Effects of Al(III) and Nano-Al13 Species on Malate Dehydrogenase Activity

The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al13 can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al13 concentration increase. Our study also found that the effects of Al(III) and Al13 on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules

Mathematical Model of Ethanol Metabolism in Liver

A lumped mathematical model of liver metabolism is presented to analyze the effect of ethanol on metabolic processes of 24 hr fasted rats. The model is developed in two parts. In the first part individual kinetic models for important regulatory steps in the liver metabolic pathways are developed and in second part transport and mass balance equations in the two well mixed domains: tissue and blood, are developed to calculate intermediate metabolite concentrations and fluxes in response to the changes in ethanol and lactate concentrations in the perfusion medium. Part of the model without ethanol metabolism has been validated and published in Chalhoub et al, 2007. The focus of this effort was to illustrate the effect of ethanol metabolism on gluconeogenesis from lactate. The kinetic models developed for phosphofructokinase and fructose 1,6 bisphosphatase have been independently validated with data from the literature, whereas the results of the comprehensive lumped model are compared with the data from Krebs et al (1969). While the lumped model show many important characteristics of ethanol metabolism and predicts the flux of glucose production in the same range, two major contradictions of the simulated results with experimental data are observed. These shortcomings are discussed with appropriate reasoning. The model presented in this thesis is expected to improve the understanding on the effects of ethanol metabolism and provide a practical tool to address alcohol related health issue

Genetic and morphological variation of the lanternfish Lampanyctodes hectoris (Myctophiformes: Myctophidae) off southern Africa

Genetic and morphological variation within the southern African population of Lampanyctodes hectoris was analysed. A total of 15 enzymes, encoding 22 isozyme loci, was examined (n = 327); seven were polymorphic. The percentage of polymorphic loci ranged from 13.6 to 27.3%. The mean heterozygosity was generally low and ranged from 0.003 to 0.005. Genetic divergences between specimens from four areas along the west coast of southern Africa were negligible, genetic distance values (D) ranging from 0 to 0.00011. The results showed the population of L. hectoris to be genetically invariant. Principal Component Analysis was performed separately on ratios of 13 morphometric and 6 meristic variables (n = 446), and there was evidence of extensive overlap between fish from all areas. However, discriminant analysis suggested some morphological variability within this population. Although genetically the population of L. hectoris seems to be homogenous, morphologically it appears to be variable. A combination of these results suggests that there is no clear genetic basis for the slight morphological differentiation within the population. Keywords: Agulhas Bank, Benguela Current, lanternfish, Namibia, population geneticsAfrican Journal of Marine Science 2002, 24: 193–20

Isozyme polymorphism and inheritance in Hatiora and Schlumbergera (Cactaceae).

Isozyme analysis was used to identify clones, measure levels of genetic variation within groups of clones, and analyze mating systems in two Cactaceae genera--Hatiora and Schlumbergera. Isozymes were extracted from phylloclades and pollen and were separated by polyacrylamide gel electrophoresis. The inheritance of aspartate aminotransferase (AAT), glucose-6-phosphate isomerase (GPI), malate dehydrogenase (MDH), phosphoglucomutase (PGM), and triosephosphate isomerase (TPI) was examined in Hatiora. Six loci (Aat-1, Gpi-1, Mdh-1, Pgm-1, Pgm-2, and Tpi-2) were analyzed, and results were generally as expected for single loci with codominant alleles. For all six isozyme loci segregation distortion was observed in at least one segregating family. Aat-1 was linked with Pgm-1 (26 cM), but the other isozyme loci assorted independently. The inheritance of leucine aminopeptidase (LAP), phosphoglucomutase (PGM), and shikimate dehydrogenase (SKD) was investigated in Schlumbergera. Three loci were analyzed (Lap-1, Pgm-1, and Skd-1), and results were generally as expected for single loci with codominant alleles. Significant segregation distortion was observed in at least one segregating family for all three isozyme loci. Disturbed segregation at Lap-1 was due to tight linkage (7 cM) with the locus controlling gametophytic self-incompatibility (S). All three loci assorted independently of each other. In a third study, a Hatiora germplasm collection composed of 49 clones was assayed for AAT, GPI, LAP, MDH, PGM, SKD, and TPI. Thirteen putative loci and 42 putative alleles were identified, and 9 of the 13 loci (69%) were polymorphic. Twenty-two clones (45%) could be distinguished solely on the basis of their isozyme profiles, but the other 27 clones shared isozyme profiles with one to five other clones. Thirteen modern H. x graeseri cultivars exhibited less genetic diversity than 40 H. gaertneri, H. x graeseri, and H. rosea clones representing older and modern cultivars plus field-collected specimens. The difference in genetic diversity was primarily attributed to a loss of alleles during breeding. In a fourth study, a Schlumbergera germplasm collection composed of 59 clones was assayed for AAT, GPI, LAP, MDH, PGM, SKD, and TPI. Twelve putative loci and 36 putative alleles were identified, and 10 of the 12 loci (83%) were polymorphic. Forty-one clones (69%) could be distinguished solely on the basis of their isozyme profiles, but the other 18 clones shared isozyme profiles with one or two other clones. Forty-two commercial clones of S. truncata, S. x buckleyi, and S. x exotica exhibited less genetic diversity than 14 field-collected clones of S. kautskyi, S. opuntioides, S. orssichiana, S. russelliana, and S. truncata. The difference in genetic diversity was attributed to limited sampling from wild populations and loss of alleles during breeding

Structure-guided Inhibitor Design of Mycobacterium Tuberculosis Drug Targets from Central Carbon Metabolism

A key determinant of the pathogenicity of M. tuberculosis (Mtb) is its ability to conserve energy and resources by altering its intermediate metabolism response to host-derived hostile conditions. Growing evidence supports that the central carbon metabolism (CCM) of M.tuberculosis including glycolysis, gluconeogenesis, the pentose phosphate pathway, and the TCA cycle, is regulated to allow for simultaneous utilization of carbohydrates and fatty acids-derived carbon sources. The genes involved in CCM represent attractive anti TB drug-targets. Although the role of Mtb phosphoenolpyruvate carboxykinase (PEPCK) in gluconeogenesis pathway and pyruvate kinase (PykA) enzymes in glycolysis pathway are relevant for the M. tuberculosis pathogenesis, targeting these enzymes for the drug development is not straight forward because of the human orthologs existence. Here, X-ray crystallographic, biochemical, and inhibitory studies of Mtb PEPCK and PykA highlight distinct features of the pathogenic’s enzyme that differ from those of the host orthologs’ and provide opportunities to develop selective and potent inhibitors. Structural data of Mtb PEPCK has revealed that the conformation changes of the flexible loops in response to substrates binding differ from the one reported for host versions of the enzyme. GTP-competitive inhibitors of human cytosolic PEPCK bind to Mtb PEPCK in similar fashion, but the discovery of two unique small pockets in Mtb has created an opportunity for the design of a selective Mtb PEPCK inhibitor. By using structure-guided medicinal chemistry for GTP-competitive inhibitor series with PEPCK, we are able to improve the inhibitory effect of these inhibitors against the enzyme, creating inhibitors with a nano-molar range IC50 and develop 10 fold selective inhibitors against Mtb PEPCK over human PEPCK. Structural study of Mtb PykA also demonstrated that adenosine mono-phosphate (AMP) is an allosteric effector, and we identified the binding mode and interaction of AMP in the allosteric binding site for the first time. Screening diverse compound libraries gave us insight into the potential scaffolds for the development of more potent pathogen-specific inhibitors. The structural and inhibitor studies of Mtb PEPCK and PykA have allowed a better understanding of their role in gluconeogenesis and glycolysis, and provide a framework for the development of selective inhibitors

Understanding and exploiting fungal PKS-NRPS biosynthesis in Pyricularia and Magnaporthe species

The fungus Pyricularia oryzae is a major pathogen of rice (Oryzae sativa) that causes substantial loss of yields every year. Remarkably, certain rice strains carrying the Pi33 gene are resistant to strains of P. oryzae encoding a PKS-NRPS called the avirulence conferring enzyme1 (ACE1). By heterologous expression studies in Aspergillus oryzae, more insights into the early biosynthetic steps of this ACE1 metabolite were obtained. O-Methyl-L-tyrosine 71 was identified as the preferred substrate for the adenylation domain of the ACE1 NRPS, indicating that the OME1 gene (O-methyltransferase) belongs to the ACE1 cluster. However, even after all biosynthetic genes of the ACE1 cluster were co-expressed; the produced compound was 88 - a shunt intermediate. In-vitro enzyme assays confirmed that reduction of the aldehyde 90 to the alcohol 88 is catalysed by a native A. oryzae enzyme at an early biosynthetic step; thus stalling the biosynthesis. Ectopic expression of BC1 (transcription factor from the ACE1 BGC) in P. oryzae did not lead to the production of ACE1-related compounds. However, the compound hinnulin A 105 was formed, which belongs to the class of DHN-melanins. A putative partial BGC potentially involved in the biosynthesis of 105 was validated by RT-PCR and a possible biosynthetic pathway was proposed. Another cytochalasan BGC was previously demonstrated to be responsible for the production of pyrichalasin H 50 in Magnaporthe grisea and now revealed to be homologous to the ACE1 BGC. Thus, the 50 BGC was used as model system to elucidate the function of two potential key enzymes in cytochalasan biosynthesis: an αβ-hydrolase (HYD) and a putative Diels-Alderase (pDA). Targeted gene deletion experiments in M. grisea revealed that the HYD PyiE is involved in early steps during the biosynthesis of 50, but its exact role remained elusive. The pDA PyiF was shown to be involved in the intramolecular [4+2] cycloaddition forming 72. Complementation studies with ORF3 from the ACE1 BGC indicated a similar catalytic function of both enzymes. By recombinant gene expression studies in E. coli soluble ORFZ (HYD) and ORF3 (pDA) protein was obtained. In-vitro assays are underway to gain further insights into their biosynthetic role