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The bulk of ATP used by many cells, to maintain homeostasis, is produced by the re-oxidation of the reduced electron carriers, NADH and FADH2, within the mitochondrial oxidative phosphorylation pathway. A large percentage of these two reduced electron carriers are generated by the oxidation of pyruvate in the TCA cycle. The fate of pyruvate depends on the cell energy charge. In liver, intestine, and kidney when the energy charge is high, pyruvate is directed toward gluconeogenesis. However, when the energy charge is low, pyruvate is preferentially oxidized to CO2 and H2O in the TCA cycle, with generation of 15 equivalents of ATP per pyruvate. The enzymatic activities of the TCA cycle (and of oxidative phosphorylation) are located within the inner mitochondrial matrix and inner mitochondrial membrane.
The transport of pyruvate into the mitochondria involves two distinct sets of transport complexes. The outer mitochondrial membrane transports pyruvate into the intermembrane compartment via the action of a voltage-gated porin complex. Transport of pyruvate across the inner mitochondrial membrane requires the mitochondrial pyruvate carrier (MPC) complex. The MPC is a heterotetrameric complex composed of subunits encoded by the MPC1 and MPC2 genes. The MPC1 gene is located on chromosome 6q27 and is composed of 7 exons that generate two alternatively spliced mRNAs encoding MPC1 isoform 1 (109 amino acids) and MPC1 isoform 2 (66 amino acids). The MPC2 gene is located on chromosome 1q24 and is composed of 7 exons that generate two alternatively spliced mRNAs that both encode the same 127 amino acid protein.back to the top
When transported into the inner mitochondrial matrix, pyruvate encounters two principal metabolizing enzymes: pyruvate carboxylase, PC (a gluconeogenic enzyme) and pyruvate dehydrogenase (PDH), the first enzyme of the PDHc. With a high cell-energy charge, co-enzyme A (CoA) is highly acylated, principally as acetyl-CoA, and able to obligately activate pyruvate carboxylase, directing pyruvate toward gluconeogenesis. When the energy charge is low, CoA is not acylated, therefore, pyruvate carboxylase is inactive, and pyruvate is preferentially metabolized via the PDHc and the TCA cycle to CO2 and H2O. The acetyl-CoA produced by the PDHc enters the TCA cycle and the reduced electron carriers (NADH and FADH2) that are generated during the oxidative reactions can then be used to drive ATP synthesis via oxidative phosphorylation.
The PDHc is comprised of multiple copies of three distinct enzyme activities: pyruvate dehydrogenase (PDH, identified as the E1 component), dihydrolipoamide S-acetyltransferase, (DLAT, identified as the E2 component) and dihydrolipoamide dehydrogenase, (DLD, identified as the E3). The PDH activity (E1) is a heterotetrameric complex composed of two α (alpha) and two β (beta) subunits. The α subunits are primarily encoded by the PDHA1 gene located on chromosome Xp22.1 and is composed of 12 exons that generate four alternatively spliced mRNAs. The predominant PDHA1 derived mRNA (isoform 1) encodes a precursor protein of 390 amino acids. Another PDH α-subunit gene (PDHA2) encodes a testis-specific isoform. The β subunits are encoded by the PDHB gene located on chromosome 3p21.1–p14.2 and is composed of 11 exons that generate two alternatively spliced mRNAs. The DLAT gene is located on chromosome 11q23.1 and is composed of 14 exons that encode a 647 amino acid protein. The DLD gene is located on chromosome 7q31–q32 and is composed of 14 exons that generate four alternatively spliced mRNAs with the largest mRNA encodong a protein of 509 amino acids. The DLD encoded proteins function within the PDHc as homodimeric units. The DLD encoded proteins are also functional components of the 2-oxoglutarate dehydrogenase complex (more commonly known as α-ketoglutarate dehydrogenase) of the TCA cycle described below, and of the branched-chain keto-acid dehydrogenase complex (BCKD, an amino acid metabolism enzyme complex).
In addition to the complexity of the PDHc at the level of the total number of protein subunits (30 subunits of E1, 60 subunits of E2, and 12 subunits of E3), the full activity of the PDHc requires five different vitamin-derived coenzymes: CoA, NAD+, FAD, lipoic acid and thiamine pyrophosphate (TPP). A mnemonic that helps one remember these five required co-factors/coenzymes is Tender (TPP) Loving (lipoic acid) Care (CoA) For (FAD) Nancy (NAD+). Three of the coenzymes of the PDHc are tightly bound to enzymes of the complex (TPP, lipoic acid and FAD) and two are employed as carriers of the products of PDHc activity (CoA and NAD+). Two additional dehydrogenase complexes require these same five coenzymes: the 2-oxoglutarate dehydrogenase complex and the BCKD complex.
The pathway of PDHc-mediated oxidation of pyruvate to acetyl-CoA is diagrammed below:
Flow diagram depicting the overall activity of the PDHc. During the oxidation of pyruvate to acetyl-CoA and CO2, by the PDH activities, the electrons flow from pyruvate to the lipoamide moiety of DLAT, then to the FAD cofactor of DLD and finally to reduction of NAD+ to NADH. The acetyl group is linked to coenzyme A (CoASH) in a high energy thioester bond. The acetyl-CoA then enters the TCA cycle for complete oxidation to CO2 and H2O.
The first enzyme of the PDHc is PDH itself (E1) which oxidatively decarboxylates pyruvate. During the course of the reaction the acetyl group derived from decarboxylation of pyruvate is bound to TPP. The next reaction of the complex is the transfer of the two carbon acetyl group from acetyl-TPP to lipoic acid, the covalently bound coenzyme of DLAT. The transfer of the acetyl group from acyl-lipoamide to CoA results in the formation of 2 sulfhydryl (–SH) groups in lipoate requiring re-oxidation of the disulfide (-S–S-) form to regenerate lipoate as a competent acyl acceptor. The enzyme DLD, with FAD as a cofactor, catalyzes that oxidation reaction. The final activity of the PDHc is the transfer of reducing equivalents from the FADH2 of DLD to NAD+. The fate of the NADH is oxidation via mitochondrial electron transport, to produce approximately three three equivalents of ATP:
The net result of the reactions of the PDHc are:
The reactions of the PDHc serve to interconnect the metabolic pathways of glycolysis, gluconeogenesis and fatty acid oxidation to the TCA cycle. The activity of the PDHc is also important in regulating the flux from glucose to malonyl-CoA which is required for the de novo synthesis of fatty acids. Acetyl-CoA, produced in the mitochondria via the action of the PDHc, will be transported out into the cytosol in the form of citrate when the energy charge of the cell rises. In the cytosol, the citrate is hydrolyzed by ATP-citrate lyase (gene symbol: ACLY) yielding oxaloacetate and acetyl-CoA. In the cytosol the acetyl-CoA can be converted to malonyl-CoA via the action of acetyl-CoA carboxylase (ACC). This malonyl-CoA serves as the precursor for the synthesis of fatty acids. Accumulation of cytosolic malonyl-CoA, as will happen in energy rich conditions, partitions cytosolic fatty acids away from the oxidizing machinery of the mitochondria by inhibiting carnitine palmitoyltransferase I (CPT I). This effect of malonyl-CoA, derived from acetyl-CoA, couples increased rates of glucose oxidation to inhibition of fatty acid oxidation. There is no pathway in mammals for the conversion of acetyl-CoA to glucose and therefore, the activity of the PDHc is highly regulated by a variety of allosteric effectors and by covalent modification. The importance of the PDHc to the maintenance of homeostasis is evident from the fact that although diseases associated with deficiencies of the PDHc have been observed, affected individuals often do not survive to maturity. Since the energy metabolism of highly aerobic tissues such as the brain, skeletal muscle, and the heart is dependent on normal conversion of pyruvate to acetyl-CoA, aerobic tissues are most sensitive to deficiencies in components of the PDHc. Most genetic diseases associated with PDHc deficiency are due to mutations in either of the two subunits (α or β) of the functional PDH heterotetramer. The main pathologic result of such mutations is moderate to severe cerebral lactic acidosis and encephalopathies.
Factors regulating the activity of the PDHc. PDH activity is regulated by its state of phosphorylation, being most active in the dephosphorylated state. Phosphorylation of PDH is catalyzed by four specific PDH kinases, designated PDK1, PDK2, PDK3, and PDK4. The activity of these kinases is enhanced when cellular energy charge is high which is reflected by an increase in the level of ATP, NADH and acetyl-CoA. Conversely, an increase in pyruvate strongly inhibits PDH kinases. Additional negative effectors of PDH kinases are ADP, NAD+ and CoASH, the levels of which increase when energy levels fall. The regulation of PDH phosphatases (PDPs) is less well understood but it is known that Mg2+ and Ca2+ activate the enzymes and that they are targets of insulin action. In adipose tissue, insulin increases PDH activity and in cardiac muscle PDH activity is increased by catecholamines. Green arrows denote activation effects, whereas red T-lines denote inhibitory actions.
The activity of PDH (E1) is rapidly regulated by phosphorylation and dephosphorylation events that are catalyzed by PDH kinases (PDKs) and PDH phosphatases (PDPs), respectively. Phosphorylation of PDH results in inhibition of activity, whereas, dephosphorylation increases it. The PDKs have been shown to be tightly bound the the lipoyl domains of E2, anchoring the kinases to the PDHc. On average, there are two to three PDKs bound to each PDHc.
Phosphorylation of the α subunit of PDH (E1α; encoded by the PDHA1 gene) occurs at three different serine residues termed site 1 (Ser-264), site 2 (Ser-271), and site 3 (Ser-203). Phosphorylation of any of these three serine residues results in the inhibition of the PDHc. However, data show that phosphorylation of site 1 occurs most rapidly and site 3 being the least rapidly phosphorylated. Given these differences in phosphorylation rate, site 1 is of primary importance for the acute regulation of the PDHc activity. Four PDK isozymes have been identified in humans: PDK1, PDK2, PDK3, and PDK4. The PDK1 gene is located on chromosome 2q31.1 and is composed of 24 exons that generate two alternatively spliced mRNAs encoding two distinct protein isoforms. The PDK2 gene is located on chromosome 17q21.33 and is composed of 14 exons that generate four alternatively spliced mRNAs encoding three distinct protein isoforms. The PDK3 gene is located on the X chromosome (Xp22.11) and is composed of 12 exons that generate two alternatively spliced mRNAs encoding two distinct protein isoforms. The PDK4 gene is located on chromosome 7q21.3 and is composed of 11 exons that generate encode a 411 amino acid protein.
Analysis of the patterns of expression of the PDKs shows that PDK2 is the most widely expressed with the highest levels of expression seen in heart, liver, and kidney. PDK1 expression is highest in the heart and skeletal muscle. PDK3 expression predominates in lung and testis. PDK4 expression is high in heart, liver, pancreatic islets, and kidney and each of these tissues are capable of high rates of fatty acid oxidation and also express high levels of the transcription factors PPARα and PPARδ which are known to be critical regulators of the expression of genes encoding lipid metabolizing enzymes.
Utilizing mutant forms of PDH it has been possible to identify which sites are phosphorylated by which PDK. All four PDKs have been shown to be able to phosphorylate sites 1 and 2. Site 3 is only phosphorylated by PDK1. PDK4 has a much higher preference for phosphorylation of site 2 when compared to the activities of PDK1, PDK2, and PDK3 at this site. Data from these types of experiments indicates that the activity of PDK2 is necessary for short-term inhibition of the PDHc via phosphorylation of site 1. On the other hand, PDK1 and PDK4 appear to be more important for allowing multisite phosphorylation which in turn prevents reactivation of the PDHc by PDP.
Regulation of the activity of the various PDKs is manifest with isoform specificity. Pyruvate, generated from glycolysis, exerts differential inhibitory actions on the various PDKs. PDK2 is the most sensitive to inhibition by pyruvate, whereas, PDK4 is relatively insensitive. As the ATP/ADP ratios falls (i.e. increases in ADP levels) ADP will exert an allosteric inhibition on the activity of the PDKs. ADP exerts its allosteric inhibition of PDKs both independently and in synergy with pyruvate inhibition. In contrast to the acute inhibition of PDKs by pyruvate, acute activation occurs via products of the PDHc as well as by fatty acid oxidation, namely acetyl-CoA and NADH. PDK2 is the most sensitive to increases in the level of acetyl-CoA in comparison to the other PDK isoforms. The ratio of NADH to NAD+ also exert isoform-specific allosteric regulation of PDKs. PDK4 is highly responsive (activated) to an increased NADH/NAD+, whereas, PDK2 is much less sensitive to this activation.
Two genetically and biochemically distinct PDP isoforms (PDP1 and PDP2) have been characterized. The PDP1 is a Mg2+-dependent and Ca2+-stimulated protein serine phosphatase of the protein phosphatase 2C (PP2C) superfamily that is found bound to the inner mitochondrial membrane. Both the PDP1 and PDP2 phosphatase are heterodimeric enzymes composed of a catalytic subunit (PDPc) and a regulatory subunit (PDPr). There are two different catalytic subunit genes in humans identified as the PDP1 gene and the PDP2 gene. The PDP1 encoded protein (also known as PPM2C) is a component of the PDP1 phosphatase complex, whereas the PDP2 encoded protein (also known as PPM2C2) is a component of the PDP2 phosphatase complex. The PDP1 gene is located on chromosome 8q22.1 and is composed of 6 exons that generate four alternatively spliced mRNAs encoding two distinct protein isoforms. The PDP2 gene is located on chromosome 16q22.1 and is composed of 3 exons that encode a 529 amino acid protein. The PDP regulatory subunit is encoded by the PDPR gene which is located on chromosome 16q22.1 and is composed of 24 exons that generate four alternatively spliced mRNAs that encode three distinct precursor isoforms.
The PDP1 phosphatase complex is widely distributed and is considered a major regulator of the activity of the PDH complex. The mechanism by which Ca2+ stimulates PDPc activity is by increasing its interaction with the PDH complex while also decreasing the Km for Mg2+ of the catalytic subunits of the PDP1 complex. Calcium ion also increases the association of PDP1 catalytic subunits with the phosphorylated E1α subunit of the PDHc. In contrast, PDPr lowers the sensitivity of catalytic subunits to Mg2+. Both PDP1 components are targeted by insulin, which enhances PDPc activity and decreases PDPr negative control, followed by enhanced PDPc sensitivity to Mg2+ and improved overall PDP1 efficiency. In addition, the level of PDP1 activity is affected by pathology with decreased levels seen in diabetes. The activity of the PDP2 phosphatase is less well understood of the two PDP complexes but has been shown to consist of a catalytic subunit that is insensitive to Ca2+ and is 10-fold less sensitive to Mg2+ than the PDPc of PDP1. However, PDP2 is also considered a target of insulin action and the level of PDP2 activity is decreased in diabetes and during starvation.
Two products of the complex, NADH and acetyl-CoA (both of which are also products of fatty acid oxidation), are negative allosteric effectors on the non-phosphorylated and most active form of PDH. These effectors reduce the affinity of the enzyme for pyruvate, thus limiting the flow of carbon through the PDH complex. In addition, as indicated above, NADH and acetyl-CoA are powerful positive effectors on the activity of PDK2 and PDK4. Since NADH and acetyl-CoA accumulate when the cell energy charge is high, it is not surprising that high ATP levels also up-regulate PDK activity, reinforcing down-regulation of PDH activity in energy-rich cells. Note, however, that pyruvate is a potent negative effector on all PDKs, with the result that when pyruvate levels rise, active PDH will be favored even with high levels of NADH and acetyl-CoA.
Concentrations of pyruvate which maintain PDH in the active form are sufficiently high so that, in energy-rich cells, the allosterically down-regulated, high Km form of PDH is nonetheless capable of converting pyruvate to acetyl-CoA. With large amounts of pyruvate in cells having high energy charge and high NADH, pyruvate carbon will be directed to the two main storage forms of carbon (glycogen via gluconeogenesis and fat production via fatty acid synthesis) where acetyl-CoA is the principal carbon donor.back to the top
The TCA cycle showing enzymes, substrates and products. The GTP generated during the succinate thiokinase (succinyl-CoA synthetase) reaction is equivalent to a mole of ATP by virtue of the presence of nucleoside diphosphokinase. The generation of this GTP is referred to a substrate-level phosphorylation. The 3 moles of NADH and 1 mole of FADH2 generated during each round of the cycle feed into the electron transport chain (ETC) of the oxidative phosphorylation pathway. Each mole of NADH leads to 3 moles of ATP and each mole of FADH2 leads to 2 moles of ATP. Therefore, for each mole of pyruvate which enters the TCA cycle, 12 moles of ATP can be generated.
Citrate synthase (gene symbol: CS) catalyzes the first reaction of the TCA cycle which involves the condensation of the methyl carbon of acetyl-CoA with the keto carbon (C-2) of oxaloacetate (OAA). The CS gene is located on chromosome 12q13.3 and is composed of 12 exons that generate two splice variants. These differentially spliced mRNAs are referred to as CSa and CSb. The CSa mRNA lacks exon 2 and encodes a protein of 466 amino acids. The CSb mRNA contains exon 2 and encodes a protein of 400 amino acids. The major CS mRNA found in hepatocytes is the CSa form.
The citrate synthase reaction is often considered the rate-limiting step of the TCA cycle, however, greater control over the overall rate of the cycle is exerted at the isocitrate dehydrogenase catalyzed reaction. The standard free energy of the citrate synthase reaction, –8.0 kcal/mol, drives it strongly in the forward direction. Since the formation of OAA from its precursor, malate, is thermodynamically unfavorable, the highly exergonic nature of the citrate synthase reaction is of central importance in keeping the entire cycle going in the forward direction, since it drives oxaloacetate formation by mass action principals.
When the cellular energy charge increases, the rate of flux through the TCA cycle will decline leading to a build-up of citrate. Excess citrate is used to transport acetyl-CoA carbons from the mitochondrion to the cytoplasm where they can be used for fatty acid and cholesterol biosynthesis. Citrate is transported across the inner mitochondrial membrane by the tricarboxylate transporter encoded by the SLC25A1 gene. Additionally, the increased levels of citrate in the cytoplasm activate the key regulatory enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase (ACC) while at the same time citrate allosterically inhibits the rate-limiting enzyme of glycolysis, PFK-1.
The isomerization of citrate to isocitrate by aconitase is stereospecific, with the migration of the –OH from the central carbon of citrate (formerly the keto carbon of OAA) being always to the adjacent carbon which is derived from the methylene group (–CH2–) of OAA. The stereospecific nature of the isomerization determines that the CO2 lost, as isocitrate is oxidized to succinyl-CoA, is derived from the oxaloacetate used in citrate synthesis.
Humans contain two distinctly different aconitase enzymes, aconitase 1 and aconitase 2. One is a soluble form found within the cytosol and is involved in the iron-mediated regulation of protein synthesis, specifically the translation of the ferritin and the transferrin receptor mRNA (see the Protein Synthesis page). The soluble aconitase 1 is encoded by the ACO1 gene located on chromosome 9q21.1 which encodes a protein of 889 amino acids. The other enzyme (aconitase 2) is the mitochondrial enzyme involved in the TCA cycle. The mitochondrial aconitase 2 is encoded by the ACO2 gene located on chromosome 22q13.2 which encodes a protein of 780 amino acids.
Mitochondrial aconitase 2 is one of several mitochondrial enzymes known as non-heme-iron proteins. These proteins contain inorganic iron and sulfur, known as iron sulfur centers, in a coordination complex with sulfhydryls of cysteine residues in the protein. There are two prominent classes of non-heme-iron complexes, those containing two equivalents each of inorganic iron and sulfur, Fe2S2, and those containing 4 equivalents of each, Fe4S4. Aconitase 2 is a member of the Fe4S4 class. Its iron sulfur centers are often designated as Fe4S4Cys4, indicating that 4 cysteine sulfur atoms are involved in the complete structure of the complex. In iron sulfur compounds the iron is generally involved in the oxidation-reduction events.
There are three different IDH enzymes in humans identified as IDH1, IDH2, and IDH3 with only the IDH3 isoform being involved in the TCA cycle. Isocitrate is oxidatively decarboxylated to 2-oxoglutarate (α-ketoglutarate) by isocitrate dehydrogenase, IDH. It is generally considered that control of carbon flow through the TCA cycle is regulated at IDH by the powerful negative allosteric effectors NADH and ATP and by the potent positive effectors; isocitrate, ADP and AMP. From the latter it is clear that cell energy charge is a key factor in regulating carbon flow through the TCA cycle.
The IDH1 and IDH2 enzymes are NADP-dependent enzymes. There are three distinct NAD+-dependent IDH enzymes expressed in humans. The NAD+-dependent isoforms are all commonly referred to as IDH3. These IDH isoforms are the primary mitochondrial enzymes responsible for oxidative decarboxylation of isocitrate within the context of the TCA cycle. The isocitrate dehydrogenase reaction is the rate-limiting step, as well as the first NADH-yielding reaction of the TCA cycle. The CO2 produced by the IDH3 reaction is from the original C-1 carbon of the oxaloacetate used in the citrate synthase reaction. Each of the functional IDH3 enzymes is a heterotetramer composed of three different subunits identified as the α, β, and γ subunits. The primary composition of IDH3 is α2β1γ1. The α subunit is encoded by the IDH3A gene located on chromosome 15q25.1–q25.2 and is composed of 12 exons that encode a protein of 366 amino acids. The β subunit is encoded by the IDH3B gene located on chromosome 20p13 and is composed of 14 exons that generate three alternatively spliced mRNAs, each of which encode a distinct protein isoforms. IDH3B isoform a is a 385 amino acid precursor protein, isoform b is a 383 amino acid precursor protein, and isoform d is a 376 amino acid precursor protein. The γ subunit is encoded by the IDH3G gene located on chromosome Xq28 and is composed of 12 exons that generate two alternatively spliced mRNAs encoding proteins of 393 amino acids (isoform a) and 380 amino acids (isoform b).
Both IDH1 and IDH2 enzymes utilize NADP+ as their cofactor and generate NADPH via oxidative decarboxylation of isocitrate outside the context of the TCA cycle. The IDH1 is a homodimeric enzyme encoded by the IDH1 gene located on chromosome 2q33.3 and is composed of 12 exons that generate three alternatively spliced mRNAs, all of which encode the same 414 amino acid protein. IDH1 is found primarily in the cytosol and the peroxisomes. The IDH2 enzyme is also a homodimeric enzyme but is located in the mitochondria. IDH2 is encoded by the IDH2 gene located on chromosome 15q26.1 and is composed of 12 exons that generate three alternatively spliced mRNAs, each of which encode a distinct protein isoform. The primary function of IDH1 and IDH2 is to serve as producers of NADPH to enhance the cellular responses to oxidative stress and the generation of reactive oxygen species, ROS. The principal sources of NADPH are from the glucose-6-phosphate dehydrogenase (G6PD) reaction in the pentose phosphate pathway and via malic enzyme which is involved in the mobilization of mitochondrial acetyl-CoA to the cytosol. The significance of IDH1 and IDH2 produced NADPH can be made clear by pointing out that cells with low level IDH1 and IDH2 expression are more sensitive to oxidative stress than cells with higher levels of expression of these two enzymes.
2-Oxoglutarate (also called α-ketoglutarate) is oxidatively decarboxylated to succinyl-CoA by the 2-oxoglutarate dehydrogenase complex, OGDH. The OGDH complex is commonly referred to as the α-ketoglutarate dehydrogenase (α-KGDH) complex. This reaction generates the second TCA cycle equivalent of CO2 and NADH. As with the PDH complex described above, the reactions of the OGDH complex proceed with a large negative standard free energy change. The OGDH complex is very similar to the PDH complex in the intricacy of its protein makeup (E1, E2, and E3 activities), cofactors (TPP, lipoic acid, CoA, FAD, and NAD+), and its mechanism of action. Although the E1 activity of the OGDH complex is not subject to covalent modification as are the E1 components of the PDHc, allosteric regulation of the OGDH complex is indeed effected. The enzyme complex is also regulated energy charge, the NAD+/NADH ratio, and effector activity of substrates and products.
The three activities of OGDH complex are identified as oxoglutarate dehydrogenase (E1k), dihydrolipoamide S-succinyltransferase (DLST or E2k), and dihydrolipoamide dehydrogenase (DLD or E3). The oxoglutarate dehydrogenase subunits of the OGDH complex are encoded by the OGDH gene located on chromosome 7p14-p13 and is composed of 26 exons that generate three alternatively spliced mRNAs that encode three distinct isoforms (isoform 1: 1023 amino acid precursor; isoform 2: 427 amino acid precursor; isoform 3: 1019 amino acid precursor). The DLST subunits are encoded by the DLST gene located on chromosome 14q24.3 and is composed of 15 exons that generate two alternatively spliced mRNAs encoding isoform 1 (453 amino acid precursor) and isoform 2 (166 amino acid precursor). The DLD subunits of the OGDH complex are the same as those found in the PDH complex, i.e. encoded by the same gene.
Succinyl-CoA and 2-oxoglutarate are also important metabolites outside the TCA cycle. In particular, 2-oxoglutarate represents a key anaplerotic metabolite linking the entry and exit of carbon atoms from the TCA cycle to pathways involved in amino acid metabolism. 2-Oxoglutarate is also important for driving the malate-aspartate shuttle. Succinyl-CoA, along with glycine, contributes all the carbon and nitrogen atoms required for the synthesis of heme and for non-hepatic tissue utilization of ketone bodies.
The malate/aspartate shuttle. This shuttle is the principal mechanism for the movement of reducing equivalents (in the form of NADH; highlighted in the red boxes) from the cytoplasm to the mitochondria. The glycolytic pathway is a primary source of cytoplasmic NADH. Within the mitochodria the electrons of NADH can be coupled to ATP production during the process of oxidative phosphorylation. The electrons are "carried" into the mitochondria in the form of malate. Cytoplasmic malate dehydrogenase (MDH) reduces oxaloacetate (OAA) to malate while oxidizing NADH to NAD+. Malate then enters the mitochondria where the reverse reaction is carried out by mitochondrial MDH. Movement of mitochondrial OAA to the cytoplasm to maintain this cycle requires it be transaminated to aspartate (Asp, D) with the amino group being donated by glutamate (Glu, E). The Asp is then transported out of the mitochondria and enters the cytoplasm. The deamination of glutamate generates 2-oxoglutarate, 2-OG, (α-ketoglutarate) which is transported out of the mitochondria to the cytoplasm. All the participants in the cycle are present in the proper cellular compartment for the shuttle to function due to concentration dependent transport. When the energy level of the cell rises the rate of mitochondrial oxidation of NADH to NAD+ declines and therefore, the shuttle slows. GAPDH is glyceraldehyde-3-phosphate dehydrogenase. AST is aspartate transaminase. SLC25A11 is the malate transporter and SLC25A13 is the aspartate/glutamate transporter.
The conversion of succinyl-CoA to succinate, by succinyl-CoA synthetase (SCS), involves the use of the high-energy thioester of succinyl-CoA to drive synthesis of a high-energy nucleotide phosphate (GTP). This process is referred to as substrate-level phosphorylation. In this process, a high energy enzyme-phosphate intermediate is formed, with the phosphate subsequently being transferred to GDP. Mitochondrial GTP is used in a trans-phosphorylation reaction catalyzed by the mitochondrial enzyme nucleoside diphosphokinase to phosphorylate ADP, producing ATP and regenerating GDP for the continued operation of succinyl-CoA synthetase.
Succinyl-CoA synthetase is a heterodimeric enzyme composed of an α (alpha) and a β (beta) subunit. The α-subunit is invariant in all forms of SCS and is encoded by the SUCLG1 (succinate-CoA ligase) gene. The SUCLG1 gene is located on chromosome 2p11.2 and is composed of 9 exons encoding a protein of 346 amino acids. The β-subunit of SCS determines the nucleotide specificity of the enzyme complex. There are two β-subunit genes encoding the A-beta and G-beta subunits. As indicated by the designations, the A-beta subunit determines specificity for ADP/ATP and the G-beta for GDP/GTP. The A-beta subunit is encoded by the SUCLA2 gene located on chromosome 13q14.2 and is composed of 11 exons encoding a protein of 463 amino acids. The G-beta subunit is encoded by the SUCLG2 gene located on chromosome 3p14.1 and is composed of 14 exons encoding a protein of 440 amino acids.
The multi-enzyme complex called succinate dehydrogenase (SDH) catalyzes the oxidation of succinate to fumarate with the sequential reduction of enzyme-bound FAD to FADH2 as well as the associated non-heme-iron. The SDH complex is the same as complex II of the electron transport chain (ETC) of oxidative phosphorylation. In mammalian cells, the electron acceptor is coenzyme Q (CoQ; written as CoQ10 to depict the major form in human cells), a mobile carrier of reducing equivalents that is restricted by its lipophilic nature to the lipid phase of the inner mitochondrial membrane and by its association with subunits of the SDH complex. The SDH complex is the only enzymatic activity of the TCA cycle that is embedded within the inner mitochondrial membrane, all of the other enzymes reside within the matrix of the mitochondria.
The SDH complex is composed of four different proteins encoded by four different nuclear genes identified as SDHA, SDHB, SDHC, and SDHD. The SDH complex is the only component of the mitochondrial electron transport chain (ETC) that is composed solely of proteins encoded by nuclear genes. In addition to these four proteins, that constitute the functional SDH complex (oxidative phosphorylation complex II), at least two additional proteins are required for proper assembly of the complex. These assembly factors are called SDH complex assembly factor 1 (SDHAF1) and 2 (SDHAF2). The A subunit of SDH is a flavoprotein encoded by the SDHA gene located on chromosome 5p15 which is composed of 15 exons the generate two alternatively spliced mRNAs (isoform 1: 664 amino acids; isoform 2: 616 amino acids). The SDHA encoded protein is the enzymatic activity that carries out the oxidation of succinate to fumarate with the simultaneous reduction of FAD to FADH2. The B subunit of SDH is an iron-sulfur protein encoded by the SDHB gene located on chromosome 1p36.1–p35 which is composed of 8 exons encoding a precursor protein of 280 amino acids. The SDHB encoded protein contains three distinct Fe-S (iron-sulfur) centers that facilitate the transfer of electrons from FADH2 to ubiquinone (coenzyme Q: CoQ). The Fe-S centers of SDHB consist of a 2Fe-2S center, which is near the FAD binding site, a 4Fe-4S center, and a 3Fe-4S center. The C subunit of SDH is an integral membrane protein encoded by the SDHC gene located on chromosome 1q23.3 which is composed of 7 exons encoding a protein of 150 amino acids. The D subunit of SDH is also an integral membrane protein encoded by the SDHD gene located on chromosome 11q235 which is composed of 6 exons encoding a protein of 85 amino acids. The inner mitochondrial membrane embedded SDHC and SDHD encoded proteins (referred to as the membrane domain of the SDH complex) anchor the SDHA and SDHB subunits to the inner mitochondrial membrane. The SDHC and SDHD subunits also facilitate interactions with CoQ and electron exchange from the Fe-S centers of the SDHB subunit. The membrane domain of the SDH complex contains a heme b moiety that is bound at the interface between the SDHC and SDHD subunits. The binding of CoQ to the SDHC and SDHD membrane domain occurs on the matrix side of the inner mitochondrial membrane.
The catalytic activity of the SDH complex is controlled by post-translational modifications that include phosphorylation and acetylation. In addition, the catalytic activity of the SDHA subunit is allosterically inhibited by oxaloacetate. As succinate levels rise the inhibitory oxaloacetate is displaced, thereby activating the SDH complex. Phosphorylation of the SDH complex has been shown to occur at two tyrosine residues via the action of FGR kinase, a SRC family member tyrosine kinase. However, these results were obtained via in vitro experiments and the identity of the kianse(s) that phosphorylate the SDH complex in vivo is still unknown. However, the significance of tyrosine phosphorylation, to the activity of the SDH complex, was demonstrated when it was shown that blocking the activity of the mitochondrial tyrosine phosphatase, PTPMT1, results in hyperphosphorylation and activation of the SDH complex. Reversible lysine acetylation of the SDHA subunit has also been shown to be responsible for the attenuation of succinate oxidation to fumarate. When acetylated the activity of SDHA is reduced primarily due to reduction in substrate entry into the enzyme active site. Multiple Lys (K) residues in the SDHA subunit have been shown to acetylated through the use of mass spec. However, similar to the situation for possible SDH kinases, no definitive SDH acetylase has been characterized. The principal enzyme responsible for the deacetylation of the SDHA subunit has been defined. One of the sirtuin family of deacetylases, SIRT3, has been shown to be the major mitochondrial deacetylase controlling the level of SDHA acetylation.
The fumarate hydratase-catalyzed reaction is specific for the trans form of fumarate. The result is that the hydration of fumarate proceeds stereospecifically with the production of L-malate. The fumarate hydratase gene (symbol: FH) is located on chromosome 1q42.1 and is composed of 11 exons that encode two distinct proteins from a single mRNA. The two forms of fumarate hydratase result from the single FH mRNA as a result of alternative translational start site utilization. One form of the enzyme is localized to the cytosol while the TCA cycle enzyme is localized to the mitochondria. The mitochondrial version of fumarate hydratase (a 510 amino acid protein) is extended at the N-terminus due to the use of an alternative AUG start codon relative to AUG start codon used to produce the cytosolic form of the enzyme. The AUG start codon used for the cytosolic fumarate hydratase makes this version of the enzyme 43 amino acids shorter than the mitochondrial version.
Mitochondrial malate dehydrogenase (MDH) catalyzes the "final" reaction of the TCA cycle. L-malate is the specific substrate for MDH. The forward reaction of the cycle, the oxidation of malate, yields oxaloacetate (OAA). In the forward direction the reaction has a standard free energy of about +7 kcal/mol, indicating the very unfavorable nature of the forward direction. As noted earlier, the citrate synthase reaction that condenses oxaloacetate with acetyl-CoA has a standard free energy of about –8 kcal/mol and is responsible for pulling the MDH reaction in the forward direction. The overall change in standard free energy change is about –1 kcal/mol for the conversion of malate to oxaloacetate and on to succinate.
There are two malate dehydrogenases in humans encoded by two different genes. Both forms of the enzyme are required for the operation of the malate-aspartate shuttle. The cytosolic form of MDH is encoded by the MDH1 gene. The MDH1 gene is located on chromosome 2p13.3 and is composed of 11 exons encoding a protein of 353 amino acids. The TCA cycle MDH is a mitochondrial enzyme encoded by the MDH2 gene. The MDH2 gene is located on chromosome 7cen-q22 and is composed of 10 exons encoding a protein of 296 amino acids.
The overall stoichiometry of the TCA cycle is:
At least three of the genes that encode TCA cycle enzymes, or protein subunits of enzyme complexes, have been shown to manifest mutations in certain types of familial cancer syndromes. Mutations in two of the three isocitrate dehydrogenase (IDH) genes expressed in humans have been identified to harbor specific mutations and these mutations are correlated to the development of certain forms of cancer. The most common forms of cancer in which mutations in the IDH1 and IDH2 genes have been discovered are gliomas in the brain. As indicated in the descriptions of the enzymes and enzyme complexes of the TCA cycle, the succinate dehydrogenase (SDH) complex is composed of four subunits encoded by four distinct genes, SDHA, SDHB, SDHC, and SDHD. Mutations in all four of these genes are associated with several familial cancer syndromes. In addition to the four functional components of the SDH complex, mutations in the SDH assembly factor gene, SDHAF2, are also found in certain forms of cancers. Although mutations in the SDH subunit assembly factor gene, SDHAF1, have not been associated with cancer syndromes, they are associated with mitochondrial deficiency syndromes. The SDH complex gene associated cancers include paragangliomas, pheochromocytomas, gastrointestinal stromal tumors (GIST), and an aggressive form of renal cancer identifed as SDH-RCC (SDH-deficient renal cell carcinoma). Cancer syndromes have also been asociated with mutations in the fumarate hydratase (FH; also called fumarase) gene. The FH gene mutation associated cancers are cutaneous and uterine leiomyomas and an aggressive form of type 2 papillary renal cancer. The renal papillary carcinoma is characterized by a metabolic shift to aerobic glycolysis and glutamine-dependent reductive carboxylation. Details of the TCA cycle gene mutations and their associations to cancer syndromes will be discussed in the order in which the enzymes appear within the context of the TCA cycle. It is important to note that succinate dehydrogenase is also complex II of the oxidative phosphorylation pathway and contributing to metabolic shifts seen in SDH-deficient cancers are deficiencies in mitochondrial energy generation via oxidative phosphorylation.
As indicated earlier, both IDH1 and IDH2 enzymes utilize NADP+ as their cofactor and generate NADPH via oxidative decarboxylation of isocitrate outside the context of the TCA cycle. The primary function of IDH1 and IDH2 is to serve as producers of NADPH to enhance the cellular responses to oxidative stress and the generation of reactive oxygen species, ROS. The principal sources of NADPH are from the glucose-6-phosphate dehydrogenase (G6PD) reaction in the pentose phosphate pathway and via malic enzyme which is involved in the mobilization of mitochondrial acetyl-CoA to the cytosol. The significance of IDH1 and IDH2 produced NADPH can be made clear by pointing out that cells with low level IDH1 and IDH2 expression are more sensitive to oxidative stress than cells with higher levels of expression of these two enzymes.
Clinical significance is associated with the IDH1 and IDH2 genes as there has been a close association made between mutations in these two genes and the development of certain forms of cancer, specifically gliomas of the brain. All of the mutations in IDH1 that are associated with gliomagenesis are missense mutations that alter the normal Arg residue at position 132 to a histidine (R132H). The mutations in IDH2 are also single amino acid missense mutations at the comparable Arg residue at position 172. The characterized mutations in IDH1 and IDH2 that are associated with the early onset of gliomagenesis result in altered activities of the encoded proteins. The altered activity leads to the production of 2-hydroxyglutarate (2-HG) and the loss of NADPH production. The R132H mutation in IDH1 and the similar mutation in IDH2 result in a loss of isocitrate binding while at the same time altering the catalytic activity of the enzyme such that it oxidizes 2-oxoglutarate (α-ketoglutarate), its normal product, to 2-HG without NADPH production. Indeed, in gliomas with IDH1 mutations the levels of 2-HG are elevated relative to normal cells. Increases in 2-HG levels coupled with decreases in 2-oxoglutarate levels results in competitive inhibition of several important 2-oxoglutarate-dependent dioxygenases such as DNA demethylases and histone demethylases that are involved in regulating chromatin structure, and prolyl hydroxylase domain (PDH) containing enzymes such as those that are involved in cellular responses to hypoxia.
Mutations in IDH2 are much less common than mutations in IDH1 and the appearance of the mutations in these two genes are mutally exclusive meaning that a glioma with mutated IDH1 does not also harbor a mutant IDH2 gene and visa versa. Although associated with gliomagenesis, gliomas that contain mutant IDH1 or IDH2 genes are associated with a better prognosis than gliomas with wild-type IDH1 or IDH2. In addition to gliomas, the same IDH1 missense mutation is found in acute myelogenous leukemia, cholangiocarcinoma, cartilaginous tumors, prostate cancer, papillary breast carcinoma, acute lymphoblastic leukemia, angioimmunoblastic T-cell lymphoma, and primary myelofibrosis.
Mutations in the genes encoding the four proteins comprising the SDH complex result in a pathophysiological state defined as pseudo-hypoxia. This pathophysiologic state leads to a subsequent increase in angiogenesis, via activation of the HIF-1 hypoxia response pathway, resulting in the ability of tumors to acquire nutrients encouraging their aberrant growth. Activation of the HIF-1 pathway in SDH-deficient tumors is most likely the result of accumulating levels of succinate which is then transported into the cytosol via the action of the mitochondrial dicarboxylate transporter. Accumulation of cytosolic succinate inhibits the 2-oxoglutarate-dependent dioxygenases [prolyl hydroxylase domain (PDH) enzymes] responsible for the hydroxylation of the α-subunit (HIF1α) of HIF-1. The hydroxylation of HIF1α normally occurs continuously under normoxic conditions which stimulates ubiquitination and proteosomal degradation of HIF1α, thereby restricting the transcription factor function of HIF-1. The loss of HIF1α hydroxylation results stabilization of the protein and consequent activation of the HIF-1 pathway in the absence of hypoxia. Activation of HIF-1 in the absence of hypoxia leads to increased angiogenesis within the SDH-deficient tumor environment promoting the growth of the tumor cells. Because loss of function of any one of the four subunits of the SDH complex results in the progression to cancer, all four genes encoding proteins of the complex (SDHA, SDHB, SDHC, and SDHD) have been classified as tumor suppressors.
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the gastrointestinal tract. Until very recently the vast majority of GISTs were associated with mutations resulting in the activation of the KIT gene encoded protein (75% to 80%). Additional activating mutations, associated with GIST, although not as common as the KIT mutations, are those in the PDGF receptor A (PDGFRA: 5% to 8%) gene. Less than 10 years age a new class of GIST was identified that is due to mutations in several of the genes encoding protein components of the SDH complex. The initially characterized SDH-deficient GISTs were found to be associated with mutations in the SDHB, SDHC, and SDHD genes. More recently SDHA deficient GISTs have been characterized. The SDH-deficient GISTs are wild type for both the KIT and PDGFRA genes. The SDH-deficient tumors occur exclusively in the stomach and commonly metastasize to lymph nodes. These SDH-deficient tumors are also charactreized by having a unique epithelioid morphology often with a plexiform and multinodular growth pattern and many of the tumors also overexpress the insulin-like growth factor 1 receptor (IGFR1) gene. Since their initial characterization, the SDH-deficient GISTs have been shown to account for up to 7.5% of these forms of cancer in adult patients and represent the vast majority these types of tumors that are found in childhood.
Mutations in all four SDH complex genes, as well as the SDHAF2 gene, are associated with additional forms of cancer. These additional forms are defined as the hereditary paraganglioma and pheochromocytoma syndrome (HPGL/PCC). Paragangliomas (PGLs) are rare tumors derived from the paraganglia. The paraganglia are neuroendrocrine tissues that are symmetrically distributed along the paravertebral axis from the base of the skull and the neck to the pelvis. In the adult that major paraganglionic organs are the carotid body and the adrenal medulla. The carotid body represents a small cluster of chemoreceptors and associated supporting cells that runs along the bottom of the bifurcation of the carotid artery. The primary function of the carotid body is to detect changes in the partial pressures of O2 and CO2 within the arterial blood, but is also involved in the sensation of pH and temperature. The adrenal medulla secretes the catecholamines, epinephrine and norepinephrine, in response to stress-mediated stimulation of preganglionic sympathetic neurons. Paragangliomas are primarily found in the head and neck region, particularly in the carotid bodies. Paragangliomas arising from chromaffin cells of the adrenal medulla are defined as pheoochromocytomas and they characteristically hypersecrete catecholamine. The paraganglioma-pheochromocytoma syndrome (HPGL/PCC) was originally identifed as being caused by mutations in the SDHB, SDHC, and SDHD genes and later to be associated with SDHA and SDHAF2 gene mutations.
Similar to the alterations that occur in metabolic programs in SDH-deficient cancers, FH-deficient cells exhibit a substantial metabolic reorganization. FH-deficient tumor cells satisfy their need for high rates of ATP production by developing high rates of glucose uptake and higher rates of glucose oxidation via glycolysis. This form of metabolic reprogramming in cancer cells (as well as that occuring in normal rapidly proliferating cells) is referred to as the Warburg effect. The reduced entry of glycolytic intermediates into the TCA cycle, due to FH deficiency, results in diversion of glucose to lactate as well as into other metabolic pathways of biomass production. All of these shifts in metabolism are not unique to FH-deficient tumors but are a common occurrence in most tumors. As indicated in the Glycolysis page and in the section above on SDH-deficient tumors, the common changes in metabolic profiles in cancer cells is, in part, the result of the activation of the hypoxia induced factor 1 (HIF-1) transcriptional program.
When fumarate accumulates in the mitochondria of FH-deficient cells the metabolite is transported to the cytosol by the dicarboxylate transporter. Within the cytosol the accumulating fumarate, similar to the effect of accumulating succinate, results in the inhibition of 2-oxoglutarate-dependent dioxygenases that are responsible for the hydroxylation of the α-subunit (HIF1α) of the HIF-1 complex. The HIF1α proline hydroxylases are commonly referred to as the prolyl hydroxylase domain (PDH) enzymes. The hydroxylation of HIF1α presents binding sites for the VHL ubiquitin ligase which then ubiquinates HIF1α resulting in its proteosomal degradation. Inhibition of the PDH enzymes by fumarate results in loss of HIF1α turnover which then allows the HIF-1 complex to migrate to the nucleus where a program of genes are activaed that are normally responsible for cell survival in response to hypoxia. One of the major responses to HIF-1 transcriptional activity is increased angiogenesis allowing tumor cells higher rates of access to biomolecules for growth. Additional metabolic changes in FH-deficient tumors have been shown to be the result of reduced activation and reduced expression of the master metabolic regulatory kinase, AMPK. The loss of AMPK activity results in increased rates of lipid and protein synthesis in the tumor cells.
Mutations in the fumarate hydratase (FH: also called fumarase) gene have been shown to be associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Leiomyoma is a form of smooth muscle tumor that can develop in any organ but is most commonly found in the uterus, esophagus, and small intestine. Uterine fibroids are a common form of leiomyoma of the uterine smooth muscle that are benign. HLRCC is an autosomal dominant hereditary cancer syndrome that is characterized by a predisposition to the development of cutaneous and uterine leiomyomas and a very aggressive form of papillary kidney cancer. HLRCC-associated renal tumors readily metastasize to both regional and distant lymph nodes making this form of renal cancer significantly more aggressive than other forms of genetically defined renal cancer. Mutations resulting in loss of function of fumarate hydratase are the primary genetic alterations associated with HLRCC. The FH mutations found in HLRCC are missense, frameshift, partial deletions, or complete deletions. Because loss of function of the FH gene results in the progression to cancer, the gene has been classified as a tumor suppressor.back to the top
There is no direct hormonal regulation of TCA cycle activity, substrate availability and allosteric regulation of enzyme activity controls the rate of flux through the cycle. Fuel enters the TCA cycle primarily as acetyl-CoA, albeit several intermediates in the cycle are replenished via the process referred to as anapleurosis. The generation of acetyl-CoA from carbohydrates is a major control point of the cycle. This is the reaction catalyzed by the PDH complex.
By way of review, the PDH complex is inhibited by acetyl-CoA and NADH and activated by non-acetylated CoA (CoASH) and NAD+. The pyruvate dehydrogenase activities of the PDH complex are regulated by their state of phosphorylation. This modification is carried out by specific kinases (PDK1–PDK4) and the phosphates are removed by a specific phosphatase (PDH phosphatase, PDP). The phosphorylation of PDH inhibits its activity and, therefore, leads to decreased oxidation of pyruvate. PDKs are activated by NADH and acetyl-CoA and inhibited by pyruvate, ADP, CoASH, Ca2+ and Mg2+. PDP, in contrast, is activated by Mg2+ and Ca2+.
Since three reactions of the TCA cycle as well as PDH utilize NAD+ as co-factor it is not difficult to understand why the cellular ratio of NAD+/NADH has a major impact on the flux of carbon through the TCA cycle. Substrate availability can also regulate TCA flux. This occurs at the citrate synthase reaction as a result of reduced availability of oxaloacetate. Product inhibition and energy state also controls the TCA flux. The major control point in the TCA cycle occurs at the IDH3 catalyzed reaction. Both NADH and ATP allosterically inhibit the enzyme. The IDH3 catalyzed reaction is, therefore, considered the rate-limiting reaction of the cycle. Other points of control include citrate inhibition of citrate synthase and NADH and ATP allosteric inhibition of 2-oxoglutarate dehydrogenase (α-ketoglutarate dehydrogenase). Several key enzymes of the TCA cycle are also regulated allosterically by Ca2+, ATP, and ADP.back to the top


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Monoposto SUNBAC Nova Awards
£1,500 Worth of Awards to be Won Annually by New Race Drivers
Monoposto Racing Club has been leading the way in organising low cost single seater racing for its members for over 50 years. The Monoposto Championship is the largest single seater championship in the UK with typical entry numbers in the 50+ range.
Monoposto Racing Club has combined forces with SUNBAC to run the Monoposto SUNBAC Nova Awards, which are specifically aimed at encouraging new drivers. SUNBAC – the Sutton Coldfield & North Birmingham Automobile club – has been historically important in introducing new competitors to motor sport. Although SUNBAC is no longer active in Motorsport organisation their major support for the Nova Awards means that these now offer a long-term platform to help new drivers develop their careers.
Started in 2011, as part of the Monoposto Championship, the Awards have been for the first and second place drivers on Championship points won in any Mono class, who start their season as "novices". The Awards provide two free twin header Monoposto Race entries in the following season for the winner, and one free twin header race entry for the runner up. The total value of the Awards is approximately £1000 - £1500 each year; arguably the most valuable prize fund in club level single seater racing.
The Monoposto SUNBAC Nova Awards will be open to any full Monoposto member, who starts their Monoposto Championship year still needing to be observed and to obtain some or all of the initial 6 signatures on their competition licence Upgrade Card. To be eligible for the Monoposto SUNBAC Nova Awards, a competitor will therefore start their Mono Championship campaign as a novice race driver as defined by the MSA, with the car carrying the compulsory black/yellow cross.
The Monoposto SUNBAC Nova Awards are a positive recognition that Monoposto is a key entry point into single seater racing in the UK, and one which also enables racers to progress to faster cars as they gain experience.
Former Monoposto Chairman Nick Harrison commented "Anyone thinking of racing a single seater season could buy a competitive car, ready to race for less than £8,000, and with race entries among the lowest in club racing we are going a long way to achieving our goal, to make single seater racing an affordable and enjoyable experience. We are delighted by SUNBAC's support, the Awards will keep this famous name to the fore in club motorsport in a very appropriate way, and enable us to extend the scheme for the next five years"
Past Winners:
2016 - 1st - James Harris (F3), 2nd - Alex Fores (1800)
2015 - 1st - Kyle Cutts (Moto), 2nd - Martin Wright (Moto)
2014 - 1st - Kevin Waring (Moto), 2nd - Kevin Couling (2000 Classic)
2013 - 1st - Daryl Jones (2000 Classic), 2nd - Dave Gillett (2000 Classic)
2012 - 1st - Luke Rosewell (1600), 2nd - Adrian Heath (1600)
2011 - 1st - Marcus Sheard (1800), 2nd - Mat Jordan (1600)

subset: commoncrawl


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Posted November 28th, 2007, 01:28 PM
Planning a cruise to Alasksa...seem to remember people talking about a certian time of year when flying little black bugs are a problem. What is the time of year that bugs can be a problem? Thanks in advance.
Welcome to Cruise Critic! If you'd like to participate on our forums by joining in the conversation, please Register Now! Be sure to visit our FAMOUS Roll Call forums, where you can meet other cruisers sailing with you and share a tour or shore excursion and SAVE MONEY!


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Separate names with a comma.
Discussion in 'Acekard' started by steve007, Feb 16, 2010.
Pikachu-Colored Pichu" Distro rom does not work at all tell me not available i im geting upset >_
Set the date of your DS back to February 14th and try again
thats retarted but ok ill try that
It'll work for sure.
o so i can only get one and thats it ? how do i get more
I don't know, maybe creating another save game and then by trading it into the old one? Pokesav? Try some things out.
um the deixsues when did he come out i need to know what date and year i need jerichi
Try setting the date of your DS forward to February 27th for Jirachi.
Not really - the distribution is based on Time Agreed events with the stores.
I am guessing you meant "retarded" as well... I feel your browser's spellcheck needs fixing.
Pichu - Jan 23rd, 2010 to Feb 15th, 2010 inclusive
Jirachi - Feb 20th, 2010 to Mar 14th, 2010 inclusive
There's also other distribution carts I've found online, here's the dates needed.
Shaymin - February 8 to 14, 2009.
Dragonite - November 8 to 9, 2008.
Deoxys - June 20 to 22 and June 27 to 29, 2008.
how do you use these dates sry
change the date on your ds
Shit, here we go again Read the distrubution thread (any of them, for that matter). All you need to do is alter the date of your DS console to the corresponding date(s) shown by Rydian. Put the distribution rom on another flashcart, put that cart in another DS console & run the .nds file. It will then look for another DS console that is running the Pokemon game and send it as a mystery gift.....and before you ask, yes, you need 2 consoles.
but im missing theses ?
Jirachi - Feb 20th, 2010 to Mar 14th, 2010 inclusive
Dragonite - November 8 to 9, 2008
i need there roms ?
alright thanks for th einfo
but there in there as well right ? or no you need to find them on the net ?
The jirachi one is the same as the pichu one, that one is two in one cart.
The others, you'll just have to google, AND NO YOU WILL NOT GET ANY HELP HERE.
I think I did you a grave injustice a couple of weeks ago....keep up the good work. Hammer any rom / link talk, whenever you see it. I don't even care if they reckon they didn't mean it....hammer 'em!


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Board of Directors
JOE THORP - Chairman - MBA, BA, FAICD, FAIM, CPMgr
Joe is the Chief Executive of TechInSA. TechInSA works closely with the high-tech industry to bring innovative South Australian products and technologies to global markets. TechInSA is the South Australian Government's innovation agency, tasked with helping to grow the high-tech sector in South Australia through customised programs including the South Australian Early Commercialisation Fund.
Joe was previously Managing Director of Hoshizaki Lancer Pty Ltd – a world leader in beverage dispensing technology based in Adelaide, with offices in all states, NZ, Hong Kong and India. Joe is also a Director of A Noble & Son Ltd, Director of Royal Guide Dogs Australia, Governor of St Peter's College, and a member of St Peter's Cathedral Council. Joe's work has been recognised with awards for training, manufacturing, innovation, export, product excellence and environmental impact. Joe joined the Board in 2011, and was appointed Chairman in 2013.
BRUCE IND - Deputy Chairman - B.Sc, B.Ed, Grad Dip T, Grad Dip Ins Comp., B.Ed Admin
Prior to his appointment to the Board in 2004, Bruce served on the Client Advisory Committee as Chair for four years. Bruce began his career as a secondary teacher in regional South Australia before moving into information services in the early 1990's. He also serves on the Australian Blindness Forum and on a number of committees within the Uniting Church. He brings an interest in, and a commitment to Quality Assurance, as well as experience with technology and governance. Bruce is a client of Guide Dogs, having received Orientation and Mobility Training with cane and dog through the organisation. Bruce joined the Board in 2004, and was appointed Deputy Chairman in 2013.
HELEN SUMMERS - Master of Optometry; Grad Cert. Ocular Therapeutics; FAICD; Fellow ACBO
Helen has a private practice in Darwin, with visiting practices in remote areas of NT. She works with adults and children with reading and learning difficulties, Neuro-optometry, Indigenous Health, Glaucoma and Macular Management. She is a Director of Eyecare Plus - a group of 180 independent optometry practices, Chairman of the Australian Optometric Panel and mentor for Autism Mentors Group NT. Helen won Telstra Business Woman of the Year 2014 and Telstra Northern Territory Business Woman of the Year. Helen joined the Board in 2005.
MICHAEL McGREGOR - BA, Psychology Major, FAICD
Michael is Director of McGregor Campbell Group. He is a registered Psychologist with extensive experience in the field of Organisational Psychology. For 11 years to 2010, he was State Manager SA & NT for Australia's largest provider of EAP Counselling and Workplace Trauma Services. He is a Fellow of the AICD and a Member of AHRI and the Australian Psychological Society. He is also a qualified mediator and an Associate Member of the Institute of Arbitrators and Mediators Australia. Michael joined the Board in 2013.
JAN TURBILL - B. Beh. Sc., Psychology Major
Jan has more than 30 years' experience in the advertising and marketing industries in Adelaide and Perth, having owned and managed companies since 1987 with a staff of 30-50 people. Jan has her own market research consultancy Intuito Pty Ltd. She is a specialist market researcher, market planner, facilitator of planning workshops, and is a marketing and communication strategist and direct marketing and Customer Relationship Management (CRM) programs expert. Jan joined the Board in October 2013.
GERRY CAWSON - LLB (Hons), GAICD
Gerry is currently a Legal Director heading the Corporate team at Kain C+C Lawyers. With more than 20 years of business experience, Gerry is highly regarded for his commercial skills. In 2013 Gerry was named by Best Lawyers Australia as "Best Commercial Lawyer - Adelaide" and in 2010 and 2011 he was recommended by Doyles' Guide to the Australian Legal Profession as a 'pre-eminent 'lawyer in South Australia in the area of mergers and acquisitions. In addition to numerous Board and committee roles over the years, Gerry has advised Boards and senior executives of Fortune 500 and ASX 50 companies while working for "top tier" law firms in London, Hong Kong, Sydney and Adelaide. Gerry is experienced in technical and practical application of corporate governance principles for organisations in the commercial and not-for-profit spheres. Gerry joined the Board in December 2013.
JOHN OLIVER - (B Commerce, Grad Dip Banking Management, GAICD, Snr Fellow FINSIA)
John is the Chief Executive Officer of HomeStart Finance. He has extensive experience in retail banking at Bendigo, Adelaide and Commonwealth Banks. He brings to Guide Dogs SA/NT high level planning skills in developing strategy and executing tactics designed to achieve corporate objectives. He is a Fellow of the Financial Services Institute of Australasia (FINSIA) and is a graduate of the Australian Institute of Company Directors (AICD). John joined the Board in 2014.
PROFESSOR ELIZABETH ANN FARMER - B.Sc (Hons), MBBS, PhD, FRACGP, MAICD
Professor Farmer is a registered medical practitioner and Honorary Professor at three Australian Universities. An independent consultant in the health sector, she has particular interest in innovation and systems change; policy and governance; accreditation; quality assurance; strategy and program evaluation; health sciences education and rural regional and remote health care issues. Currently, Professor Farmer also serves the Australian Medical Council as a Technical Advisor Research and Development, Chair of the National Prevocational Standards Accreditation Committee and member of the Board of Examiners. A recipient of the Vice Chancellor's Award for Excellence and Innovation in Education at Flinders University, she also received the inaugural Heaslip Fellow Scholarship in Medical Education at Flinders. Professor Farmer joined the Board in 2014.
MATT LANG – B.Ec, MBA (Exec), GAICD, CA, AICPA
Matt is the Chief Financial Officer for Discovery Parks and Director of a number of aviation companies in Australia and the UK. He worked with BAE Systems in defence and began his career with PricewaterhouseCoopers as a Chartered Accountant. He has extensive experience in strategy, finance, accounting and enterprise risk, and has worked extensively in the USA and UK. He joined the board in 2010.


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Original paper
A new quantitative air-lift sampler for collecting invertebrates designed for operation in deep, fast-flowing gravel bed rivers
Pehofer, Harald E.
Large Rivers Vol. 11 No. 2 (1998), p. 213 - 232
20 references
published: Nov 10, 1998
ArtNo. ESP142011502007, Price: 29.00 €
Conventional methods for collecting benthos from gravel substratum usually fail when quantitative samples have to be taken in deep and large fast-flowing rivers. As a purchased air-lift sampler did not perform satisfactorily under the special conditions encountered in alpine gravelbed rivers, a new sampler was designed. This sampler was used successfully on the armed gravel substratum of rivers with current velocities of >2 m/s and depths of 1.5 to 6 m. To operate the sampler a stable floating platform or a boat large enough to accommodate sampler, generator, compressor and the crew needed for handling (3-5 people, depending on the conditions in the river) is needed. Sampling is possible without anchoring, provided the sampling craft is able to hold its position in the current for the time needed to retrieve a sample (normally 10 to 20 seconds). Quantitative samples can be collected from sediments containing predominantly sand (> 100µm the mesh size of the collecting net) to cobble nearly the diameter of the suction pipe (10 cm). In fine muddy sediment the collecting cylinder penetrates very fast and the collecting nets tend to clog rapidly and to rupture by the abruptly growing pressure. Therefore, in fine mud an adequate core sampler normally is preferable. Penetration depth into the substratum is controlled by the time the sampler is left in operation. In our studies mean penetration depths usually lay around 20-25 cm. Sampling efficiency of the new air-lift is excellent: When compared to other deep water samplers (Petersen grab, slurp gun, deep water freeze corer) the air-lift samples regularly yielded by far the highest benthos numbers. Regarding sampler precision, when stratified sampling techniques were employed, the sample variances with the air-lift samples usually lay in the range of those achieved with stratified core samples from lakes or with stratified HESS-samples from gravel bars. A relative measure of precision (95 % confidence intervals expressed as a proportion of the mean values), calculated for the air-lift samples from two impoundments, showed values between 42 and 99 % for the abundance of total macrobenthos. Comparison of a deep mid-channel site and an adjacent shallow gravel bar, while yielding similar overall benthos densities, showed significant differences in the faunal composition of the two sites, emphasizing the importance of the deep main-channel areas for quantitative assessment of the benthic biota of a large river.
benthos • sediment • gravel bed river • invertebrate • large river


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Sports | iPhone, iPad
Sports | iPhone, iPad
Sports | iPhone, iPad
The best of the University of Virginia is now at your fingertips with the Official U.Va. iPhone app. Rely on "The Good Old App†to stay connected wherever you go. With only a tap you can access news, events, life on Grounds, live sports scores, and much more. The Official Virginia iPhone app has something for everyoneâ€â€students, faculty and staff, prospective students, alumni, parents, friends and fans. Features: - Maps & Augmented Reality: Search for buildings on Grounds, use GPS to pinpoint your location, or use Augmented Reality to identify that building over there with the dome and the columns. - Athletics: Get scores, schedules, videos, photos and news. Who is #21? View her photo and complete bio on your iPhone. A big football fan? Always have access to live scores. - Week in Photos: Curious about U.Va. life, both on Grounds and around the world? Keep up to date with Week in Photos. - Courses: Search for classes and find out where and when they meet and who will be teaching, then tap to add to your list of favoritesâ€â€all while you wait for the bus. - Directory: Search for students, faculty and staff, and then it's one tap to call or email them. - News: Keep on top of current events by reading the latest news from UVA Today, Virginia Magazine and the Cavalier Daily. - UVaClubs: Looking for alumni or an event in your state or abroad? Select your location for a full list of Clubs and events wherever you are. - Alumni: Stay on top of Homecomings and Reunions schedules, and your classmates in the Class Notes section of UVa Magazine. - Admissions: Interested in attending U.Va.? Use the app to keep track of deadlines, get the inside story by reading Dean J's Blog, plan a visit, or see when U.Va. will be in your neighborhood. - Health System: Need to make an appointment? Visiting a friend or family member? Get all the information you needâ€â€parking, phone numbers, services, visiting hours. - Student Support: Need a safe ride home, someone to talk to, or medical advice--a menu of support services is at your fingertips. - Fight Songs: A single tap lets everyone know you bleed orange and blue as your iPhone plays "The Good Old Song.†Have another favorite? No problem, change your default to one of eight UVA classics. - Alerts: At your option, receive push notifications for new UVA Today stories, final scores, or major Athletics news.
Version: 2.5
Size: 12.85 MB
Price: 0,00 €
Day of release: 0000-00-0


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With 802.11n offering performance and security on par with cabled Ethernet, why wouldnt you embrace wireless? This, anyway, is the vision of WLAN equipment vendors. The most telling slogan is Motorolas: Wireless by default, wired by exception. The truth, though, is this all-wireless vision takes some parsing.
All-wireless is comingif youre talking about the end-user perspective. No one is proposing wireless data centers. Ethernet to the desktop isnt dead yet, especially since theres no reason to tear out wiring already in place. Yet, as far as new equipment goes, its slowly being phased out.
Theres no reason that client devices shouldnt be wireless, said Craig Mathias, a principal at Farpoint Group, a wireless advisory firm. You can argue that even a good part of LAN infrastructure will be wireless eventually, especially with mesh architectures, but one thing that Wi-Fi still doesnt achieve is Gigabit-speed throughput.
Which means wires are here to stay, albeit in a pared back roll. If you need those thick pipes for specialized applications, such as video editing or CAD, youll still be running cable for a long time to come. Even things like continuous backup and disaster recovery would overwhelm Wi-Fi, but those IT-specific tasks should have little bearing on the typical end user.
Is 802.11n Even a Standard Yet?
Technically, no. 802.11n is still not ratified by the IEEE, although draft four was passed in May and most of the work left to do is fairly obscure, such as dynamic frequency selection. If youre confused, youre not alone, especially since 802.11n products have been shipping for some time now. Officially, those are pre-standard products, but theyve been approved by the Wi-Fi Alliance, which stepped in to speed up the process.
Wi-Fi Alliance approval means these products have reached a de facto standards threshold. They must interoperate, and theyll be easily upgraded as the official IEEE standard evolves.
Despite a history of 802.11 standards in-fighting, most CIOs shouldnt worry too much about the nitty gritty details of 802.11n. The real question, especially in tough economic times is, Why do it? said Chris Roeckl, VP of Marketing for AirMagnet, a provider of WLAN assurance tools. The answer will surprise most people. If youre talking about investing in new equipment, wireless is much more cost effective than wiring a new desktop.
Security and Throughput
Lets face it 802.11 has a checkered past within the enterprise. First, there was WEP (Wired Equivalent Privacy). Then there were the various 802.11 flavors that never quite interoperated as well as they were supposed to. Then there were backwards compatibility issues, with .11b clients killing .11g throughput. Then there were the competing infrastructure philosophies on how to control and manage WLANs; with people spending vast amounts of time and energy debating the merits of switches vs. gateways vs. whatever other architecture vendors dreamed up.
No wonder plenty of CIOs (and even whole industries) are wary of wireless. Today, when we get called into financial organizations, its to make sure there is no wireless, Roeckl said. However, 802.11n is light years beyond its troubled .11a/b/g predecessors. 802.11n brings two fundamental shifts. First, its safe enough. Second, its fast enough.
Compared to wired infrastructure, Roeckl argues that 802.11n is actually more secure than a wired environment. Encryption schemes like WPA (Wi-Fi Protected Access) are extremely advanced; rogue AP detection comes standard with most enterprise WLAN management suites; and VPNs add another layer of protection. Ethernet, by contrast, doesnt have these protections at the transport layer. (Since Ethernet is physically contained within buildings, you could argue that the transport-layer protections are things like security desks and identification badges.)


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Members of the City Council are:
Back Row (left to right): Council Member TJ Thomson, Council Member Ben Quintana, Council President Pro Tem Lauren McLean, Council Member Scot LudwigFront Row (left to right): Council President Elaine Clegg, Mayor David H. Bieter, Council Member Maryanne Jordan
Phone: (208) 384-4422
Email the Mayor
Email the City Council
150 N Capitol Blvd
Boise, ID 83702
William L. Bones
Chief of Police
Contact: BPD Media Relations Office
Boise Police officers responded to the scene of a reported attempted carjacking that occurred near the intersection of Kootenai Street and Hilton Street at 7:43 a.m. Reports indicate a male adult ran out into the street in front of a moving vehicle, forcing it to stop. The suspect then opened the driver side door and demanded that the victim give him the vehicle and other personal belongings. The victim refused, and the suspect punched the victim multiple times. The victim was able to fight the suspect off, causing him to flee southbound through an alley towards Overland Road.
The suspect is described as a light-skinned male adult, clean shaven with a skinny face, wearing dark clothing. He is approximately 6'0" tall, of slender build, between the ages of 25-30. Anyone with any information about the suspect in the area of the crime Monday morning is asked to call non-emergency dispatch at 377-6790 or Crime Stoppers at 343-COPS.


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At Gemvara, we believe that the jewelry you wear should be one of a kind, like you. That's why we make each piece to order, one at a time, just the way you want it. Choose Green Amethyst and Rose Gold or Tanzanite and Platinum: it's your choice. Mix and match gems and precious metals for a look that's exactly you. Gemvara uses only the highest quality materials to craft your Hanukkah Gift Ideas: Sterling Silver Blue Topaz Jewelry design with meticulous attention to detail. Our precious metals are recycled or responsibly mined. The ancient Greeks believed that topaz has the power to increase strength and make its wearer invisible in times of emergency. Blue topaz is a December birthstone, the gem for the 4th anniversary gem, and the Texas state gem.A noble metal with a cool white tone, sterling silver develops a patina over time. If you prefer a bright finish, polish will restore its shine.Our wide selection of Hanukkah Gift Ideas: Sterling Silver Blue Topaz Jewelry by talented designers means you can find jewelry that's exactly your style. You can also shop by style, choosing our Family, Edwardian, or Pastels Collections. Gemvara's fine quality Hanukkah Gift Ideas: Sterling Silver Blue Topaz Jewelry is designed to be passed down for generations to come, crafted by hand in the United States.
We can help right away. Chat with a jewelry expert now!


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Via Rail increases security following 'unfounded' threat
CTVNews.ca Staff
Published Wednesday, March 16, 2016 9:12AM EDT
Via Rail has added increased police and canine patrols at certain rail stations, following what the company called an "unfounded" threat.
In an email to CTV News, Via Rail Spokesperson Malcolm Andrews said the threat appears to be unsubstantiated, and that the company has already notified police and employees about the situation.
"For some time now, passenger transportation companies globally have been the target of unsubstantiated threats against their facilities," Andrews said. "A threat of this nature was received at VIA Rail recently and the police were informed."
In addition to the additional police and canine patrols, Andrews said employees were reminded to stay vigilant, and Via's Police Service was working with law enforcement to keep passengers safe.
A spokesperson from Metrolinx, the transit company in charge of regional transportation in the Greater Toronto Area, confirmed on Wednesday that there was an increased security presence at Toronto's Union Station.
Metrolinx spokesperson Anne Marie Aikins said the City of Toronto, Via Rail, Metrolinx and the Toronto Transit Commission were working together with Toronto police on the increased security measures.
Aikins said the extra precautions were expected to last at least until the end of the day.
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Even for circus collectors, only a privileged few will ever own an original painting by one of the great masters, but original circus art and limited-edition prints are readily available.
I own several pieces of original, circus art. One of my favorites is a block print of a performing elephant, received as a gift in the early 1960s. At the time I was the TV editor at the St. Petersburg Times. At Christmastime, we had a "white elephant" gift exchange. My name happened to be drawn by feature writer, Lorna Carroll. Knowing my love for the circus, she decided to give me an elephant print. It had been a wedding present to her from the artist Beryl Garrett. Lorna laughingly said she hated the print, but thought I would enjoy it. Recently, I've tried to find information about the artist—with no success. I seem to recall Beryl Garrett being a local St. Petersburg artist who was the wife of George Garrett, a news editor at the St. Petersburg Times.
But what about famous paintings? How can we see and enjoy them? Aside from traveling all over the world to museums, we can usually find them on the printed pages of books and magazines. Circus-themed paintings are often used as the focus for books and magazine articles. Below are some of the books featuring the circus in art:
More than 20 years ago, I began compiling a computerized index of magazine articles about the circus. One of my early WorthPoint stories, "Magazine Articles Chronicle Circus Life of Yesterday and Today," was about that list. The list revealed a number of articles on circus themed artwork:
American Artist – Apr 1958 – Mostly Under Canvas (Bill Ballantine)
American Artist – Jan 1968 – Donald Putnam's wild west and circus scenes
American Artist – Jul 1989 – The Evolution of a Painting . . . (Milwaukee Parade)
American Artist – Mar 1992 – Coming Soon (RBBB Mural)
Antiquarian, The – Jan 1932 – An Artist At The Circus
Art Digest – Mar 15, 1946 – Exhibition of Circus Paintings at Kootz Gallery
Art Digest – Nov 1, 1947 – Kosa Clowns at Cowie Galleries
Art Digest – Feb 15, 1948 – Circus In Art
Art Digest – Nov 1, 1950 – Sport and Circus at Michigan University
Art In America – Oct 1964 – Calder's Circus
Art In America – Jul 1969 – Buffalo Bill Museum In Cody, Wy
ARTnews – Jun 1948 – The Greatest Show Down South
Art News – Dec 15, 1941 – Kuhn Under the Big Top
Art News – Apr 15, 1942 – San Francisco: A Circus Show
Art News – May 1, 1943 – Walt Kuhn Clowns in a Great Tradition
Art News – Mar 1946 – Circus Paintings at Kootz Gallery
Art News – Mar 1992 – It's Stupendous! It's Sensational! It's Calder's Circus
Artforum – Oct 1971 – Harlequins, Saltimbanques, Clowns and Fools (Picasso)
Life – Nov 23, 1936 – Curry Of Kansas (Circus Paintings)
Life – Jun 15, 1942 – Paintings at Andover (Painting "Outside the Big Tent")
Life – Nov 29, 1943 – Speaking of Pictures (Paintings of John Steuart Curry)
Life – Feb 3, 1958 – High Cost of a Clown (Painting The White Clown by Walt Kuhn)
Theatre Arts – May 1946 – Circus Paintings
You can purchase the entire 51-page Circus Article Index, listing nearly 3,000 articles through WorthPoint.
The circus, world-wide, will always be a subject for artists of all ages, giving the rest of us enjoyment as we pursue our individual collections.
Larry Kellogg is a Worthologist specializing in circus memorabilia.
WorthPoint—Discover Your Hidden Wealth


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What have in common the enchanting Lisbon, the international Geneva and our Bel Paese? Probably not much, except for being all part of our beloved Old Continent. But recently, these three places have been mentioned for some important news regarding the food industry.
- The Geneva Act of the Lisbon Agreement on Appellations of Origin and Geographical Indications
On 20 May 2015, a Diplomatic Conference at the WIPO in Geneva adopted a new Act which revised and expanded the Lisbon Agreement for the Protection of Appellations of Origin and their International Registration.
"Appellations of origin" are geographical names that identify a product originating therein, whose quality and characteristic are due exclusively or essentially to the geographical environment.
The Lisbon Agreement, signed in 1958, offers a means of obtaining protection for an appellation of origin (AO) through a single registration process (administered by the International Bureau of WIPO), and ensures that in all contracting States, appellations of origin receive protection when they are protected in their Country of origin. As of today, 28 states, including of course Italy, are party to the agreement and 1000 appellations of origin have been registered so far; about 100 of them are Italian.
Some examples of Italian appellations of origin registered under the Lisbon Agreement are: Grana Padano, Parmigiano Reggiano, Prosciutto di Parma, Aceto Balsamico di Modena, Pomodoro di Pachino, Brunello di Montalcino.
The Geneva Act now allows the international registration of geographical indications (GIs), in addition to appellations of origin.
A "geographical indication" is a sign used on goods that have a specific geographical origin and possess qualities, reputation or characteristics that are essentially attributable to that place of origin. It is similar to an appellation of origin, but requires less stringent criteria for usage since the link with the place of origin is less strong. Appellations of origin can be therefore considered a special kind of GIs.
Another important change introduced by the Geneva Act concerns safeguards for prior trademark rights. Where the law of a contracting State provides that a prior trademark may not entitle its owner to prevent a registered appellation of origin or geographical indication from being granted protection or used in that contracting State, they shall coexist. On the contrary, a prior registration of an AO or a GI shall prevent a subsequent registration of the sign as a trademark.
- "The Extraordinary Italian Taste"
A few days after the adoption of the Geneva Act, the Italian Government presented at EXPO Milan "The Extraordinary Italian Taste", a trademark that – according to a statement from the Italian Ministry of Agriculture – shall be used to distinguish Italian food products at an international level and will help to challenge the so called "Italian sounding", i.e. foreign-made goods that cash in on Italian food's fame and popularity by using names that make them sound Italian (e.g. Parmesan).
Click here to view image.
"The Extraordinary Italian Taste" has no legal link with GIs and AOs, although it shares with them the purpose to identify the geographical origin of a product and the qualitative link between the product and its place of origin.
It is not even a "collective trademark", i.e. a trademark registered by associations and used to distinguish the goods and services of their members pursuant the associations regulations.
It is something different that should not overlap with any of the abovementioned rights. This is because – differently from other similar trademarks as the famous Swiss flag displayed on chocolates and cheeses – it won't be used on single products.
As a part of a wider special plan for the internationalization of the Italian products ("Decreto Sblocca Italia"), it is an "umbrella" trademark (an Italian trademark application has already been filed), owned by the Italian Trade Agency and the Ministry of Agriculture that will serve to identify Italian food products in international exhibitions, and will also be used for promotional and in-store activities abroad, in communication campaigns on TV, traditional media, the Internet and social media. See this video for a nice presentation of the trademark.
Will these initiatives serve to protect the uniqueness of Italian – and not only – food products and to protect consumers from deceptive initiatives? Hopefully yes, but it will be interesting to see how they will develop in the next months.


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Quick Details
- Type: Cartridge Chip, toner chip
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- Place of Origin: Guangdong, China (Mainland)
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- Compatible brand: OEM For xerox
- Commodity: toner chip for xerox color 800
- Warranty: 12 months
- Replacement: 1:1
- Defect rate: 0.1%
Packaging & Delivery
|Packaging Details
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why our product have so high quality?
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The orders chip for xerox color 1000 toner will be shipped immediately within 2 work days after payment has been received or confirmed. DHL, EMS, UPS, TNT, FEDEX...whatever shipping you prefer.
Shipping Terms : reset chip for xerox color 800
1. Ship Via: China Post Air Mail, EMS, DHL, UPS, TNT, FEDEX.
2. Ship out: 3-5 days upon received your payment.
3. Transit Time:China Post Air Mail: approx 25-38 working days.
EMS: approx 5-15 working days.
DHL, UPS, TNT,FEDEX: approx 3-8 working days.
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If you have any requirements or questions on our 3610 toner chip,feel free to contact me at any time.thanks!
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text:
VideoHive Icon Reveal 134778
After Effects Project Files \ Logo Stings \ 3D Object
Icon Reveal 134778 by uniquefx @ VideoHive
After Effects Project Files \ Logo Stings \ 3D Object
After Effects Version: CS4, CS5 File Size: 26mb Requires Plugins: Yes Resolution: 1920x1080
Icon Reveal introduces your corporate or personal logo with a very stylish and unique fly through animation.
Trapcode Form plugin required.
Logo placeholder = 500500.
Icon placeholder = 300300.
Use you own icons.
Video tutorial included.
Icons not included but can be purchased here.
Icons used in the tutorial can be downloaded here.
Technical specifications:
16:9 Square Pixels.
25 Frames Per Second.
Adobe After Effects CS4 Project File.
About the author:
Hello everyone! I am Esko Ahonen – also known as uniquefx around Videohive.
I'm a motion designer from Finland with many years of experience in graphics and animation.I hope you will find my files useful and helpful to you.
I take good care of my customers and answer every e-mail i get.If you are new to Adobe After Effects and using project templates, feel free to contact me with your questions.
Social links:
Download Link


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The Biology, Chemistry, Mathematics/Computer Science, and Physics Deparments
invite applications for Summer Research Stipends
- Austin College biology, chemistry, mathematics, computer science, or physics students
- No previous research experience required.
Deadline for applications -- Monday, March 27, 2017 by 5:00 p.m.
Initial Notification of awards -- Tuesday, April 4, 2017
Final notification Monday, April 10, 2017.
Students receive a competitive stipend plus 1/2 the cost of AC student housing if they choose to live on campus.
Note: Students choosing to live off-campus will not receive a housing allowance. Stipend amounts for individual students in different labs may vary depending on the length of period worked (see below) and the sources of funding available.
Expectations of stipend recipients
- Enthusiasm for original research
- 6 to 10 weeks in residence (typically the months of June and July)
- At least 40 hours per week
- A willingness to adapt your schedule to the needs of the research project
- No concurrent jobs or summer courses
- Biology & chemistry students are required to make a presentation to the Biology or Chemistry Department during the subsequent Fall semester
Application Procedure
Attend presentations by Austin College faculty on potential research projects.
- Biology Department: Thursday, March 2, 4:30 p.m., IDEA Center 127
- Chemistry Department: Wednesday, March 8, 4:30 p.m., IDEA Center 310
- Mathematics, Computer Science, & Physics Departments: contact individual faculty below
- Review faculty areas of research interest (see list below) regarding potential topics. Please note, faculty in the Department of Biology and Drs. Hebda and Richardson request potential applicants meet with them prior to applying. These meetings should be scheduled as soon as possible.
- Interested students are encouraged to attend the Austin College Scholarship Conference (ACSC), March 24-25, 2017 to discuss with current students work ongoing in prospective labs.
- Complete and submit the Online Application.
Selection procedure
Successful candidates will be selected based on 1) Quality of application statement (and any other written material requested by your proposed sponsor); 2) Courses taken; 3) Austin College record; and 4) Available research positions with faculty members.
Potential Sponsors for Summer 2017
Department of Biology
Lance Barton — Cell & Cancer Biology
Loriann Garcia — Plant-Insect Interactions
Wayne Meyer – Bird Behavior
Ernesto Perez – Drosophila Molecular Genetics
Departments of Mathematics & Computer Science
Jack Mealy – Staircase Metric Geometry
Department of Chemistry
Andy Carr — Organic Chemistry & Materials Science
Ryan Felix — Organic/Inorganic Chemistry
Jim Hebda – Biochemistry
John Richardson — Biochemistry
Brad Smucker — Inorganic Chemistry
Lindsay Zack – Astrochemistry
Department of Physics
No sponsors in 2017—check back in 2018!
This program is funded by sources including the Brittain Biology Student Research Endowment, The Andy and Narcadean Buckner Biology Scholars Program, the Howard McCarley Biology Research Endowment, and the Welch Foundation.
For further information, contact Dr. Jessica Healy (biology), x2338, IC 222.


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$649,000 Offers From
|1 | 2 | 3 | 4 | 5 | Next >>
|1 of 32
46 Ferndale Corner, Mindarie, WA 6030
4 Bed | 2 Bath | 2 Car
Property ID: WA2832818
Great value, double storey home in quiet street location, with ocean views and close to schools and in sought after beachside suburb. Call to view today!
* Double doors to tiled entry hall
* Formal dining
* Understairs storage cupboard
* Kitchen, family and meals inc TV point and reverse cycle airconditioning
* Kitchen inc breakfast bar, double bowl s/s sink, s/s gas cooktop, s/s retractable rangehood, walk in pantry, microwave recess, s/s electrical wall oven, double fridge recess
* Double 2nd, 3rd and 4th bedrooms inc double BIR's
* Double built in linen cupboard
* 2nd WC
* 2nd bathroom
* Laundry inc built in overhead cupboards, built in bench with s/s tub and cupboards
* Double garage inc remote door and rear roller door with rear access
* Master bedroom inc retreat, reverse cycle airconditioning, ensuite with bath, WIR, balcony with ocean views
* Feature wraparound patio over paved and timber decked outdoor entertaining area inc cafe blinds
* Landscaped, paved and auto reticulated gardens inc water feature
* Gas storage hot water system
* Built in 2000
* 540 square metre block
View CENTURY 21 Gold Key Realty
Contact Agent:
Justin O'Connell
Mobile: 0407577865
Email: Email Me
Open Times:
1. Sunday November 26, from 1:45 PM to 2:15 PM


text:
Contact Info / Websites
- Skype
- mrtonie2000
Entry #1
I want to take this 3d thing seriously now since lots of people are encouraging me to continue this. i make animations when im bored, and now i think i have an oppurtunity of entertaining the whole community of newgrounds with my content, you'll see alot more from me. Cheers, friends :D
- 3 comments
You must be logged in to comment on this post.
2014-06-08 18:25:35
Stay awesome!
2014-06-09 10:28:28
Woohoo! BTW, you say it freezes when you try to update the video - does it freeze during the upload process and never finish uploading, or is it stuck on the part where it's in the conversion queue?
I finally found out the situation tom, it was actually my video file that got corrupted when i rendered it in my program. so it wouldnt upload. my whole project got deleted suddenly so i cant make any further updates on the video. its not NGs fault. :)
2014-08-17 03:06:38
Kewl! Go 4 it!!!