Identification of potential modulators for human GPD1 by docking-based virtual screening, molecular dynamics simulations, binding free energy calculations, and DeLA-drug analysis.

Cytosolic Glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays a pivotal role in regulating the Embden-Meyerhof glucose glycolysis pathway (E-M pathway), as well as in conditions such as Huntington's disease, cancer, and its potential role as a specific marker for Dormant Glioma Stem Cells. In this study, we conducted virtual screening using the ZINC database ( http://zinc.docking.org/ ) and the GPD1 structure to identify potential GPD1 modulators. The investigation involved screening active candidate ligands using ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) parameters, combined with molecular docking, pose analysis, and interaction analysis based on Lipinski and Veber criteria. Subsequently, the top 10 ligands were subjected to 200 ns all-atom molecular dynamics (M.D.) simulations, and binding free energies were calculated. The findings revealed that specific residues, namely TRP14, PRO94, LYS120, ASN151, THR264, ASP260, and GLN298, played a crucial role in ensuring system stability. Furthermore, through a comprehensive analysis involving molecular docking, molecular M.D., and DeLA-Drug, we identified 10 promising small molecules. These molecules represent potential lead compounds for developing effective therapeutics targeting GPD1-associated diseases, thereby contributing to a deeper understanding of GPD1-associated mechanisms. This study's significance lies in identifying key residues associated with GPD1 and discovering valuable small molecules, providing a foundation for further research and development.

Amperometric triglyceride bionanosensor based on nanoparticles of lipase, glycerol kinase, glycerol-3-phosphate oxidase.

The nanoparticles (NPs) aggregates of lipase from porcine pancreas, glycerol kinase (GK) from Cellulomonas sp. and glycerol-3-phosphate oxidase (GPO) from Aerococcus viridanss were prepared by desolvation and glutaraldehyde crosslinking and functionalized by cysteamine. These enzyme nanoparticles (ENPs) were characterized by transmission electron microscopy (TEM) and Fourier transform infra red (FTIR) spectroscopy. The functionalzed ENPs aggregates were co-immobilized covalently onto polycrystalline Au electrode through thiolated bond. An improved amperometric triglyceride (TG) bionanosensor was constructed using this ENPs modified Au electrode as working electrode. Biosensor showed optimum current at 1.2 V within 5s, at pH 6.5 and 35 °C.A linear relationship was obtained between current (mA) and triolein concentration in lower concentration range,10-100 mg/dL and higher concentration range, 100-500 mg/dL. Limit of detection (LOD) of bionanosensor was 1.0 µg/ml. Percent analytical recovery of added trolein (50 and 100 mg/dL) in serum was 95.2 ± 0.5 and 96.0 ± 0.17. Within and between batch coefficients of variation (CV) were 2.33% and 2.15% respectively. A good correlation (R2 = 0.99) was obtained between TG values in sera measured by present biosensor and standard enzymic colorimetric method with the regression equation: y= (0.993x + 0.967). ENPs/Au electrode was used 180 times over a period of 3 months with 50% loss in its initial activity, when stored dry at 4 °C.

Identification and Characterization of Heptaprenylglyceryl Phosphate Processing Enzymes in Bacillus subtilis.

In Archaea, ether lipids play an essential role as the main building blocks of the cellular membrane. Recently, ether lipids have also been discovered in the domain of Bacteria, and the key enzymes that catalyze their synthesis, glycerol-1-phosphate dehydrogenase and heptaprenylglyceryl phosphate synthase, have been described. In Bacillales, heptaprenylglyceryl phosphate does not become linked to a second polyprenyl moiety like ether lipids in Archaea but is dephosphorylated and acetylated. Here, we report on the enzymes that catalyze these reactions. We enriched the phosphatase activity from a B. subtilis cell extract and suppose that dephosphorylation is catalyzed by the phosphatase PhoB or by any other phosphatase in an unspecific manner. By screening a B. subtilis knock-out library for deficiency in acetylation, the yvoF gene product was identified to be the acetyltransferase. The acetyl-CoA-dependent enzyme YvoF is a close relative of maltose O-acetyltransferase (MAT). Its catalytic properties were analyzed and compared with MAT. YvoF and MAT partially overlap in substrate and product range in vitro, but MAT is not able to complement the yvoF knock-out in vivo.

Rational Design of Nanoparticle Platforms for "Cutting-the-Fat": Covalent Immobilization of Lipase, Glycerol Kinase, and Glycerol-3-Phosphate Oxidase on Metal Nanoparticles.

The aggregates of nanoparticles (NPs) are considered better supports for the immobilization of enzymes, as these promote enzyme kinetics, due to their unusual but favorable properties such as larger surface area to volume ratio, high catalytic efficiency of certain immobilized enzymes, non-toxicity of some of the nanoparticle matrices, high stability, strong adsorption of the enzyme of interest by a number of different approaches, and faster electron transportability. Co-immobilization of multiple enzymes required for a multistep reaction cascade on a single support is more efficient than separately immobilizing the corresponding enzymes and mixing them physically, since products of one enzyme could serve as reactants for another. These products can diffuse much more easily between enzymes on the same particle than diffusion from one particle to the next, in the reaction medium. Thus, co-immobilization of enzymes onto NP aggregates is expected to produce faster kinetics than their individual immobilizations on separate matrices. Lipase, glycerol kinase, and glycerol-3-phosphate oxidase are required for lipid analysis in a cascade reaction, and we describe the co-immobilization of these three enzymes on nanocomposites of zinc oxide nanoparticles (ZnONPs)-chitosan (CHIT) and gold nanoparticles-polypyrrole-polyindole carboxylic acid (AuPPy-Pin5COOH) which are electrodeposited on Pt and Au electrodes, respectively. The kinetic properties and analytes used for amperometric determination of TG are fully described for others to practice in a trained laboratory. Cyclic voltammetry, scanning electron microscopy, Fourier transform infra-red spectra, and electrochemical impedance spectra confirmed their covalent co-immobilization onto electrode surfaces through glutaraldehyde coupling on CHIT-ZnONPs and amide bonding on AuPPy/Pin5COOH. The combined activities of co-immobilized enzymes was tested amperometrically, and these composite nanobiocatalysts showed optimum activity within 4-5s, at pH 6.5-7.5 and 35°C, when polarized at a potential between 0.1 and 0.4V. Co-immobilized enzymes showed excellent linearity within 50-700mg/dl of the lipid with detection limit of 20mg/dl for triolein. The half life of co-immobilized enzymes was 7 months, when stored dry at 4°C which is very convenient for practical applications. Co-immobilized biocatalysts measured triglycerides in the sera of apparently healthy persons and persons suffering from hypertriglyceridemia, which is recognized as a leading cause for heart disease. The measurement of serum TG by co-immobilized enzymes was unaffected by the presence of a number of serum substances, tested as potential interferences. Thus, co-immobilization of enzymes onto aggregates of NPs resulted in improved performance for TG analysis.

Two glycerol 3-phosphate dehydrogenase isogenes from Candida versatilis SN-18 play an important role in glycerol biosynthesis under osmotic stress.

Two isogenes of glycerol 3-phosphate dehydrogenase (GPD) from Candida versatilis SN-18 were cloned and sequenced. These intronless genes (Cagpd1 and Cagpd2) were both predicted to encode a 378 amino acid polypeptide, and the deduced amino acid sequences mutually showed 76% identity. Interestingly, Cagpd1 and Cagpd2 were located tandemly in a locus of genomic DNA within a 262 bp interval. To our knowledge, this represents a novel instance of isogenic genes relating to glucose metabolism. The stress response element (STRE) was found respectively at -93 to -89 bp upstream of the 5'end of Cagpd1 and -707 to -703 bp upstream of Cagpd2, indicating that these genes are involved in osmotic stress response. In heterologous expression using a gpd1Δgpd2Δ double deletion mutant of Saccharomyces cerevisiae, Cagpd1 and Cagpd2 transformants complemented the function of GPD, with Cagpd2 being much more effective than Cagpd1 in promoting growth and glycerol synthesis. Phylogenetic analysis of the amino acid sequences suggested that Cagpd1p and Cagpd2p are NADP(+)-dependent GPDs (EC 1.1.1.94). However, crude enzyme extract from Cagpd1 and Cagpd2 transformants showed GPD activity with only NAD(+) as cofactor. Hence, both Cagpd1p and Cagpd2p are likely NAD(+)-dependent GPDs (EC 1.1.1.8), similar to GPDs from S. cerevisiae and Candida magnoliae.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney.

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made possible the measuring of cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor are coated with a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

The branched mitochondrial respiratory chain from Debaryomyces hansenii: components and supramolecular organization.

The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV2, I-IV, III2-IV4, V2, I-III2, I-III2-IV, I-III2-IV2, I-III2-IV3 and I-III2-IV4. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.

Molecular cloning and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene from Penicillium expansum PE-12.

Penicillium expansum produces large amounts of lipase, which is widely used in laundry detergent and leather industry. We isolated the glyceraldehyde-3-phosphate dehydrogenase gene (PeGPD) from P. expansum PE-12 through reverse transcriptase PCR and 5'-3' rapid amplification of cDNA ends (RACE-PCR). The gene is 1266 bp long, including an ORF of 1014 bp, encoding a polypeptide chain of 337 amino acids. A phylogenetic tree based on GPD proteins showed that P. expansum is close to Aspergillus species, but comparatively distant from P. marneffei. Southern blot results revealed a single copy of PeGPD, and expression analysis gave evidence of high expression levels. PeGPD genes have potential for genetic engineering of P. expansum for industrial lipase production.

Lab-on-a-chip for analysis of triglycerides based on a replaceable enzyme carrier using magnetic beads.

In this paper an enzyme-carrier-based microfluidic chip coupled with a gold nanoband microelectrode as electrochemical detector for Triglyceride (TG) determination was developed by co-immobilized lipase, Glycerokinase (GK) and glycerol-3-phosphate oxidase (GPOx) on chitosan/Fe(3)O(4) composite nanoparticles with a shell-core structure, which combined the advantageous features of microfluidic chips technology with magnetic beads. This procedure enabled the easy renewal of the microchip enzyme carrier after each determination in a highly reproducible manner. Several operational parameters such as working potential, buffer pH, adenosine triphosphate concentrations (ATP, mM), separation voltage and temperature were evaluated and optimized. The performance of enzyme-carrier-based microfluidic chip for TG determination was modulated by changing the length of enzyme carrier from 1.0 to 3.0 cm, and the linear ranges were changed from 0-4.0 mM to 0-10.0 mM with the detection limits from 15 µM to 6.0 µM. The enzyme carrier remained its 70% activity after 40 days storage. This system was successfully employed for on-line detection of TG in serums. The experimental results demonstrated that this enzyme carrier using magnetic beads based microfluidic chip provided a relatively simple, sensitive, miniature, and replaceable means for the accurate determination of TG in serum.

Cloning and characterization of CmGPD1, the Candida magnoliae homologue of glycerol-3-phosphate dehydrogenase.

Glycerol-3-phosphate dehydrogenase (GPDH) plays a central role in glycerol metabolism. A genomic CmGPD1 gene encoding NADH-dependent GPDH was isolated from Candida magnoliae producing a significant amount of glycerol. The gene encodes a polypeptide of 360 amino acids, which shows high homology with known NADH-dependent GPDHs of other species. The CmGPD1 gene was expressed in recombinant Escherichia coli with the maltose-binding protein (MBP) fusion system and purified to homogeneity using simple affinity chromatography. The purified CmGpd1p without the MBP fusion displayed an apparent molecular mass of 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The CmGpd1p enzyme exhibited a K(cat)/K(m) value of 195 min(-1) mM(-1) for dihydroxyacetone phosphate whereas K(cat)/K(m) for glycerol-3-phosphate is 0.385 min(-1) mM(-1). In a complementation study, CmGpd1p rescued the ability of glycerol synthesis and salt tolerance in a Saccharomyces cerevisiae GPD1DeltaGPD2Delta mutant strain. The overall results indicated that CmGPD1 encodes a functional homologue of S. cerevisiae GPDH.

Increased expression of mitochondrial glycerophosphate dehydrogenase and antioxidant enzymes in prostate cancer cell lines/cancer.

The involvement of mitochondrial glycerophosphate dehydrogenase (mGPDH) has previously been established in the production of ROS in prostate cancer cell lines (LNCaP, DU145, PC3 and CL1). The current study demonstrates that the mRNA level of mGPDH in prostate cancer cells is 3.3-8.9-fold higher compared to the normal prostate epithelial cell line, PNT1A. This is consistent with the enzymatic activity and protein level of mGPDH. However, cytochrome c oxidase (COX) activity is 2.9-3.2-fold down-regulated in androgen-independent prostate cancer cell lines. The level of antioxidant enzymes, catalase, MnSOD and CuZnSOD are up-regulated in prostate cancer cell lines. Furthermore, it was observed that the activity of mGPDH is significantly higher in liver tissues from all mice with cancer compared to liver tissues from control mice. These data suggest that the up-regulation of mGPDH, due to a highly glycolytic environment, contributes to the overall increase in ROS generation and may result in the progression of the cancer.

Peptergents: peptide detergents that improve stability and functionality of a membrane protein, glycerol-3-phosphate dehydrogenase.

Toward enhancing in vitro membrane protein studies, we have utilized small self-assembling peptides with detergent properties ("peptergents") to extract and stabilize the integral membrane flavoenzyme, glycerol-3-phosphate dehydrogenase (GlpD), and the soluble redox flavoenzyme, NADH peroxidase (Npx). GlpD is a six transmembrane spanning redox enzyme that catalyzes the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate. Although detergents such as n-octyl-beta-D-glucpyranoside can efficiently solubilize the enzyme, GlpD is inactivated within days once reconstituted into detergent micelles. In contrast, peptergents can efficiently extract and solubilize GlpD from native Escherichia coli membrane and maintain its enzymatic activity up to 10 times longer than in traditional detergents. Intriguingly, peptergents also extended the activity of a soluble flavoenzyme, Npx, when used as an additive. Npx is a flavoenzyme that catalyzes the two-electron reduction of hydrogen peroxide to water using a cysteine-sulfenic acid as a secondary redox center. The lability of the peroxidase results from oxidation of the sulfenic acid to the sulfinic or sulfonic acid forms. Oxidation of the sulfenic acid, the secondary redox center, results in inactivation, and this reaction proceeds in vitro even in the presence of reducing agents. Although the exact mechanism by which peptergents influence solution stability of Npx remains to be determined, the positive effects may be due to antioxidant properties of the peptides. Peptide-based detergents can be beneficial for many applications and may be particularly useful for structural and functional studies of membrane proteins due to their propensity to enhance the formation of ordered supramolecular assemblies.

Isolation and properties of cytoplasmic alpha-glycerol 3-phosphate dehydrogenase from the pectoral muscle of the fruit bat, Eidolon helvum.

Cytoplasmic alpha-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was 59,500 +/- 650 daltons; its subunit size was estimated to be 35,700 +/- 140 by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were 3.9 +/- 0.7 mM, 0.65 +/- 0.05 mM, 0.26 +/- 0.06 mM, and 0.005 +/- 0.0004 mM for L-glycerol-3-phosphate, NAD(+), DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were 2.30 +/- 0.21 mM and 0.20 +/- 0.01 mM for L-glycerol-3-phosphate and NAD(+), respectively. The turnover number, k(cat), of the forward reaction was 1.9 +/- 0.2 x 10(4)s(-1). The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that alpha-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.

Redox regulation of cytosolic glycerol-3-phosphate dehydrogenase: Cys(102) is the target of the redox control and essential for the catalytic activity.

Cytosolic glycerol-3-phosphate dehydrogenase (cG3PDH) occupies the branch point between the glycolytic pathway and triglyceride biosynthesis. However, the regulatory mechanism of the cG3PDH activity has remained obscure. Here we report that cG3PDH is efficiently inhibited by modification of the thiol group through a redox mechanism. In this study, we found that sodium selenite and nitric oxide (NO) donors such as S-nitroso-N-acetylpenicillamine and 3-morpholinosydnonimine inhibited cG3PDH activity, and that similar effects could be achieved with selenium metabolites such as selenocysteine and selenomethionine. Furthermore, we found that reducing agents, such as dithiothreitol and beta-mercaptoethanol, restored the cG3PDH activity suppressed by selenite and NO both in vitro and in cultured cells. Buthionine sulfoximine depleted levels of both reduced glutathione and the oxidized form but had no effect on the suppression of cG3PDH activity by selenite in cultured cells. Moreover, thiol-reactive agents, such as N-ethylmaleimide and o-iodosobenzoic acid, blocked the enzyme activity of cG3PDH through the modification of redox-sensitive cysteine residues in cG3PDH. The inhibitor of NO synthase, L-N(G)-nitro-arginine, restored the cG3PDH activity inhibited by NO in cultured cells, whereas the inhibitor of guanylyl cyclase, 1H-[1,2,4] oxadiazole[4,3-alpha] quinoxalin-1-one (ODQ), has no effect. NO directly inhibits cG3PDH activity not via a cGMP-dependent mechanism. Finally, using site-directed mutagenesis, we found that Cys(102) of cG3PDH was sensitive to both selenite and NO. From the results, we suggest that cG3PDH is a target of cellular redox regulation.

Adenosine analogues as inhibitors of Trypanosoma brucei phosphoglycerate kinase: elucidation of a novel binding mode for a 2-amino-N(6)-substituted adenosine.

As part of a project aimed at structure-based design of adenosine analogues as drugs against African trypanosomiasis, N(6)-, 2-amino-N(6)-, and N(2)-substituted adenosine analogues were synthesized and tested to establish structure-activity relationships for inhibiting Trypanosoma brucei glycosomal phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycerol-3-phosphate dehydrogenase (GPDH). Evaluation of X-ray structures of parasite PGK, GAPDH, and GPDH complexed with their adenosyl-bearing substrates led us to generate a series of adenosine analogues which would target all three enzymes simultaneously. There was a modest preference by PGK for N(6)-substituted analogues bearing the 2-amino group. The best compound in this series, 2-amino-N(6)- [2''(p-hydroxyphenyl)ethyl]adenosine (46b), displayed a 23-fold improvement over adenosine with an IC(50) of 130 microM. 2-[[2''-(p-Hydroxyphenyl)ethyl]amino]adenosine (46c) was a weak inhibitor of T. brucei PGK with an IC(50) of 500 microM. To explore the potential of an additive effect that having the N(6) and N(2) substitutions in one molecule might provide, the best ligands from the two series were incorporated into N(6),N(2)-disubstituted adenosine analogues to yield N(6)-(2''-phenylethyl)-2-[(2'' -phenylethyl)amino]adenosine (69) as a 30 microM inhibitor of T. brucei PGK which is 100-fold more potent than the adenosine template. In contrast, these series gave no compounds that inhibited parasitic GAPDH or GPDH more than 10-20% when tested at 1.0 mM. A 3.0 A X-ray structure of a T. brucei PGK/46b complex revealed a binding mode in which the nucleoside analogue was flipped and the ribosyl moiety adopted a syn conformation as compared with the previously determined binding mode of ADP. Molecular docking experiments using QXP and SAS program suites reproduced this "flipped and rotated" binding mode.

Aedes aegypti in Tahiti and Moorea (French Polynesia): isoenzyme differentiation in the mosquito population according to human population density.

Genetic differences at five polymorphic isoenzyme loci were analyzed by starch gel electrophoresis for 28 Aedes aegypti samples. Considerable (i.e., high Fst values) and significant (i.e., P values >10(-4)) geographic differences were found. Differences in Ae. aegypti genetic structure were related to human population densities and to particularities in mosquito ecotopes in both Tahiti and Moorea islands. In highly urbanized areas (i.e., the Papeete agglomeration), mosquitoes were highly structured. Recurrent extinction events consecutive to insecticidal treatments during dengue outbreaks tend to differentiate mosquito populations. In less populated zones (i.e., the east coast of Moorea and Tahiti), differences in ecotope characteristics could explain the lack of differentiation among mosquitoes from rural environments such as the east coast of Tahiti where natural breeding sites predominate. When the lowest populated zones such as Tahiti Iti and the west coast of Moorea are compared, mosquito are less differentiated in Moorea. These results will be discussed in relation to the recent findings of variation in mosquito infection rates for dengue-2 virus.

Population structure and genetic divergence in Anopheles nuneztovari (Diptera: Culicidae) from Brazil and Colombia.

Anopheles nuneztovari is considered an important vector of human malaria in several localities in Venezuela and Colombia. Its status as a vector of human malaria is still unresolved in areas of the Brazilian Amazon, in spite of have been found infected with Plasmodium sp.. For a better understanding of the genetic differentiation of populations of A. nuneztovari, electrophoretic analysis using 11 enzymes was performed on four populations from Brazil and two from Colombia. The results showed a strong differentiation for two loci: alpha-glycerophosphate dehydrogenase (alpha-Gpd) and malate dehydrogenase (Mdh) from 16 loci analyzed. Diagnostic loci were not detected. The populations of A. nuneztovari from the Brazilian Amazon showed little genetic structure and low geographic differentiation, based on the F(IS) (0.029), F(ST) (0.070), and genetic distance (0.001-0.032) values. The results of the isozyme analysis do not coincide with the indication of two lineages in the Amazon Basin by analysis of mitochondrial DNA, suggesting that this evolutionary event is recent. The mean F(ST) value (0.324) suggests that there is considerable genetic divergence among populations from the Brazilian Amazon and Colombia. The genetic distance among populations from the Brazilian Amazon and Colombia is ranges from 0.047 to 0.148, with the highest values between the Brazilian Amazon and Sitronela (SIT) (0.125-0.148). These results are consistent with those observed among members of anopheline species complexes. It is suggested that geographic isolation has reduced the gene flow, resulting in the genetic divergence of the SIT population. Dendrogram analysis showed three large groups: one Amazonian and two Colombia, indicating some genetic structuring. The present study is important because it attempted to clarify the taxonomic status of A. nuneztovari and provide a better understanding of the role of this mosquito in transmission of human malaria in northern South America.

Hippocampal responses to corticosterone and stress, one of which is the 35,000 M(r) protein, glycerol phosphate dehydrogenase.

Previously, the synthesis of a hippocampal 35,000 M(r) protein increased in response to glucocorticoid treatment and a variety of stressors. We now show by immunoprecipitation that this cytosolic protein is glycerol 3-phosphate dehydrogenase (E.C.1.1.1.8; GPDH). In addition, four polypeptides encoded by glucocorticoid-induced mRNAs co-migrated with hippocampal protein synthetic products on two-dimensional polyacrylamide gels, including 35,000 M(r) protein of approximately pl 6.3, that had previously been identified as GPDH by hybrid-selection with a GPDH cDNA clone. The 35,000 M(r) in vitro translation product was also immunoprecipitated with the GPDH antibody. Using radiolabeled hippocampal slices and two-dimensional gel analysis, a 35,000 M(r) polypeptide of approximately pl 6.4 increased five-fold after 30 min of intermittent tail-shock. This protein was found predominantly in the 20,000 x g pellet and did not immunoprecipitate with the GPDH antibody. However, a 35,000 M(r) polypeptide was also found in the cytosol as a minor component after stress, which did immunoprecipitate with the GPDH antibody. Therefore, there are at least two shock-induced 35,000 M(r) proteins, one of which is GPDH. These results establish that increases in GPDH mRNA prevalence and protein synthesis occur in response to both glucocorticoids and stress in the adult rat hippocampus. Based on the increased enzyme activity seen in the nervous system in response to glucocorticoids, dietary restriction, and nerve injury, the induction of GPDH may have functional consequences in cellular adaptation to stress.

Molecular cloning of human mitochondrial glycerophosphate dehydrogenase gene: genomic structure, chromosomal localization, and existence of a pseudogene.

cDNA of mitochondrial glycerophosphate dehydrogenase (mGPDH), a defect of which is a possible cause of non-insulin dependent diabetes mellitus, was cloned from a human insulinoma cDNA library. The deduced amino acid sequence showed 91% and 92% homology with those of rat and mouse mGPDH, respectively. The mGPDH gene was mapped to chromosome 2q23 by FISH analysis. Genomic clones for mGPDH were then isolated using mouse mGPDH cDNA and PCR products of human mGPDH cDNA as probes. Genomic structure was studied by sequencing the exon-intron boundaries and by PCR amplification of intronic regions using genomic clones as templates. The human mGPDH gene was shown to be composed of 15 coding exons, containing a (CA)n repeat region inside the gene, which was not polymorphic in the Japanese population. Genomic cloning also identified a pseudogene located on chromosome 19q13.4. These results provide information useful for analyzing the mGPDH gene in patients with non-insulin dependent diabetes mellitus.

Cloning and characterization of the NAD-linked glycerol-3-phosphate dehydrogenases of Trypanosoma brucei brucei and Leishmania mexicana mexicana and expression of the trypanosome enzyme in Escherichia coli.

A polyclonal antiserum raised against the purified glycosomal glycerol-3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b. brucei expression library. This cDNA was subsequently used to obtain genomic clones containing glycerol-3-phosphate dehydrogenase genes. Two tandemly arranged genes were detected in these clones. Characterization of one of the genes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37,651 Da and a calculated net charge of +8. Using the T.b. brucei gene as a probe, a corresponding glycerol-3-phosphate dehydrogenase gene was also identified in a genomic library of Leishmania mexicana mexicana. The L.m. mexicana gene codes for a polypeptide of 365 amino acids, with a molecular mass of 39,140 Da and a calculated net charge of +8. The amino-acid sequences of both polypeptides are 63% identical and carry a type-1 peroxisomal targeting signal (PTS1) SKM and -SKL at their respective C-termini. Moreover, the L.m. mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence. Subcellular localisation analysis showed that in L.m. mexicana the glycerol-3-phosphate dehydrogenase activity co-fractionated both with mitochondria and with glycosomes. This is not the case in T. brucei, where the enzyme is predominantly glycosomal. The two trypanosomatid sequences resemble their prokaryotic homologues (32-36%) more than their eukaryotic counterparts (25-31%) and carry typical prokaryotic signatures. The possible reason for this prokaryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase is discussed.