxylase are reported. Crystals grew in space group P6(1), with unit-cell parameters a = b = 210.5, c = 100.7 A, and diffracted to a d spacing of 2.0 A. Crystals of SeMet-labelled protein were used to collect a three-wavelength multiple anomalous dispersion (MAD) data set around the Se K edge.
Alpha-actinin forms antiparallel homodimers that cross-link actin filaments from adjacent sarcomeres within the Z-discs of striated muscle. The N-terminal actin-binding domain (ABD) is composed of two calponin homology (CH) domains followed by four spectrin-like repeats and a calmodulin-like EF-hand domain at the C-terminus. The ABD of human alpha-actinin crystallizes in space group P2(1), with unit-cell parameters a = 101.9, b = 38.4, c = 154.9 A, beta = 109.2 degrees. A complete native data set from a native crystal was collected extending to 2.0 A resolution and a single-wavelength anomalous dispersion (SAD) data set to 2.9 A resolution was collected from a selenomethionine-labelled microcrystal using the microfocusing beamline ID-13 at the ESRF. Analysis of the anomalous contribution shows a rapid decrease in the sigma(normal)/sigma(anomal) ratio owing to radiation damage.
Transthyretin is a tetrameric plasma protein associated with two forms of amyloid disease. The structure of the highly amyloidogenic transthyretin triple mutant TTRG53S/E54D/L55S determined at 2.3 A resolution reveals a novel conformation: the beta-slip. A three-residue shift in beta strand D places Leu-58 at the position normally occupied by Leu-55 now mutated to serine. The beta-slip is best defined in two of the four monomers, where it makes new protein-protein interactions to an area normally involved in complex formation with retinol-binding protein. This interaction creates unique packing arrangements, where two protein helices combine to form a double helix in agreement with fiber diffraction and electron microscopy data. Based on these findings, a novel model for transthyretin amyloid formation is presented.
Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3'-triiodo-l-thyronine (T(3)) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine, T(4)). Human TTR has higher affinity for T(4) than T(3), whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 A resolution and bound to ligands T(3) and T(4), both at 1.9 A resolution. The apo structure is similar to human TTR with structural changes only at beta-strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR.T(4) complex shows the T(4)-binding site to be similar but not identical to human TTR, whereas the TTR.T(3) complex shows the I3' halogen situated at the site normally occupied by the hydroxyl group of T(4). The significantly wider entrance of the hormone-binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T(3) and T(4).
The Y114C mutation in human transthyretin (TTR) is associated with a particular form of familial amyloidotic polyneuropathy. We show that vitreous aggregates ex vivo consist of either regular amyloid fibrils or disordered disulfide-linked precipitates that maintain the ability to bind Congo red. Furthermore, we demonstrate in vitro that the ATTR Y114C mutant exists in three forms: one unstable but nativelike tetrameric form, one highly aggregated form in which a network of disulfide bonds is formed, and one fibrillar form. The disulfide-linked aggregates and the fibrillar form of the mutant can be induced by heat induction under nonreduced and reduced conditions, respectively. Both forms are recognized by the amyloid specific antibody MAB(39-44). In a previous study, we have linked exposure of this epitope in TTR to a three-residue shift in beta-strand D. The X-ray crystallographic structure of reduced tetrameric ATTR Y114C shows a structure similar to that of the wild type but with a more buried position of Cys10 and with beta-mercaptoethanol associated with Cys114, verifying the strong tendency for this residue to form disulfide bonds. Combined with the ex vivo data, our in vitro findings suggest that ATTR Y114C can lead to disease either by forming regular unbranched amyloid fibrils or by forming disulfide-linked aggregates that maintain amyloid-like properties but are unable to form regular amyloid fibrils.
The human plasma protein transthyretin (TTR) is a highly stable soluble homotetrameric protein. Still, conformational changes in the wild type protein can lead to self-assembly into insoluble amyloid fibrils. In addition, 74 point mutations are known to enhance amyloid formation causing familial amyloidotic polyneuropathy (PAP). Alignment of TTR sequences from twenty different species shows that only six of these mutations occur as natural amino acids in other organisms. In this paper we analyse the distribution of FAP mutations within the three-dimensional structure of TTR. Contradictory to what might be expected from protein stability studies, the mutations are not restricted to structurally rigid parts of the molecule, nor are they concentrated at the monomer interaction sites.
The Drosophila Minutes are haploinsufficient mutations that are defective in ribosomal protein (rp) production, resulting in short, thin bristles, delayed development and recessive lethality. In a Minute fly, the amount of rp gene messenger RNA (mRNA) is reduced to >or=50% of the normal amount of gene product, and becomes rate limiting for ribosome biogenesis, cell proliferation and growth. Haploinsufficiency increases the vulnerability to complete loss of gene function (homozygous null state) if hit by a second mutation. Because of the homozygous lethality, it has only been possible to study the effects of Minute mutations in heterozygous animals. To be able to study the consequences of a loss-of-function of an rp gene (0%>mRNA<50%) in developing and differentiated cells we used heritable RNA interference (RNAi) in combination with the yeast GAL4/UAS binary system to spatiotemporally knock down the ribosomal protein L14 (RpL14) gene. We show, at the RNA and phenotypic levels, that RNAi efficiently reduces RpL14 gene expression throughout development, causing lethality and distinct and dramatic somatic anomalies in both developing and differentiated cells.
The prokaryotic signal recognition particle Ffh and its receptor FtsY allow targeting of proteins into or across the plasma membrane. The targeting process is GTP dependent and the two proteins constitute a distinct GTPase family. The receptor FtsY is composed of A and NG domains where the NG's GTPase domain plays a critical role in the targeting process. In this study, we describe two X-ray structures determined independently of each other of the NG domain of FtsY from Mycoplasma mycoides (MmFtsY). The two structures are markedly different in three of the nucleotide-binding segments, GI (P-loop), GII, and GIII, making only one of the structures compatible with nucleotide binding. Interestingly, the two distinct conformations of the nucleotide-binding segments of MmFtsY are similar to the apo- and ADP-loaded forms of certain ATPases. The structure of the extended interface between the A and NG domains of MmFtsY provides new insights into the role of the A domain for phospholipid interaction.
The prokaryotic signal recognition particle (SRP) pathway comprises two proteins, Ffh and FtsY, homologous to the SRP54 and SRalpha proteins in the more complex eukaryotic system. All four proteins are part of a unique subfamily of GTPases. Four truncated versions of the 412 amino-acid FtsY receptor protein from Mycoplasma mycoides have been cloned, expressed in Escherichia coli and purified. Purified proteins from all constructs and the full-length FtsY protein were subjected to crystallization trials. Crystals were obtained for the construct which comprised residues 98-412 corresponding to the conserved NG-domain (residues 194-497 in E. coli). A native data set at 1.9 A resolution has been collected at 100 K using synchrotron radiation. The crystals belong to the space group P2(1)2(1)2, with unit-cell parameters a = 68.7, b = 101.1, c = 42.5 A and one molecule in the asymmetric unit.
The signal-recognition particle (SRP) is a ubiquitous protein-RNA complex that targets proteins to cellular membranes for insertion or secretion. A key player in SRP-mediated protein targeting is the evolutionarily conserved core consisting of the SRP RNA and the multidomain protein SRP54. Communication between the SRP54 domains is critical for SRP function, where signal sequence binding at the M domain directs receptor binding at the GTPase domain (NG domain). These SRP activities are linked to domain rearrangements, for which the role of SRP RNA is not clear. In free SRP, a direct interaction of the GTPase domain with SRP RNA has been proposed but has never been structurally verified. In this study, we present the crystal structure at 2.5-A resolution of the SRP54-SRP19-SRP RNA complex of Methanococcus jannaschii SRP. The structure reveals an RNA-bound conformation of the SRP54 GTPase domain, in which the domain is spatially well separated from the signal peptide binding site. The association of both the N and G domains with SRP RNA in free SRP provides further structural evidence for the pivotal role of SRP RNA in the regulation of the SRP54 activity.
Proper assembly of large protein-RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein-RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-A resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs.
The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein. It associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. In mammalian cells, the SRP consists of a 7S RNA and six protein components. The S domain of SRP comprises the 7S.S part of RNA bound to SRP19, SRP54 and the SRP68/72 heterodimer; SRP54 has the main role in recognizing signal sequences of nascent polypeptide chains and docking SRP to its receptor. During assembly of the SRP, binding of SRP19 precedes and promotes the association of SRP54 (refs 4, 5). Here we report the crystal structure at 2.3 A resolution of the complex formed between 7S.S RNA and SRP19 in the archaeon Methanococcus jannaschii. SRP19 bridges the tips of helices 6 and 8 of 7S.S RNA by forming an extensive network of direct protein RNA interactions. Helices 6 and 8 pack side by side; tertiary RNA interactions, which also involve the strictly conserved tetraloop bases, stabilize helix 8 in a conformation competent for SRP54 binding. The structure explains the role of SRP19 and provides a molecular framework for SRP54 binding and SRP assembly in Eukarya and Archaea.
Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)(1) reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and beta-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The beta-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.
The crystal structure of carbonic anhydrase from Neisseria gonorrhoeae has been solved to a resolution of 1.78 A by molecular replacement using human carbonic anhydrase II as a template. After refinement the R factor was 17.8% (Rfree=23.2%). There are two molecules per asymmetric unit (space group P21), but they have essentially identical structures. The fold of the N. gonorrhoeae enzyme is very similar to that of human isozyme II; 192 residues, 74 of which are identical in the two enzymes, have equivalent positions in the three-dimensional structures. This corresponds to 85% of the entire polypeptide chain of the bacterial enzyme. The only two cysteine residues in the bacterial enzyme, which has a periplasmic location in the cell, are connected by a disulfide bond. Most of the secondary structure elements present in human isozyme II are retained in N. gonorrhoeae carbonic anhydrase, but there are also differences, particularly in the few helical regions. Long deletions in the bacterial enzyme relative to human isozyme II have resulted in a considerable shortening of three surface loops. One of these deletions, corresponding to residues 128 to 139 in the human enzyme, leads to a widening of the entrance to the hydrophobic part of the active site cavity. Practically all the amino acid residues in the active site of human isozyme II are conserved in the N. gonorrhoeae enzyme and have similar structural positions. However, the imidazole ring of a histidine residue, which has been shown to function as a proton shuttle in the catalytic mechanism of the human enzyme, interacts with an extraneous entity, which has tentatively been identified as a 2-mercaptoethanol molecule from the crystallization medium. When this entity is removed by soaking the crystal in a different medium, the side-chain of His66 becomes quite mobile. The structure of a complex with the sulfonamide inhibitor, acetazolamide, has also been determined. Its position in the active site is very similar to that observed in human carbonic anhydrase II.
Self-assembly of the human plasma protein transthyretin (TTR) into unbranched insoluble amyloid fibrils occurs as a result of point mutations that destabilize the molecule, leading to conformational changes. The tertiary structure of native soluble TTR and many of its disease-causing mutants have been determined. Several independent studies by X-ray crystallography have suggested structural differences between TTR variants which are claimed to be of significance for amyloid formation. As these changes are minor and not consistent between the studies, we have compared all TTR structures available at the protein data bank including three wild-types, three non-amyloidogenic mutants, seven amyloidogenic mutants and nine complexes. The reference for this study is a new 1.5 A resolution structure of human wild-type TTR refined to an R-factor/R-free of 18.6 %/21.6 %. The present findings are discussed in the light of the previous structural studies of TTR variants, and show the reported structural differences to be non-significant.
Conformational changes in native and variant forms of the human plasma protein transthyretin (TTR) induce several types of amyloid diseases. Biochemical and structural studies have mapped the initiation site of amyloid formation onto residues at the outer C and D beta-strands and their connecting loop. In this study, we characterise an engineered variant of transthyretin, Ala108Tyr/Leu110Glu, which is kinetically and thermodynamically more stable than wild-type transthyretin, and as a consequence less amyloidogenic. Crystal structures of the mutant were determined in two space groups, P2(1)2(1)2 and C2, from crystals grown in the same crystallisation set-up. The structures are identical with the exception for residues Leu55-Leu58, situated at beta-strand D and the following DE loop. In particular, residues Leu55-His56 display large shifts in the C2 structure. There the direct hydrogen bonding between beta-strands D and A has been disrupted and is absent, whereas the beta-strand D is present in the P2(1)2(1)2 structure. This difference shows that from a mixture of metastable TTR molecules, only the molecules with an intact beta-strand D are selected for crystal growth in space group P2(1)2(1)2. The packing of TTR molecules in the C2 crystal form and in the previously determined amyloid TTR (ATTR) Leu55Pro crystal structure is close-to-identical. This packing arrangement is therefore not unique in amyloidogenic mutants of TTR.
Drosophila melanogaster exhibits two expression-regulating systems that target whole, specific chromosomes: the dosage compensation system whereby the male-specific lethal complex doubles transcription of genes on the male X-chromosome and the chromosome 4-specific protein Painting of fourth, POF. POF is the first example of an autosome-specific protein and its presence raises the question of the universality of chromosome-specific regulation. Here we show that POF and heterochromatin protein 1 (HP1) are involved in the global regulation of the 4th chromosome. Contrary to previous conclusions, Pof is not essential for survival of diplo-4th karyotype flies. However, Pof is essential for survival of haplo-4th individuals and expression of chromosome 4 genes in diplo-4th individuals is decreased in the absence of Pof. Mapping of POF using chromatin immunoprecipitation suggested that it binds within genes. Furthermore, we show that POF binding is dependent on heterochromatin and that POF and HP1 bind interdependently to the 4th chromosome. We propose a balancing mechanism involving POF and HP1 that provides a feedback system for fine-tuning expression status of genes on the 4th chromosome.
Two specific chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of Fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. Here we use chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional "peak" in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. Our results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, our results contradict transposable elements as a major nucleation site for HP1 on the fourth chromosome.
The modified nucleoside 2-thiocytidine (s(2)C) has so far been found in tRNA from organisms belonging to the phylogenetic domains Archaea and BACTERIA: In the bacteria Escherichia coli and Salmonella enterica serovar Typhimurium, s(2)C is present in position 32 of only four tRNA species-, and. An in-frame deletion of an S. enterica gene (designated ttcA, for "two-thio-cytidine") was constructed, and such a mutant has no detectable s(2)C in its tRNA. The TtcA protein family is characterized by the existence of both a PP-loop and a Cys-X(1)-X(2)-Cys motif in the central region of the protein but can be divided into two distinct groups based on the presence and location of additional Cys-X(1)-X(2)-Cys motifs in terminal regions of the sequence. Mutant analysis showed that both cysteines in this central conserved Cys-X(1)-X(2)-Cys motif are required for the formation of s(2)C. The DeltattcA1 mutant grows at the same rate as the congenic wild-type strain, and no growth disadvantage caused by the lack of s(2)C was observed in a mixed-population experiment. Lack of s(2)C32 did not reduce the selection rate at the ribosomal aminoacyl-tRNA site (A-site) for at any of its cognate CGN codons, whereas A-site selection at AGG by was dependent on the presence of s(2)C32. The presence of s(2)C32 in peptidyl- or in peptidyl- interfered with decoding in the A-site. The presence of s(2)C32 in decreased the rate of translation of the CGA codon but not that of the CGU codon.
The hereditary transthyretin (TTR) amyloidoses are rare, and in severe cases, fatal disorders caused by mutations in the TTR gene. The clinical picture is diverse, involving neuropathies and myopathies, and mainly depends on the causative mutation and the sites and rates of amyloid deposition. The ultimate aim of the field of research presented in this thesis is to prevent TTR amyloid disease. To reach this ambitious goal, a thorough understanding of the normal as well as the pathological properties of the protein is essential. Here, comparisons between TTR from humans and other species may provide valuable information.
The three-dimensional structure of TTR from Gilthead sea bream (Sparus aurata) was determined at 1.75 Å resolution by X-ray crystallography, and was found to be structurally similar to human TTR. However, significant differences were observed in the area at and around β-strand D, an area believed to dissociate from the structure prior to amyloid formation, thereby allowing the β-strands A and B to participate in polymerization. During evolution, the preference of TTR for the thyroid hormones, 3,5,3'-triiodo-L-thyronine (T3) and 3,5,3',5'-tetraiodo-L-thyronine (T4), has shifted. While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles, where T3 is the main ligand. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution, as well as the complex of human TTR with T3. A significantly wider entrance and narrower thyroid hormone binding channel suggest a structural explanation to the differences in thyroid hormone preference between human and piscine TTR.
The Tyr114Cys substitution in TTR is associated with severe systemic amyloidosis. The mutation introduces a second cysteinyl group in the TTR monomer, and has been shown to inhibit the formation of fibril formation in vitro, promoting the formation of disulfide-bonded amorphous aggregates. To deduce the role of intermolecular disulfide bonds in fibril formation we characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular disulfide bond at position 114 enhances the exposure of Cys10, which promotes the formation of additional intermolecular disulfide-linked assemblies. Also, we were able to isolate a disulfide-linked dimeric form of this mutant that formed protofibrils in vitro, suggesting the architecture of TTR amyloid may be the result of different underlying structures rather than that of a highly stringent assembly.
We have also been able to successfully adapt a method of protein pre-heating to enable crystallization, thereby succeeding in a particularly problematic protein crystallization experiment. By heating the protein solution, we succeeded in separating several forms of protein micro-heterogeneities from the properly folded protein species, thereby allowing the growth of well diffracting crystals.
The Tyr114Cys substitution in the human plasma protein transthyretin leads to a particularly aggressive form of familial amyloidotic polyneuropathy. In a previous study we demonstrated that ATTR Tyr114Cys forms intermolecular disulfide bonds, which partly impair fibril formation and result in a more amorphous morphology. Apart from the introduced cysteinyl group in position 114, the native sequence contains one cysteine located at position 10. To deduce the role of intermolecular disulfide bridging in fibril formation we generated and characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular cysteine bridge at position 114 enhances the exposure of cysteine 10, thereby facilitating additional intermolecular cysteine assemblies. We also purified a disulfide-linked dimeric form of TTR Cys10Ala/Tyr114Cys, which was recognized by the anti-TTR amyloid-specific monoclonal antibody MAb (39-44). Moreover, this dimeric molecule can form protofibrils indistinguishable from the fibrils formed under reducing conditions, as judged by atomic force microscopy. Assuming that both molecules of the dimer are part of the core of the fibril, the assembly is incompatible with a preserved native or near-native dimeric interphase. Our findings raise the question of whether TTR-amyloid architecture is indeed the result of one highly stringent assembly of structures or if different fibrils may be built from different underlying structures.
The use of high temperatures in the purification procedures of heat-stable proteins is a well established technique. Recently, rapid pre-heat treatment of protein samples prior to crystallization trials was described as a final polishing step to improve the diffraction properties of crystals [Pusey et al. (2005), Prog. Biophys. Mol. Biol. 88, 359-386]. The present study demonstrates that extended high-temperature incubation (328 K for 48 h) of the highly amyloidogenic transthyretin mutant TTR G53S/E54D/L55S successfully removes heterogeneities and allows the reproducible growth of well diffracting crystals. Heat treatment might be applied as an optimization method to other cases in which the protein/biomolecule fails to form diffracting crystals.
Over the past 100 years Drosophila has been developed into an outstanding model system for the study of evolutionary processes. A fascinating aspect of evolution is the differentiation of sex chromosomes. Organisms with highly differentiated sex chromosomes, such as the mammalian X and Y, must compensate for the imbalance in gene dosage that this creates. The need to adjust the expression of sex-linked genes is a potent force driving the rise of regulatory mechanisms that act on an entire chromosome. This review will contrast the process of dosage compensation in Drosophila with the divergent strategies adopted by other model organisms. While the machinery of sex chromosome compensation is different in each instance, all share the ability to direct chromatin modifications to an entire chromosome. This review will also explore the idea that chromosome-targeting systems are sometimes adapted for other purposes. This appears the likely source of a chromosome-wide targeting system displayed by the Drosophila fourth chromosome
Transthyretin (TTR) is one of several proteins involved in amyloid disease in humans. Unknown conformational changes of the native state of TTR result in aggregation of TTR molecules into amyloid fibrils, which accumulate in extracellular tissues. This may result in different clinical symptoms, e.g. polyneuropathy or cardiomyopathy, depending on their site of accumulation.
Our long-term goal is to identify structural changes associated with amyloid formation. For this work, structural characterization of TTR from other species than human may provide valuable information.
The three-dimensional X-ray crystallographic structure of TTR from sea bream (Sparus aurata) was determined at 1.75 Å resolution. Human and sea bream TTR were found to be structurally very similar. However, interesting differences were present in the area at and around -strand D, which in fish forms an extended loop region. Interestingly, this area is believed to dissociate from the structure prior to amyloid formation, to allow -strands A and B to participate in polymerization.
During evolution, TTR from different species have developed differences in preference to their natural ligands, the thyroid hormones 3,5,3'-triiodo-L-thyronine (T3) and 3,5,3',5'-tetraiodo-L-thyronine (T4). While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution. A significantly wider entrance and narrower thyroid hormone binding channel provide a structural explanation to the differences in thyroid hormone preference between human and piscine TTR.
In a separate work, we identified a novel protein family with structural similarity to TTR, which we named the transthyretin-related protein (TRP) family. To attain information about this protein family, we cloned, expressed, purified and characterized TRP from Escherichia coli (EcTRP). Furthermore, we solved the structure of EcTRP to 1.65 Å resolution. As predicted, EcTRP and human TTR are structurally very similar. Interesting structural differences are found in the area corresponding to the thyroid hormone binding site in TTR, which due to its amino acid conservation within the TRP family we identified as a putative ligand-binding site in TRPs. The function of the TRP is not known, however, recent studies suggest that it might be involved in purine catabolism.
It has been shown that partial acid denaturation of human TTR results in amyloid-fibril formation. Interestingly, we have shown that sea bream TTR also forms amyloid-like fibrils in vitro, even though it shares only 52% sequence identity to human TTR. Corresponding studies on EcTRP did not generate amyloid-like fibrils. EcTRP has 30% sequence identity to human TTR. The fact that two of the proteins form amyloid fibrils and one does not means that they can serve as a model system for the study of amyloid formation. Further studies on these three proteins are currently performed to attain more information about the mechanism of amyloid formation.
The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 A resolution in the presence of zinc, and the other at 2.1 A resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.
Medin, a recently discovered 5.5 kDa peptide, is associated with amyloid deposits in the medial layer of human arteries and the prevalence is nearly 100% within individuals above 50 years. Presently, not much is known about its biochemical and biophysical properties or its pathway from soluble peptide to insoluble amyloid. Here we have characterized the behavior of medin in the presence of lipid membranes, using circular dichroism, isothermal titration calorimetry, differential scanning calorimetry, size exclusion chromatography, and atomic force microscopy (AFM). Medin was shown to exist as a monomer in solution with a predominantly random-coil structure. It binds lipid vesicles that have either a neutral or a negative surface potential. Upon association to membranes containing acidic lipids, it undergoes an electrostatically driven conformational change towards a mainly α-helical state. Prolonged incubation converts medin from an α-helical structure into an amyloid β-sheet fibrillar state as confirmed by AFM. Based on these findings, we propose a mechanism of medin-amyloid formation where medin electrostatically associates in its monomeric form to biological interfaces displaying a negative potential. This process both increases the local peptide concentration and induces an aggregation-prone α-helical fold.
The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six beta-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F' and H-H' with a prolongation of these beta-strands and, interestingly, with a possible shift in beta-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented.
AD (Alzheimer's disease) is a neurodegenerative disorder characterized by self-assembly and amyloid formation of the 39–43 residue long Ab (amyloid-b)-peptide. The most abundant species, Ab(1–40) and Ab(1–42), are both present within senile plaques, but Ab(1–42) peptides are considerably more prone to self-aggregation and are also essential for the development of AD. To understand the molecular and pathological mechanisms behind AD, a detailed knowledge of the amyloid structures of Ab-peptides is vital. In the present study we have used quenched hydrogen/deuterium-exchange NMR experiments to probe the structure of Ab(1–40) fibrils. The fibrils were prepared and analysed identically as in our previous study on Ab(1–42) fibrils, allowing a direct comparison of the two fibrillar structures. The solvent protection pattern of Ab(1–40) fibrils revealed two well-protected regions, consistent with a structural arrangement of two b-strands connected with a bend. This protection pattern partly resembles the pattern found in Ab(1–42) fibrils, but the Ab(1–40) fibrils display a significantly increased protection for the N-terminal residues Phe4–His14, suggesting that additional secondary structure is formed in this region. In contrast, the C-terminal residues Gly37–Val40 show a reduced protection that suggests a loss of secondary structure in this region and an altered filament assembly. The differences between the present study and other similar investigations suggest that subtle variations in fibril-preparation conditions may significantly affect the fibrillar architecture.
Alzheimer disease is a neurodegenerative disorder that is tightly linked to the self-assembly and amyloid formation of the 39-43-residue-long amyloid-beta (Abeta) peptide. Considerable evidence suggests a correlation between Alzheimer disease development and the longer variants of the peptide, Abeta-(1-42/43). Currently, a molecular understanding for this behavior is lacking. In the present study, we have investigated the hydrogen/deuterium exchange of Abeta-(1-42) fibrils under physiological conditions, using solution NMR spectroscopy. The obtained residue-specific and quantitative map of the solvent protection within the Abeta-(1-42) fibril shows that there are two protected core regions, Glu11-Gly25 and Lys28-Ala42, and that the residues in between, Ser26 and Asn27, as well as those in the N terminus, Asp1-Tyr10, are solvent-accessible. This result reveals considerable discrepancies when compared with a previous investigation on Abeta-(1-40) fibrils and suggests that the additional residues in Abeta-(1-42), Ile41 and Ala42, significantly increase the solvent protection and stability of the C-terminal region Lys28-Ala42. Consequently, our findings provide a molecular explanation for the increased amyloidogenicity and toxicity of Abeta-(1-42) compared with shorter Abeta variants found in vivo.
The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of II-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the II-spectrin SH3 domain.
Regulation of chromatin through histone acetylation is an important step in gene expression. The Gcn5 histone acetyltransferase is part of protein complexes, e.g., the SAGA complex, that interact with transcriptional activators, targeting the enzyme to specific promoters and assisting in recruitment of the basal RNA polymerase transcription machinery. The Ada2 protein directly binds to Gcn5 and stimulates its catalytic activity. Drosophila contains two Ada2 proteins, Drosophila Ada2a (dAda2a) and dAda2b. We have generated flies that lack dAda2b, which is part of a Drosophila SAGA-like complex. dAda2b is required for viability in Drosophila, and its deletion causes a reduction in histone H3 acetylation. A global hypoacetylation of chromatin was detected on polytene chromosomes in dAda2b mutants. This indicates that the dGcn5-dAda2b complex could have functions in addition to assisting in transcriptional activation through gene-specific acetylation. Although the Drosophila p53 protein was previously shown to interact with the SAGA-like complex in vitro, we find that p53 induction of reaper gene expression occurs normally in dAda2b mutants. Moreover, dAda2b mutant animals show excessive p53-dependent apoptosis in response to gamma radiation. Based on this result, we speculate that dAda2b may be necessary for efficient DNA repair or generation of a DNA damage signal. This could be an evolutionarily conserved function, since a yeast ada2 mutant is also sensitive to a genotoxic agent
The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that associates with ribosomes to mediate the targeting of membrane and secretory proteins to biological membranes. In higher eukaryotes, SRP biogenesis involves the sequential binding of SRP19 and SRP54 proteins to the S domain of 7S RNA. The recently determined crystal structures of SRP19 in complex with the S domain, and that of the ternary complex of SRP19, the S domain and the M domain of SRP54, provide insight into the molecular basis of S-domain assembly and SRP function.
Mammalian G9a is a histone H3 Lys-9 (H3-K9) methyltransferase localized in euchromatin and acts as a co-regulator for specific transcription factors. G9a is required for proper development in mammals as g9a-/g9a- mice show growth retardation and early lethality. Here we describe the cloning, the biochemical and genetical analyses of the Drosophila homolog dG9a. We show that dG9a shares the structural organization of mammalian G9a, and that it is a multi-catalytic histone methyltransferase with specificity not only for lysines 9 and 27 on H3 but also for H4. Surprisingly, it is not the H4-K20 residue that is the target for this methylation. Spatiotemporal expression analyses reveal that dG9a is abundantly expressed in the gonads of both sexes, with no detectable expression in gonadectomized adults. In addition we find a low but clearly observable level of dG9a transcript in developing embryos, larvae and pupae. Genetic and RNAi experiments reveal that dG9a is involved in ecdysone regulatory pathways.
Lysine methylation has important functions in biological processes that range from heterochromatin formation to transcription regulation. Here, we demonstrate that Drosophila dSet2 encodes a developmentally essential histone H3 lysine 36 (K36) methyltransferase. Larvae subjected to RNA interference-mediated (RNAi) suppression of dSet2 lack dSet2 expression and H3-K36 methylation, indicating that dSet2 is the sole enzyme responsible for this modification in Drosophila melanogaster. dSet2 RNAi blocks puparium formation and adult development, and causes partial (blister) separation of the dorsal and ventral wing epithelia, defects suggesting a failure of the ecdysone-controlled genetic program. A transheterozygous EcR null mutation/dSet2 RNAi combination produces a complete (balloon) separation of the wing surfaces, revealing a genetic interaction between EcR and dSet2. Using immunoprecipitation, we demonstrate that dSet2 associates with the hyperphosphorylated form of RNA polymerase II (RNAPII).
Background: All eukaryotic organisms need to distinguish each of their chromosomes. A few protein complexes have been described that recognise entire, specific chromosomes, for instance dosage compensation complexes and the recently discovered autosome-specific Painting of Fourth (POF) protein in Drosophila. However, no sequences have been found that are chromosome-specific and distributed over the entire length of the respective chromosome. Here, we present a new, unbiased, exhaustive computational method that was used to probe three Drosophila genomes for chromosome-specific sequences.
Results: By combining genome annotations and cytological data with multivariate statistics related to three Drosophila genomes we found sequence signatures that distinguish Muller's F-elements ( chromosome 4 in D. melanogaster) from all other chromosomes in Drosophila that are not attributable to differences in nucleotide composition, simple sequence repeats or repeated elements. Based on these signatures we identified complex motifs that are strongly overrepresented in the F-elements and found indications that the D. melanogaster motif may be involved in POF-binding to the F-element. In addition, the X-chromosomes of D. melanogaster and D. yakuba can be distinguished from the other chromosomes, albeit to a lesser extent. Surprisingly, the conservation of the F-element sequence signatures extends not only between species separated by approximately 55 Myr, but also linearly along the sequenced part of the F-elements.
Conclusion: Our results suggest that chromosome-distinguishing features are not exclusive to the sex chromosomes, but are also present on at least one autosome ( the F-element) in Drosophila.
The process of spermatogenesis is in many organisms one of the most dramatic cellular transformations - a normal round diploid cell is ultimately transformed into a needle shaped haploid cell with tightly packaged cell machinery. In Drosophila melanogaster this process involves several characteristic stages, one of these being the primary spermato-cyte stage, which is the stage prior to meiosis. This stage is characterized by a loose chromatin structure in the nucleus and exceptionally high rates of transcription and translation to produce essentially all the mRNAs and proteins that are needed later during spermatid formation. Two proteins that are expressed in high levels in primary spermatocytes are ThioredoxinT (TrxT) and Painting of fourth (POF).
Thioredoxins are small thiol proteins that reduce disulfides in other proteins, a mechanism that is utilized in many different contexts. In this thesis I show that the TrxT gene encodes a testis-specific thioredoxin that specifically associates to Y-chromosome loops in primary spermatocytes. TrxT is located right next to deadhead (dhd), a gene that encodes a female-specific thioredoxin that specifically locates to nuclei in the ovaries. A third thioredoxin in Drosophila is the ubiquitously expressed Thioredoxin-2 (Trx-2). I have found that flies lacking Trx-2 are viable but have shorter life spans than wild type flies, while over-expression of Trx-2 mediates an increased resistance to oxidative stress. Interestingly, a lack of all three thioredoxins does not result in lethality, contrary to what could be expected. All three thioredoxins are conserved among Drosophilids and the striking genomic organization of TrxT and dhd is generally conserved.
The gene name Painting of fourth originates from the finding that POF stains the 4th chromosome of Drosophila in a banded pattern on salivary gland chromosomes. I show in this thesis that POF binding to the equivalent of the 4th chromosome is conserved in genus Drosophila and that POF co-localizes with both a protein and a histone modification associated with dosage compensation in species where POF also binds the male X. POF is expressed ubiquitously in both males and females, but at very high levels in male testes. I show that POF is present in nuclei of primary spermatocytes, but also in nuclei of maturing spermatids and that a lack of POF in the male germline causes a global down-regulation of chromosome 4 genes. These results combined suggest a function of POF in the first known case of chromosome-wide gene regulation of an autosome.
So far, two thioredoxin proteins, DHD and Trx-2, have been biochemically characterized in Drosophila melanogaster. Here, with the cloning and characterization of TrxT we describe an additional thioredoxin with testis-specific expression. TrxT and dhd are arranged as a gene pair, transcribed in opposite directions and sharing a 471 bp regulatory region. We show that this regulatory region is sufficient for correct expression of the two genes. This gene pair makes a good model for unraveling how closely spaced promoters are differentially regulated by a short common control region. Both TrxT and DHD proteins are localized within the nuclei in testes and ovaries, respectively. Use of a transgenic construct expressing TrxT fused to Enhanced Yellow Fluorescent Protein reveals a clear association of TrxT with the Y chromosome lampbrush loops ks-1 and kl-5 in primary spermatocytes. The association is lost in the absence of the Y chromosome. Our results suggest that nuclear thioredoxins may have regulatory functions in the germline.
Thioredoxins are proteins that have thiol-reducing activity and a characteristic conserved active site (WCGPC). They have
several documented functions, e.g. roles in defences against oxidative stress and as electron donors for ribonucleotidereductase.
In Drosophila melanogaster there are three ''classical'' thioredoxins with the conserved active site: deadhead,
ThioredoxinT and Thioredoxin-2. Here, we report the creation of null-mutations in the Thioredoxin-2 (Trx-2) gene.
Characterization of two Trx-2 mutants indicated that Trx-2 affects the lifespan of D. melanogaster, and is involved in the
organism's oxidative stress protection system. We found that the mutants have a shorter lifespan than wild-type flies, and
thioredoxin double mutant flies showed lower tolerance to oxidative stress than wild-type flies, while flies carrying multiple
copies of a Trx-2 rescue construct showed higher tolerance. These findings suggest that Trx-2 has modest or redundant
functions in Drosophila physiology under unstressed conditions, but could be important during times of environmental
Thioredoxins are small thiol proteins that have a
conserved active site sequence, WCGPC, and reduce
disulfide bonds in various proteins using the two active site
cysteines, a reaction that oxidizes thioredoxin and renders it
inactive. Thioredoxin reductase returns thioredoxin to its
reduced, active form in a reaction that converts NADPH to
NADP+. The biological functions of thioredoxins vary
widely; they have roles in oxidative stress protection, act as
electron donors for ribonucleotide reductase, and form
structural components of enzymes. To date, three thioredoxin
genes have been characterized in Drosophila melanogaster:
the generally expressed Thioredoxin-2 (Trx-2) and the two
sex-specific genes ThioredoxinT (TrxT) and deadhead
(dhd). The male-specific TrxT and the female-specific dhd
are located as a gene pair, transcribed in opposite directions,
with only 470 bp between their transcription start points. We
show in this study that all three D. melanogaster thioredoxins
are conserved in 11 other Drosophilid species, which are
believed to have diverged up to 40 Ma ago and that Trx-2 is
conserved all the way to Tribolium castaneum. We have
found that the intriguing gene organization and regulation of
TrxT and dhd is remarkably well conserved and identified
potential conserved regulatory sequences. In addition, we
show that the 50–70 C terminal amino acids of TrxT constitute
a hyper-variable domain, which could play a role in
sexual conflict and male–female co-evolution.
Bioinformatics is a fast-developing field that make use of computational methods to analyse and structure biological data. An important branch of bioinformatics is structure and function prediction of proteins. To determine the structure of a protein is a crucial part in the characterisation of the molecule. The structure can also give clues about how the protein functions in the cell.
Since the experimental determination of a protein structure can be both difficult and time-consuming, and in some cases is impossible using current techniques, it is desirable to be able to predict the structure. If two protein sequences are very similar, it is known that they share the same structure. However, there are many proteins that share the same fold, but have no clear sequence similarity. To find these relationships, and be able to predict the structure of these proteins, so called "protein fold recognition methods" have been developed.
In this thesis, the field of bioinformatics is briefly surveyed, and two fold recognition methods are presented. Both methods use hidden Markov models (HMMs) to find related proteins, and they both exploit the fact that structure is more conserved than sequence, but in two different ways.
The first paper introduces the reader to the field of molecular biology, and also describes some common tools used for protein sequence comparison. HMMs in general are described in detail, as well as some methods for the construction of multiple structure superposition. Since 3D structure is more conserved than sequence, it is expected that a multiple sequence alignment based on a ultiple
structure superposition, is more biologically correct than an lignment based on sequence information, especially for proteins with low sequence identities.
Our structure anchored HMMs (saHMMs), which are presented in the paper, are constructed from multiple sequence alignments that are based on structural superposition. The paper also describes the selection of representatives for each protein family, that were used for the construction of the saHMMs. In this selection, no protein in a given family have a sequence identity higher than a certain threshold to any other protein in the same family. The threshold is
defined as the border to the so-called twilight zone. The saHMMs are shown to be able to find the family relationships for almost 90% of the test cases, even when the saHMMs are based on two proteins only.
The second paper describes the secondary structure HMMs (ssHMMs). These HMMs are based on an ordinary multiple sequence alignment, as well as on the secondary structures of the proteins. When a query sequence is compared to the ssHMM, a predicted secondary structure is used, and the score based on the sequence is increased or decreased depending on the match of the secondary
structures. A rigorous benchmark is also presented, and used to compare automatically generated HMMs with ordinary sequence search methods. The results show that the ordinary sequence search methods tested perform about as well as automatic HMMs built from multiple alignments. The ssHMMs, however, are better at detecting the correct fold of a protein than all the other methods
The ever increasing speed of DNA sequencing widens the discrepancy between the number of known gene products, and the knowledge of their function and structure. Proper annotation of protein sequences is therefore crucial if the missing information is to be deduced from sequence-based similarity comparisons. These comparisons become exceedingly difficult as the pairwise identities drop to very low values. To improve the accuracy of domain identification, we exploit the fact that the three-dimensional structures of domains are much more conserved than their sequences. Based on structure-anchored multiple sequence alignments of low identity homologues we constructed 850 structure-anchored hidden Markov models (saHMMs), each representing one domain family. Since the saHMMs are highly family specific, they can be used to assign a domain to its correct family and clearly distinguish it from domains belonging to other families, even within the same superfamily. This task is not trivial and becomes particularly difficult if the unknown domain is distantly related to the rest of the domain sequences within the family. In a search with full length protein sequences, harbouring at least one domain as defined by the structural classification of proteins database (SCOP), version 1.71, versus the saHMM database based on SCOP version 1.69, we achieve an accuracy of 99.0%. All of the few hits outside the family fall within the correct superfamily. Compared to Pfam_ls HMMs, the saHMMs obtain about 11% higher coverage. A comparison with BLAST and PSI-BLAST demonstrates that the saHMMs have consistently fewer errors per query at a given coverage. Within our recommended E-value range, the same is true for a comparison with SUPERFAMILY. Furthermore, we are able to annotate 232 proteins with 530 nonoverlapping domains belonging to 102 different domain families among human proteins labelled unknown in the NCBI protein database. Our results demonstrate that the saHMM database represents a versatile and reliable tool for identification of domains in protein sequences. With the aid of saHMMs, homology on the family level can be assigned, even for distantly related sequences. Due to the construction of the saHMMs, the hits they provide are always associated with high quality crystal structures. The saHMM database can be accessed via the FISH server at http://babel.ucmp.umu.se/fish/.
The FISH server is highly accurate in identifying the family membership of domains in a query protein sequence, even in the case of very low sequence identities to known homologues. A performance test using SCOP sequences and an E-value cut-off of 0.1 showed that 99.3% of the top hits are to the correct family saHMM. Matches to a query sequence provide the user not only with an annotation of the identified domains and hence a hint to their function, but also with probable 2D and 3D structures, as well as with pairwise and multiple sequence alignments to homologues with low sequence identity. In addition, the FISH server allows users to upload and search their own protein sequence collection or to quarry public protein sequence data bases with individual saHMMs. The FISH server can be accessed at http://babel.ucmp.umu.se/fish/.
MOTIVATION: Order and Disorder prediction using Conditional Random Fields (OnD-CRF) is a new method for accurately predicting the transition between structured and mobile or disordered regions in proteins. OnD-CRF applies CRFs relying on features which are generated from the amino acids sequence and from secondary structure prediction. Benchmarking results based on CASP7 targets, and evaluation with respect to several CASP criteria, rank the OnD-CRF model highest among the fully automatic server group. AVAILABILITY: http://babel.ucmp.umu.se/ond-crf/
It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.


text:
Lauren Chan
Lauren completed her articles at Ormston List Frawley LLP in March 2016 and has since joined the corporate group. She will be called to the Bar in Ontario in June 2016. Her practice focuses on general corporate and commercial transactions, and assisting in advising on mergers and acquisitions and other corporate reorganizations, securities, and private equity transactions.
Lauren studied law at the University of Bristol in England. Prior to returning to Canada and converting her law degree, Lauren earned distinctions in the postgraduate course mandatory for those seeking to become solicitors in England and Wales as well as in her masters degree in law.
Areas of Practice
- General Corporate and Commercial Transactions
- Mergers and Acquisitions
- Corporate Reorganizations
- Securities
- Private Equity Transactions
Bar Admissions
- Ontario, 2016
- University of Bristol Law School, Bristol, England
- BPP Law School, London, United Kingdom
- Legal Practice Course - 2013
- Honours: Distinction
- BPP Law School, London, United Kingdom
- LLM Professional Legal Practice - 2015
- Honours: Distinction
Pro Bono Activities
- Law Society of Upper Canada

subset: commoncrawl


text:
Let the fall buying season begin. With four albums entering the Top
10 and eight new albums in the Top 20, music stores are flooded
with red-hot records. That should come as no surprise. Rife with
superstar releases, the period from late September to the end of
the year has traditionally been the music industry's strongest
season for sales.
This year, Mariah Carey kicks off the fourth quarter
sweepstakes. To nobody's surprise, her Butterfly, debuts
at No. 1, selling 235,000 copies during the week ending September
21, according to SoundScan. The week's other Top 10 debuts go to
rapper Busta Rhymes' When Disaster Strikes (which came in
at No. 3) and Christian rockers Jars of Clay's Much Afraid
(No. 7). Aqua's Aquarium moves up to No. 10 from No.
From the top, it's Butterfly, followed by LeAnn Rimes' You Light Up My Life-Inspirational Songs (which sold
205,000 copies); When Disaster Strikes (165,000); Master
P's Ghetto D (124,000); Puff Daddy's No Way Out
(119,000); Fleetwood Mac's The Dance (103,000); Much
Afraid (100,000); Jewel's Pieces of You (91,000);
Trisha Yearwood's (Songbook) A Collection of Hits (90,000), and Aquarium (90,000).
The other acts to debut in the Top 20 are rapper Mack 10 (No.
13), R&B singer Usher (No. 14), the R& B-oriented
soundtrack to Soulfood (No. 16), country duo Brooks &
Dunn (No. 17), and comedian Adam Sandler (No. 18).
Retailers and labels are thrilled with the influx of hit
records. The only downside is that, in order to make room for the
new, several older acts are paying the price on the sales chart.
Rock acts are taking some of the hardest hits. Oasis' Be Here
Now drops from No. 2 to No. 26 in just four weeks' time.
Fellow English rockers Radiohead watch their critically acclaimed
OK Computer fall from No. 91 to No. 142 — a whopping 51
spots — in just seven days. Among the week's other casualties: the
Foo Fighters' The Colour & the Shape (No. 48 to No.
75); Meredith Brooks' Blurring the Edges (No. 52 to No.
113), and the Verve Pipe's Villains (No. 74 to No. 152).
After 28 weeks on the Top 200, U2's Pop falls off the
chart altogether.


text:
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text: The caring personnel at Mc Clure Funeral Service provide quiet and well-maintained grounds created to meet the needs of each family and to commemorate the lives of those buried at the grounds. The professional, devoted staff can assist you in making memorial service service arrangements, funeral arranging, and assist you through cremation choices. The funeral service director will certainly guide you through all facets of the program; including funeral service flowers, casket choices, appropriate music selection, and local accommodations. Trust is certainly their top concern and they specialize in tradition funerals, basic cremation, grief support, urn selection, and military memorial services which includes offering veteran burial flags.


text: "Technology is everything that doesn't work yet" – technology is only perceived as technology until it creates value and function. When the people at Ruter are working with customer services they view digitalization the same way. Digitalization is just something that doesn't work yet, until it delivers value to the customer and the organization. In this talk you'll learn about Ruter's approach to understanding customer feelings and behavior along with their quest to define "flow" in public transport services.


text:
A big part of the decision to save more money is deciding where to keep the cash you're putting away.
While a savings or money market account may seem like an obvious storage place for your dollars, another vehicle may be more appropriate, depending on your goals and time horizon.
A certificate of deposit (CD) could be a good place for your funds if you're looking to earn additional interest and it'll be a while before you need to access the money you're setting aside.
What is a CD and how does it work?
A CD is an account offered by banks and credit unions. If you visit a brokerage firm, you'll find that some offer CDs, too, from different financial institutions.
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How much interest can you earn with a CD?
CDs often come with a fixed interest rate that applies until the term ends. But some banks offer step-up or bump-up CDs, which allow the yields tied to accounts to increase, according to set intervals or one time during the term.
The amount of interest you can earn from a CD varies depending on multiple factors. Online banks tend to pay higher rates than brick-and-mortar banks. How much interest you'll earn also depends on how thirsty a certain bank or credit union is for deposits. Competition, changes in Treasury yields and economic conditions, like the rate of inflation, also come into play.
Long-term CDs typically pay higher yields than short-term CDs. To earn a higher yield, you'll usually have to open a four-year CD or five-year CD. But depending on the interest rate environment we're in, opting for a long-term CD may not make sense.
If interest rates are rising, for example, it's best to go with a short-term CD. That way, you're not missing out on better offers that will likely come as short-term interest rates increase. If the opposite is true and interest rates are expected to go down, locking in a higher rate through a long-term CD may be worth considering, depending on how soon you'll need the money you're putting away. Of course, thinking about your own financial goals and needs is better than trying to squeeze out more interest by timing the market.
Use Bankrate's CD calculator to see how much interest you can earn with a CD.
Is a CD a good investment?
CDs generally provide a safe place for storing savings. You can often calculate how much interest you'll earn in advance. And by opening a CD maturing in a year or less, you'll earn more than you would through a standard savings account. Because there are many types of CDs, there's a good chance you'll find one that meets your short-term savings needs.
However, a CD isn't necessarily the best option for everyone or every circumstance. Minimum deposits for CDs are often higher than the ones tied to savings accounts. And it all depends on what you're trying to accomplish and how quickly you need the funds in your account. If you need the money for an emergency situation, for example, you're better off keeping it in a savings account or money market account that's much more liquid.
CD yields are also low compared with what you would earn by investing in bonds or putting your money in the stock market. To earn a higher rate of interest, you'll have to aim for a riskier investment.
Are CDs safe?
Savers who keep their money in CDs usually have little to lose. At the end of your term, you can expect to get back your initial deposit plus some interest. As long as you keep your money locked up until maturity, you'll avoid the risk of losing money because of an early withdrawal.
Some types of CDs, however, are riskier than others. With a callable CD, for example, you could begin with a high yield but end up with a lower one if the bank decides to call back your CD in a declining interest rate environment. On the other hand, your CD may not be called if CD rates were to rise, meaning you would still be earning less money than others.
"Callable CDs are a 'heads I win, tails you lose' proposition," says Greg McBride, CFA, Bankrate chief financial analyst. "They exist to benefit the issuer, not the investor."
How safe it is to invest in a particular CD also depends on the financial institution offering the account. It's always best to choose a bank insured by the Federal Deposit Insurance Corp. or a credit union insured by the National Credit Union Administration. That way, your deposit is safe if your bank or credit union has to close shop.
Specialty CDs
There are many different types of CDs. Brokered CDs, for instance, can be found at a brokerage firm and may pay more interest than the CDs available at banks and credit unions. You could also invest in a zero-coupon CD offering a higher face value than what you originally paid. One big downside: You won't be paid interest until your CD matures, but you'll still be taxed annually anyway.
Other CDs offer the opportunity to earn a higher yield as time progresses. Through bump-up CDs, you can decide when to raise your interest rate. But with step-up CDs, interest rate hikes happen automatically at certain time periods during the CD term.
You won't get to bump up your interest rate when you invest in a no-penalty CD, but you will be able to withdraw your savings penalty-free as early as a week after locking up the money. Through an add-on CD, an early withdrawal penalty applies, but you'll get to continuously add money into the account and take advantage of a yield that's potentially higher than the one tied to your savings account.
Two additional types of CDs to mention include jumbo and IRA CDs. Jumbo CDs are geared toward consumers with a lot of liquid savings. Minimum deposits usually begin at $100,000 for these accounts. With IRA CDs, you get to put some of your retirement savings in a CD rather than another kind of investment account.
Are you taxed on a CD?
There are several benefits to investing in CDs. One drawback, however, is that you'll need to prepare to pay taxes on any interest that accumulates over the course of your term (even if you don't receive any regular interest payments, as is the case with zero-coupon CDs). Taxes have the potential to significantly reduce your earnings. And regardless of whether you keep your money in a CD or high-yield savings account, you'll be taxed either way.
Taxes are something you'll have to consider as you're calculating your savings and setting goals. If you're transferring interest from your CD into another account, think about setting up a separate account with funds you'll use to pay your taxes.
Learn more:
- Build a CD ladder that's right for you
- How to use the safety of CDs to boost returns on your portfolio
- Top 18 strategies for CD savers


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How to Buy Airtime from your Ecobank via Short-code
To buy airtime from your ECOBANK account, simply dial *326*Amount#
for example; you should dial *326*1000# to buy 1000 NGN Airtime.
ATTENTION: You can only dial the code from the phone line that is registered with the bank (preferably the line that receives your debit/credit alert notifications).
Got any contribution to this post? Kindly share it via comments. Thanks


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Appalachian Trail DVD
Appalachian Trail DVD
Appalachian Trail DVD
This item ships to the USA.
For more than half of the U.S. population, the Appalachian Trail is less than a day's drive away. Yet despite its proximity to many major cities, few truly know the splendor of this national treasure. National Geographic travels off the beaten track to discover the remote and often unknown corners of the 5-million-step journey.
Not available for shipment outside of the U.S. and Canada.
This product is manufactured on demand using DVD-R recordable media and is not closed-captioned. We apologize for any inconvenience this may cause.
- 1 disc; approximately 50 minutes
- Widescreen
- © 2009


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517. Nymphomaniac
© Bruce Goodman 11 March 2015
Wally watched Whitney through the window. Every day.
Every day Whitney would go for a run. Every day, as she passed Wally's house, Whitney would push back her head and thrust out her mammary glands. She did it on purpose. She knew Wally was watching.
In fact, Whitney would push back her head and thrust out her mammary glands as she passed what seemed like every second house.
My mother reckons she's a nymphomaniac, said Wally. I'm only thirteen and I hope you're impressed that I can spell 'nymphomaniac'.
And I know what nymphomaniacs are as well. Do I what! Dad told me. He said they never get any sleep.


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You need management
We see entertained people
Fighting for your right to party
It's all about the music
Ask not what good music can do for you, but what you can do to hear good music
Managing the soundtrack of your life
Your local music fix
Band management. Not man bandagement.
The future of local music.
Awesomeness in real time
Musicphiles of the world, unite!
Oh, Sleeper Down Under
Australia is bracing itself for one of the hottest tours to close out the summer as American metal giants Oh, Sleeper get set to invade our coast lines. Thanks to Taperjean Music, we can all expect an awesome mosh fest across the country as Oh, Sleeper put on shows from Melbourne up to Perth with every major city in between, plus a couple shows in New Zealand.
We are also please to announce that two of our bands on the Mannequin Republic roster have been given spots to play alongside Oh, Sleeper as they hit New South Wales and Victoria.
First check out Ghosts On Broadway as they break it down in Sydney at Live In The Wall (343 Parramatta Rd, Leichardt, NSW) on Sunday February 3 for an off the hook all ages gig. Doors open early at 2:00 pm for this special all ages afternoon show. Also on the card are For All Eternity, Storm The Sky and Elegist. Tickets are $25 through the band with a free bonus EP! See more here.
A week later, catch This Fiasco as Oh, Sleeper make their way to Melbourne as they take over the Ringwood OLP (2 Wilana Street, Ringwood, VIC) for another epic all ages event. Get there when doors open early at 2:00 pm on Sunday February 10 and catch other awesome acts For All Eternity, Storm The Sky, Bellehaven and Restless. Tickets area special price of $20 through the band. See more here.
Be part of the action as Oh, Sleeper run through Australia to promote their latest full length album, Children Of Fire, and throw down with us in Sydney and Melbourne as Ghosts On Broadway and This Fiasco tear up the stage for this hot overseas tour. We'll see you there!


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Top Five Super Green Modern Homes
Preston at Jetson Green picks his "top five super green modern homes," most of which will be known to TreeHugger readers. "They're ultra-stylish and ultra-green." One trend we like is that they are getting smaller and more urban; modern green design is no longer just the preserve of the very rich, but is becoming accessible to the moderately rich- not too many people can afford a ::Living Home but Michelle Kaufmann's new MKLotus is reasonably sized, and ::5IVE is not excessive.
With housing starts in the USA at their lowest point in ten years and the mortgage crisis expanding, the cost of construction should begin to drop as demand for construction labour and building materials falls. Perhaps the premium for good green design will fall as well. ::Jetson Green


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0 Members and 1 Guest are viewing this topic.
Surely planning permission won't be a problem
The planning board consists of a planner and Danny Murphy
I think i'd have preferred to hear a suitability study had been carried out prior to handing over £20odd million pounds
What we have paid has a planning permission clause I believe.
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The business magazine of UQ Business School
Keeping you up-to-date on the latest business thinking, research insights and expert opinion. Subscribe to get Momentum updates delivered straight to your inbox.
- 2 January 2017Employment in bars, restaurants and hotels could help young people with multiple challenges to break the cycle of despair. We just need to find the right recipe, says Dr Richard Robinson.
- 2 January 2017An expat assignment can have a damaging effect on the 'trailing spouse' – but why do some partners thrive when others sink into despair?
- 2 January 2017Innovation is the key to a better future for Australians – but we need to take pride in our past achievements to convince people of the benefits, says Professor Mark Dodgson.
- 3 October 2016Today's focus on image, brands and buzzwords may divert attention from the nuts and bolts of business. Is it time for us all to drop the hype and face reality?
- 3 October 2016Research has revealed the different approaches that managers use to deal with diversity in real life work situations – but which is the most effective?
- 3 October 2016Aristotle's writings still have relevance for business today, says Professor Bernard McKenna. Perhaps the CEOs of the big banks ought to read them.
- 1 August 2016As finance departments face ever greater reporting demands, it is time to rethink the whole finance function and find ways to cut costs and improve the quality of management information.
- 1 August 2016The Islamic finance sector has seen rapid growth since the financial crisis. But is it really less risky than conventional banking – and how will new regulations affect the industry?
- 1 June 2016From socialising with staff to devising strategies to save face, the latest article in our 'Asian century' series explores the challenges facing Western managers in China's hotels.
- 1 January 2016As a series of major transport schemes gets underway, could a new and more radical approach to project management help keep them on track?
- 1 January 2016Research has provided new insights into the way consumers view innovations – and how to command their love and respect.
- 1 January 2016Written by Professor Mark Dodgson for The Conversation (December 2015).
- 1 January 2016Social media is transforming business. It is building new kinds of communities and shaping key conversations. What are the trends? Who's doing it well? And how can you build your own online profile?
- 1 January 2016The reality of tougher trading conditions and the high Australian dollar means that SMEs will need smart and well-targeted strategies to make a dent in overseas markets. How can a small business determine a successful export strategy?
- 1 January 2016'Two-speed economy' has become a bit of a business catch phrase, some might say cliché. We talk to 20 leaders about what it means to their businesses.
- 1 January 2016Cash flow and customers – your business needs both. But in times of rapid change, just about everything else is up for grabs. What business skills will keep your company relevant? What are the capabilities of Business 3.0?
Five UQ Business School thought leaders give their take on core capabilities for competitive corporations.
- 1 January 2016Your cultural background may determine how you react to bad customer service, suggests a study led by UQ Business School and conducted across the US, Australia, Thailand and China. Companies seeking to expand internationally, beware, one culture's polite complaint can be another culture's outraged outburst.
- 1 January 2016It's time for leaders to step up. The world faces a range of complex, global challenges: resource depletion, food security, global financial instability, demographic imbalance and climate change. Business can be part of the solution, but are our business leaders up to the job? Associate Professors Bernard McKenna, David Rooney and Dr Hannes Zacher consider wisdom as a prerequisite for leadership.
- 1 January 2016Low pay is no longer just a problem for the poor – it can have a damaging effect on business and the economy too, according to a leading economist.
- 1 January 2016By Associate Professor Sunil Venaik and Dr Paul Brewer for The Conversation (January 2015)
- 1 January 2016Many of those facing redundancy in the current round of industry restructurings are the over-45s, who find it more difficult to find new jobs. How can we help them get back to work?
- 1 January 2016In sport, as well as business, improving performance is about creating an environment that enables people to grow and develop. A former Olympic coach explains how it's done.
- 1 January 2016Do X-Politicians come back as company directors?
There is life after politics, but is it in business? When politicians join company boards in Australia, the share price is more likely to drop than when a non-politician is appointed, says UQ Business School research. Don't shareholders value the expertise that our former leaders bring to the boardroom? Or perhaps it's a vote of confidence in our democracy?
- 1 January 2016Everyone knows the first thing you should do when starting a business is to write a business plan. Or is it? According to one school of thought, business plans are a waste of time because they are based on untested hypotheses. The lean startup movement has turned conventional business thinking on its head in an attempt to make the whole process of starting a company less risky.
- 1 January 2016'Greed is good' was the theme of business in the 80s, but more recently the GFC has led to calls for business to rediscover its moral purpose. Now a group of worldwide leaders including Sir Richard Branson have come together to launch the B Team, which aims to 'put people and planet alongside profit'. Can business save the world? And should we all be working towards more altruistic goals?


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Boutique insurance brokerage Oxygen Holdings to sell off all assetsPosted On: Nov. 8, 2011 12:00 AM CST
LONDON—Oxygen Holdings P.L.C. has announced that it will dispose of all its remaining assets.
The London-based boutique brokerage, which was formed in 2004, on Monday announced the sale of its music and entertainment industry insurance brokerage, Robertson Taylor Holdings Ltd., to Entertainment Insurance Partners for an undisclosed sum.
In August, Oxygen sold its London market brokerage teams to the U.K. operations of Itasca, Ill.-based Arthur J. Gallagher & Co.
Oxygen said Monday that an announcement regarding the sale of its U.K. corporate risks division was "imminent."
In a statement, Nigel Barton, CEO of Oxygen, said the brokerage had been impacted "by tough trading and some particularly adverse situations in the last few years."
He said the decision to sell the company was "also influenced by a belief that the short/medium term forecasts for the U.K. and U.S. economies, coupled with continuing weak insurance conditions, would present a very difficult environment for insurance brokers."
"The stronger insurance broking firms, both big and small, will survive this chapter and will ultimately flourish," he said. "However, we came to the sad conclusion that Oxygen's financial condition was not strong enough to withstand the chapter ahead."
He added that while it had originally been planned to sell the company in a single transaction, it became clear that the disposal of individual business units would "better achieve the optimum objective for clients and shareholders."


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During this season of giving, Adopt-A-Highway is happy to support Earth Day Network's Canopy Project. We will be donating 50 tree for each new sign sponsorship!
Happy Holidays from Adopt A Highway Litter Removal Service of America, Inc.
Adopt-A-Highway Give Back Litter Removal Sponsor-A-HighwayDec 10th, 20150 comments
© 2017-2019 Adopt-A-Highway LITTER REMOVAL SERVICE of America, Inc.
Our Name Says It All!


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Have you ever purchased something for a specific purpose that soon becomes obsolete? Me, too. I bought a cherry entertainment cabinet just a couple years before we converted to wide screen. I don't know why I didn't see that coming.
Initially, we constrained ourselves to a new unit sized to fit in the old cabinet. We quickly realized our mistake, squinting from the back of the room. Sub par! Eventually, the Hubs insisted on el grande wall-mounted flat screen. Then our good ol' fashioned TV cabinet became almost useless. Huh.
What a waste! So I began using it almost immediately for other things. As you might imagine, it wasn't long before it was holding a hodgepodge collection of media, gadgets, cables, and cast-offs. What a mess.
Last fall, inspiration struck: this classy cabinet was perfect for a hidden home office. My end goal was to make good use of every inch of the cabinet, get rid of ugly filing cabinets — stuffed to the rims — plus the pile of files accumulating near by. Finally, inspired by Ikea Hackers, I had a plan!
First, I counted and measured shelving. The good news is these old style cabinets are deep! Then I struck out on a bargain treasure hunt. I was looking for home office storage supplies I could live with a long time. They had to mix well with the few odds and ends I already owned.
At TJ Maxx, I purchased four small gray covered storage boxes in varying depths. I also purchased three sectioned organizers in classy French motif. On a whim, I snagged an open box that matched.
At Office Max, I purchased six self-assemble cardboard filing boxes; five standard size and one for legal-size paperwork. They snap together along the edges and seem pretty sturdy. I also picked up some Manila folders and hanging file folders.
I filled the small storage boxes with common supplies such as business envelopes and stamps, banking materials, small craft tools and such. The sectioned organizers worked great for all of the stuff that usually fills the junk drawer: scissors, tape, rulers, highlighters, erasers, staples, and various doo-dads. The open box was perfect for greeting cards and thank you notes.
Into one filing box fit reams of computer paper, notebook paper, photo paper, and card stock. In another, boxes of stationary and Christmas cards were tucked away. There was room enough for my paper shredder and pencil sharpener, using the handy dandy electrical connection built into the cabinet. That was the easy part.
Several months later, I had enough courage to tackle the hard part…
.. which brings us to July Fourth weekend, 2014. It took me most of three full days to process all the paperwork accumulated through neglect. So many records saved over the years have no value but each had to be reexamined. Thirty-three years of marriage (27 years in this house) raising three children through childhood to college, several major surgeries and a few minor along with a life threatening illness or two… sigh. You get the picture. Most of those records passed through my fingertips this weekend.
It might have been the most boring Independence Day holiday weekend, ever. My back ached and ached. My head spun. The only thing saving my sanity was the movie queue. It seemed like acres of paper and it probably was.
In the end, what filtered out of all of that fit easily in the four empty filing boxes. The result is a refreshingly clean and organized cabinet fulfilling its potential once again. Oh, and two empty filing cabinets in need of a new home. Plus a massive pile of paper to recycle. I have to admit to a sense of pride in finally wrestling this beastie into submission.
I made a few discoveries as well, saving some important tidbits from obscurity and identifying others that deserve no less than a toasty hot hot hot! fire pit. I will surely enjoy watching those burn while sipping on a generous pour of red. Fortnately, I know just the bottle for the occasion!
~ René Morley


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Glossary: Message
are miniature self-contained programs. The only way to interact with them is via their
member functions
which are created by the object's designer who, thereby, defines exactly how the object may be used and keeps it at arms length from the user. To underline this concept of autonomous objects, accessing them via their member functions is often referred to as sending them messages; the name of the function being the name of the message. For example: "The TObject is sent the Draw message".
See OO Concepts:
Objects & Classes
Go Back to the
The Glossary Top Page
If you have any comments about this page please send them to
Nick West


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Angelika Montalbano, Psy.D.
Dr. Montalbano is a licensed psychologist specializing in the assessment of children, adolescents and young adults.
Dr. Montalbano obtained her doctoral degree in clinical psychology from Chestnut Hill College in 2012. She has specialized training and experience with Autism spectrum evaluation, psychoeducational evaluations and ADHD assessments.
Medical School
Chestnut Hill College


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The Basics:
-Score: WPI 9, Anna Maria College 1
-Records: WPI (11-4-1), Anna Maria (2-14-1)
-Location: Worcester, Mass. (Alumni Stadium)
- The Lead: Freshman Sean Meaney (Bridgewater, MA) notched a hat-trick while junior Frank Ciliberto (Spencerport, NY) and sophomore Zach Zlotnick (Rochester, NY) each scored twice in a 9-1 victory for the WPI men's soccer team over visiting Anna Maria Sunday in Alumni Stadium. The Crimson and Gray are now 11-4-1 while the AMCATS close out the season at 2-14-1.
How It Happened:
-Meaney picked up his first collegiate goal at 69:38 and quickly collected his second at 70:42. He completed the feat with 11 minutes to play with aid by Zlotnick.
-Ciliberto scored his first of the game at 29:38 with help from senior Ryan Stokes (Fairport, NY) and knocked home a one-timer from sophomore Liam Setterlund (Holden, MA) at 63:42. Zlotnick converted a PK at 68:09 and closed out the scoring with a wide open net following a feed from Stokes at 82:04. Stokes, meanwhile, picked up his third career goal when he teamed up with classmate Anthony Vigliotta (Beverly, MA) in the 11th minute. Senior Pavolas Christidis (Fairport, NY) found an opening in the bottom right with a fraction of a second left in the first half. Sophomore Matt Mulligan (Columbia, CT) helped setup the opportunity that gave the Engineers a 3-0 lead after 45 minutes.
-Sophomore Troy Repekta (Dudley, MA) broke up the shutout for Anna Maria with a header from 10 yards out at 77:44. The AMCATS utilized a pair of goalkeepers with freshman Jeffre Donahue (Rockland, MA) stopping two shots in the start and junior Mark Robinshaw (Ashford, CT) halting one in relief.
WPI saw all four keepers take a shift. Senior Connor Hoeckele (Granby, CT) began the game and handed over duties to classmate Jordan Finzel (Northport, NY), who served for over a half hour. Sophomore DJ Newman (DeWitt, NY) and freshman Rufus Adams (Duxbury, MA) also saw action for the Engineers.
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