Evolutionary genetics of the Drosophila alcohol dehydrogenase gene-enzyme system

Evolutionary genetics embodies a broad research area that ranges from the DNA level to studies of genetic aspects in populations. In all cases the purpose is to determine the impact of genetic variation on evolutionary change. The broad range of evolutionary genetics requires the involvement of a diverse group of researchers: molecular biologists, (population) geneticists, biochemists, physiologists, ecologists, ethologists and theorists, each of which has its own insights and interests. For example, biochemists are often not concerned with the physiological function of a protein (with respect to pH, substrates, temperature, etc.), while ecologists, in turn, are often not interested in the biochemical-physiological aspects underlying the traits they study. This review deals with several evolutionary aspects of the Drosophila alcohol dehydrogenase gene-enzyme system, and includes my own personal viewpoints. I have tried to condense and integrate the current knowledge in this field as it has developed since the comprehensive review by van Delden (1982). Details on specific issues may be gained from Sofer and Martin (1987), Sullivan, Atkinson and Starmer (1990); Chambers (1988, 1991); Geer, Miller and Heinstra (1991); and Winberg and McKinley-McKee (1992).Dedicated to Professor Billy W. Geer, because of his contributions to knowledge of the biochemical genetics of Drosophila.     

A potent specific inhibitor of 6-phosphogluconate dehydrogenase ofCryptococcus neoformans and of certain other fungal enzymes

A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases fromCryptococcus neoformans andSchizophyllum commune and glucose-6-phosphate dehydrogenase fromSchizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than theCryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent.     

Structural characterization and physiological function of component B from Methanosarcina thermophila

The electron donor (component B) to the methyl coenzyme M methylreductase system from Methanosarcina thermophila was isolated as the 7-methyl derivative and characterized. Gas chromatography-mass spectrometry and ¹H NMR analyses identified this derivative as 7-methylthioheptanoylthreonine phosphate (CH₃-S-HTP), indicating that the original component B had the same structure (HS-HTP) as previously determined for component B from Methanobacterium thermoautotrophicum. The heterodisulfide of HS-HTP and coenzyme M (HS-CoM, 2-mercaptoethanesulfonate) was enzymatically reduced in cell extracts using electrons supplied by either H₂ or CO, confirming that HS-HTP was a functional molecule in M. thermophila.     

Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus: involvement of reactions of the Embden-Meyerhof pathway

The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase.Cell extracts of pyruvate-grown P.furiosus contained the following enzyme activities: phosphoenolpyruvate synthetase (0.025 U/mg, 50 °C), enolase (0.9 U/mg, 80 °C), phosphoglycerate mutase (0.13 U/mg, 55 °C), phosphoglycerate kinase (0.01 U/mg, 50 °C), glyceraldehyde-3-phosphate dehydrogenase reducing either NADP⁺ or NAD⁺ (NADP⁺: 0.019 U/mg, NAD⁺: 0.009 U/mg; 50 °C), triosephosphate isomerase (1.4 U/mg, 50 °C), fructose-1,6-bisphosphate aldolase (0.0045 U/mg, 55 °C), fructose-1,6-bisphosphate phosphatase (0.026 U/mg, 75 °C), and glucose-6-phosphate isomerase (0.22 U/mg, 50 °C). Kinetic properties (V _max values and apparent K _m values) of the enzymes indicate that they operate in the direction of sugar synthesis. The specific enzyme activities of phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (NADP⁺-reducing) and fructose-1,6-bisphosphate phosphatase in pyruvate-grown P. furiosus were by a factor of 3, 10 and 4, respectively, higher as compared to maltose-grown cells suggesting that these enzymes are induced under conditions of gluconeogenesis. Furthermore, cell extracts contained ferredoxin: NADP⁺ oxidoreductase (0.023 U/mg, 60 °C); phosphoenolpyruvate carboxylase (0.018 U/mg, 50 °C) acts as an anaplerotic enzyme.Thus, in P. furiosus sugar formation from pyruvate involves reactions of the Embden-Meyerhof pathway, whereas sugar degradation to pyruvate proceeds via a modified non-phosphorylated Entner-Doudoroff pathway.     

Formation of acetyl-CoA in Zymomonas mobilis by a pyruvate dehydrogenase complex

In the gram negative, obligately ethanologenic bacterium Zymomonas mobilis a pyruvate dehydrogenase complex was identified and the complex was enriched from cell extracts. This multienzyme complex is responsible for acetyl-CoA biosynthesis from pyruvate. No activities of related multienzyme complexes, 2-ketoglutarate dehydrogenase and branched chain keto acid dehydrogenase, could be detected.     

GPD1L inhibits renal cell carcinoma progression by regulating PINK1/Parkin-mediated mitophagy

Few approaches have been conducted in the treatment of renal cell carcinoma (RCC) after nephrectomy, resulting in a high mortality rate in urological tumours. Mitophagy is a mechanism of mitochondrial quality control that enables selective degradation of damaged and unnecessary mitochondria. Previous studies have found that glycerol-3-phosphate dehydrogenase 1-like (GPD1L) is associated with the progression of tumours such as lung cancer, colorectal cancer and oropharyngeal cancer, but the potential mechanism in RCC is still unclear. In this study, microarrays from tumour databases were analysed. The expression of GPD1L was confirmed by RT–qPCR and western blotting. The effect and mechanism of GPD1L were explored using cell counting kit 8, wound healing, invasion, flow cytometry and mitophagy-related experiments. The role of GPD1L was further confirmed in vivo. The results showed that GPD1L expression was downregulated and positively correlated with prognosis in RCC. Functional experiments revealed that GPD1L prevented proliferation, migration and invasion while promoting apoptosis and mitochondrial injury in vitro. The mechanistic results indicated that GPD1L interacted with PINK1, promoting PINK1/Parkin-mediated mitophagy. However, inhibition of PINK1 reversed GPD1L-mediated mitochondrial injury and mitophagy. Moreover, GPD1L prevented tumour growth and promoted mitophagy by activating the PINK1/Parkin pathway in vivo. Our study shows that GPD1L has a positive correlation with the prognosis of RCC. The potential mechanism involves interacting with PINK1 and regulating the PINK1/Parkin pathway. In conclusion, these results reveal that GPD1L can act as a biomarker and target for RCC diagnosis and therapy.     

Crystal structure analysis of amicyanin and apoamicyanin from Paracoccus denitrificans at 2.0 A and 1.8 A resolution.

The crystal structure of amicyanin, a cupredoxin isolated from Paracoccus denitrificans, has been determined by molecular replacement. The structure has been refined at 2.0 A resolution using energy-restrained least-squares procedures to a crystallographic residual of 15.7%. The copper-free protein, apoamicyanin, has also been refined to 1.8 A resolution with residual 15.5%. The protein is found to have a beta-sandwich topology with nine beta-strands forming two mixed beta-sheets. The secondary structure is very similar to that observed in the other classes of cupredoxins, such as plastocyanin and azurin. Amicyanin has approximately 20 residues at the N-terminus that have no equivalents in the other proteins; a portion of these residues forms the first beta-strand of the structure. The copper atom is located in a pocket between the beta-sheets and is found to have four coordinating ligands: two histidine nitrogens, one cysteine sulfur, and, at a longer distance, one methionine sulfur. The geometry of the copper coordination is very similar to that in the plant plastocyanins. Three of the four copper ligands are located in the loop between beta-strands eight and nine. This loop is shorter than that in the other cupredoxins, having only two residues each between the cysteine and histidine and the histidine and methionine ligands. The amicyanin and apoamicyanin structures are very similar; in particular, there is little difference in the positions of the coordinating ligands with or without copper. One of the copper ligands, a histidine, lies close to the protein surface and is surrounded on that surface by seven hydrophobic residues. This hydrophobic patch is thought to be important as an electron transfer site.     

Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release

The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h^–1 to 0.01 h^–1 when decreasing the pH from 7 to 6.8.Abbreviations D dilution rate (h^–1) - k_b specific trypan-blue dead cells appearance rate (h^–1) - k_L specific lysis rate of viable cells (h^–1) - k_d specific death rate (h-1) - LDH₀ lactate dehydrogenase activity in the feed culture medium (IU.l^–1) - LDH lactate dehydrogenase activity in the outlet culture medium (IU.l^–1) - LDH_i intracellular lactate dehydrogenase activity of viable cells (IU.10^–9 cells) - r_LDH total rate of LDH release (IU.h^–1.L^–1) - r_b transformation rate of viable cells into blue dead cells (10⁹ cells.h^–1.L^–1) - x_v viable cell concentration (10⁹ cells.l^–1) - x_b trypan-blue dead cell concentration (10⁹ cells.l^–1)     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.