Comparison of isocitrate dehydrogenase from three hyperthermophiles reveals differences in thermostability, cofactor specificity, oligomeric state, and phylogenetic affiliation

With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus. An optimum for activity at 90 degrees C or above was found for each IDH. PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9 degrees C, respectively, compared with 98.3 and 98.5 degrees C for TmIDH and AfIDH, respectively. Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH. TmIDH and ApIDH were NADP-dependent (K(m)((NADP)) of 55.2 and 44.4 microm, respectively) whereas PfIDH was NAD-dependent (K(m)((NAD)) of 68.3 microm). These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea. The homodimeric NADP-IDH present in A. pernix is the most common form of IDH known so far. The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di- and multimeric IDHs are described and discussed.     

Human glutathione transferase A1-1 demonstrates both half-of-the-sites and all-of-the-sites reactivity

A study of the kinetics of a heterodimeric variant of glutathione transferase (GST) A1-1 has led to the conclusion that, although the wild-type enzyme displays all-of-the-sites reactivity in nucleophilic aromatic substitution reactions, it demonstrates half-of-the-sites reactivity in addition reactions. The heterodimer, designed to be essentially catalytically inactive in one subunit due to a single point mutation (D101K), and the two parental homodimers were analyzed with seven different substrates, exemplifying three types of reactions catalyzed by glutathione transferases (nucleophilic aromatic substitution, addition, and double-bond isomerization reactions). Stopped-flow kinetic results suggested that the wild-type GST A1-1 behaved with half-of-the-sites reactivity in a nucleophilic aromatic substitution reaction, but steady-state kinetic analyses of the GST A1-D101K heterodimer revealed that this was presumably due to changes to the extinction coefficient of the enzyme-bound product. In contrast, steady-state kinetic analysis of the heterodimer with three different substrates of addition reactions provided evidence that the wild-type enzyme displayed half-of-the-sites reactivity in association with these reactions. The half-of-the-sites reactivity was shown not to be dependent on substrate size, the level of saturation of the enzyme with glutathione, or relative catalytic rate.     

UV-visible spectral identification of the solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid

Solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid were investigated by spectroscopy. The spectral data showed the formation of a stable organomanganese intermediate, which was responsible for the rise in the absorbance at 420 nm. This result enables unambiguous interpretation of the absorbance change at 420 nm, as the intermediate permanganate ions could be isolated on the solid supports.     

Pathogenesis of renal ischemia/reperfusion injury: lessons from knockout mice

Ischemia/reperfusion-induced acute renal failure is a common clinical problem associated with a high morbidity and mortality. Upon hypoxic injury, the depletion of ATP causes mitochondrial dysfunction, and accumulation of intracellular sodium, calcium and reactive oxygen species. Subsequently, multiple enzyme systems including proteases, nitric oxide synthases, phospholipases and endonuclease are activated and responsible for cytoskeleton disruption, membrane damage, and DNA degradation, and eventually cell death. Ischemia/reperfusion injury also activates complement, cytokines, and chemokines, which are cytotoxic themselves, but also attract leukocytes into the ischemic area to cause further damage. The vascular endothelial cell injury and dysfunction prolong ischemia and induce vascular congestion, edema, and further infiltration of inflammatory cells. Many players in renal ischemia/reperfusion injury and their mechanisms have been investigated using genetically manipulated mouse models. In this review, we focus on the information gathered from these studies. Deficiency of the Na/Ca exchanger, inducible nitric oxide synthase, Caspase-1, A3 adenosine receptor, C3, C5, C6, Factor B, or midkine protects the kidney against I/R injury. Conversely, deficiency of the interleukin-1 receptor, osteopontin, C4, or recombination activation gene-1 is not protective, while the absence of adrenomedullin or endothelin receptor B delays the recovery of ischemia/reperfusion injury. The knowledge obtained from these studies provides new direction for designing potential therapeutic agents for treating ischemia/reperfusion injury.     

The use of genetic markers to measure genomic response to selection in livestock

A method to measure genomic response to natural and artificial selection by means of genetic markers in livestock is proposed. Genomic response through several levels of selection was measured using sequential testing for distorted segregation of alleles among selected and nonselected sons, single-sperm typing, and a test with records for growth performance. Statistical power at a significance level of 0.05 was >0.5 for a marker linked to a QTL with recombination fractions 0, 0.10, and 0.20 for detecting genomic responses for gene effects of 0.6, 0.7, and 1.0 phenotypic standard deviations, respectively. Genomic response to artificial selection in six commercial bull sire families comprising 285 half-sib sons selected for growth performance was measured using 282 genetic markers evenly distributed over the cattle genome. A genome-wide test using selected sons was significant (P < 0.001), indicating that selection induces changes in the genetic makeup of commercial cattle populations. Markers located in chromosomes 6, 10, and 16 identified regions in those chromosomes that are changing due to artificial selection as revealed by the association of records of performance with alleles at specific markers. Either natural selection or genetic drift may cause the observed genomic response for markers in chromosomes 1, 7, and 17.     

Haemolymph parameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taura syndrome virus

Pacific white shrimp (Litopenaeus vannamei) were injected with Taura syndrome virus (TSV) to assess shrimp immune responses and survival. TSV-infected shrimp suffered high mortality, but mock-infected and untreated shrimp experienced no mortality. Moribund shrimp were a pale, reddish colour and were lethargic and soft-shelled. Their haemolymph was clear red and coagulated poorly. In TSV-infected shrimp, the total haemocyte count (THC), hyalinocyte and granulocyte counts, and total plasma protein decreased significantly to 21%, 24%, 17% and 56% of untreated control values, respectively. Haemocyanin decreased to 67%, and clottable proteins to 80% of control values (P< 0.01). Copper and calcium ions, haemocytic transglutaminase (TGase) activity and plasma growth inhibitory activity against Vibrio harveyi also decreased significantly. Generation of intrahaemocytic superoxide anion, O(-2), in TSV-infected shrimp was significantly greater (P< 0.05) than in both control groups, no matter whether glucan stimulated or unstimulated. But the relative increase of intrahaemocytic O(-2) generation in TSV-infected shrimp response to glucan stimulation was lower in both controls. Plasma phenoloxidase (PO) activity increased significantly in TSV-infected shrimp. The plasma bacterial agglutinin titre against E. coli and V. harveyi, growth inhibition of E. coli and the concentration of magnesium ions in TSV-infected shrimp did not change significantly.In conclusion, ten of thirteen haemolymph parameters changed significantly during the host-TSV interaction. These parameters might be valuable references of shrimp health status.     

Chalconoids from Fissistigma bracteolatum

Phytochemical studies on the leaves of Fissistigma bracteolatum yielded besides the two known compounds 2-hydroxy-3,4,6-trimethoxychalcone (1) and 5,7,8-trimethoxyflav-3-ene (2), five new chalconoids 2-hydroxy-3,4,6-trimethoxychalcene (3), 2-hydroxy-3,4,6-trimethoxydihydrochalcone (4), 2'-hydroxy-3',4',6'-trimethoxydihydrochalcone (5), 2'-hydroxy-3',4',6'-trimethoxy-beta'-methoxychalcane (6) and 2'-hydroxy-3',4',6'-trimethoxy-beta'-ethoxychalcane (7). The structures of these compounds were determined by mass and NMR spectroscopic methods.     

Fitting genetic mapping functions based on sperm typing: results for three chromosomal segments in cattle

Genetic mapping functions translate the observed recombination rate between two loci into the corresponding map distance in Morgan units. Different mapping functions give different weights to multiple crossing overs and therefore lead to different results. This points out that not every function is best suited to fit a data set. The data used in this study originated from 2214 sperm from 37 Norwegian bulls, which were genotyped for 11 markers. The optimal functions for the chromosomes 6, 23 and the sex chromosome of cattle were derived using the maximum likelihood method, the likelihood ratio test and empirical discriminant analysis. It became apparent that for each chromosome a different function fitted the data best. These were the function of Rao et al. (Human Heredity 1977, 27, 99–104) with p = 0·63 for chromosome 6, the function of Goldgar & Fain (American Journal of Human Genetics 1988, 43, 38–45) with c₀ = 0·42, c₁ = 0·47, c₂ = 0·07 and c₃ = 0·04 for chromosome 23 and the function of Felsenstein (Genetics 1979, 91, 769–75) with K = 0·23 for the sex chromosome. The well known functions of Haldane (Journal of Genetics 1919, 8, 299–309) and Kosambi (Annals of Eugenics 1944, 12, 172–5) were shown to be suboptimal in most cases. A function is said to be multilocus feasible if the evaluation of the probability of all possible recombination events does not lead to negative values. The optimal function for chromosome 23 turned out to be multilocus feasible, whereas the functions for chromosome 6 and the sex chromosome were not. The choice of the correct mapping function is shown to have a considerable impact in mapping studies, when double recombinations have to be taken into account. Since there is no unique best mapping function, it is argued that it might be useful to use a simple parametric mapping function (like the one of Felsenstein 1979) and to estimate the respective parameter specifically for a given data set.