ELISA for the detection of venoms from four medically important snakes of India

A double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed to detect Echis carinatus venom in various organs (brain, heart, lungs, liver, spleen and kidneys) as well as tissue at the site of injection of mice, at various time intervals (1, 6, 12, 18, 24 h and 12 h intervals up to 72 h) after death. The assay could detect E. carinatus venom levels up to 2.5 ng/ml of tissue homogenate and the venom was detected up to 72 h after death. A highly sensitive and species-specific avidin-biotin microtitre ELISA was also developed to detect venoms of four medically important Indian snakes (Bungarus caeruleus, Naja naja, E. carinatus and Daboia russelli russelli) in autopsy specimens of human victims of snake bite. The assay could detect venom levels as low as 100 pg/ml of tissue homogenate. Venoms were detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area and postmortem blood. In all 12 human victim cadavers tested the culprit species were identified. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart and lungs. Development of a simple, rapid and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

Nonlinear Enzyme Kinetics Can Lead to High Metabolic Flux Control Coefficients: Implications for the Evolution of Dominance

In a classic study, Kacser & Burns (1981, Genetics 97, 639-666) demonstrated that given certain plausible assumptions, the flux in a metabolic pathway was more or less indifferent to the activity of any of the enzymes in the pathway taken singly. It was inferred from this that the observed dominance of most wild-type alleles with respect to loss-of-function mutations did not require an adaptive, meaning selectionist, explanation. Cornish-Bowden (1987, J. theor. Biol. 125, 333-338) showed that the Kacser-Burns inference was not valid when substrate concentrations were large relative to the relevant Michaelis constants. We find that in a randomly constructed functional pathway, even when substrate levels are small, one can expect high values of control coefficients for metabolic flux in the presence of significant nonlinearities as exemplified by enzymes with Hill coefficients ranging from two to six, or by the existence of oscillatory loops. Under these conditions the flux can be quite sensitive to changes in enzyme activity as might be caused by inactivating one of the two alleles in a diploid. Therefore, the phenomenon of dominance cannot be a trivial ''default'' consequence of physiology but must be intimately linked to the manner in which metabolic networks have been moulded by natural selection.

Identification of actin as an estradiol 17-beta-stimulated protein in the human placenta

Addition of estradiol 17-beta to first trimester human placental minces resulted in an increased synthesis of a protein of apparent molecular weight 45 kDa. The specific involvement of estrogen in the stimulation of this protein was established by demonstrating a reduction in the level of this protein by the addition of CCS 16949 A, an inhibitor of aromatase, a key enzyme in the biosynthesis of estradiol 17-beta and ICI 182,780, an estrogen receptor antagonist. The protein was purified to homogeneity and N-terminal sequencing of two of the internal peptides obtained by enzymatic digestion of the protein, as well as the absence of a free N-terminal indicated that it could be actin. This was confirmed by Western blotting using commercially available actin antiserum. The role of estradiol 17-beta in the stimulation of actin synthesis in human placenta was also established by monitoring the quantitative inhibition of DNase I by actin.

Estimation of mineral-adhered biomass of Thiobacillusferrooxidans by protein assay-some problems and remedies

Enumeration of adhered cells of Thiobacillus ferrooxidans on sulphide minerals through protein assay poses problems due to interference from dissolved mineral constituents. The manner in which sulphide minerals such as pyrite, chalcopyrite, sphalerite, arsenopyrite and pyrrhotite interfere with bacterial protein estimation is demonstrated. Such interferences can be minimised either through dilution or addition of H2O2 to the filtrate after hot alkaline digestion of the biotreated mineral samples.

A Synthetic Model for the Inhibition of Glutathione Peroxidase by Antiarthritic Gold Compounds

In this paper, inhibition of the glutathione peroxidase activity of two synthetic organoselenium compounds, bis[2-(N,N-dimethylamino)benzyl]diselenide (5) and bis[2-(N,N-dimethylamino)benzyl]selenide (9), by gold(I) thioglucose (1), chloro(triethylphosphine)gold(I), chloro(trimethylphosphine)gold(I), and chloro(triphenylphosphine)gold(I) is described. The inhibition is found to be competitive with respect to a peroxide (H2O2) substrate and noncompetitive with respect to a thiol (PhSH) cosubstrate. The diselenide 5 reacts with PhSH to produce the corresponding selenol (6), which upon treatment with 1 equiv of gold(I) chlorides produces the corresponding gold selenolate complexes 11−13. However, the addition of 1 equiv of selenol 6 to complexes 11−13 leads to the formation of bis-selenolate complex 14 by ligand displacement reactions involving the elimination of phosphine ligands. The phosphine ligands eliminated from these reactions are further converted to the corresponding phosphine oxides (R3PO) and selenides (R3PSe). In addition to the replacement of the phosphine ligand by selenol 6, an interchange between two different phosphine ligands is also observed. For example, the reaction of complex 11 having a trimethylphosphine ligand with triphenylphosphine produces complex 13 by phosphine interchange reactions via the formation of intermediates 15 and 16. The reactivity of selenol 6 toward gold(I) phosphines is found to be similar to that of selenocysteine.

Mycobacterium tuberculosis pantothenate kinase: possible changes in location of ligands during enzyme action

The crystal structures of complexes of Mycobacterium tuberculosis pantothenate kinase with the following ligands have been determined: (i) citrate; (ii) the nonhydrolysable ATP analogue AMPPCP and pantothenate (the initiation complex); (iii) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (the end complex); (iv) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (the intermediate complex); (v) CoA; (vi) ADP prepared by soaking and cocrystallization, which turned out to have identical structures, and (vii) ADP and pantothenate. Solution studies on CoA binding and catalytic activity have also been carried out. Unlike in the case of the homologous Escherichia coli enzyme, AMPPCP and ADP occupy different, though overlapping, locations in the respective complexes; the same is true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK is substantially preformed, while that of EcPanK exhibits considerabl plasticity. The difference in the behaviour of the E. coli and M. tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action. Therefore, the results have to be treated with caution. However, the changes in the locations of ligands exhibited by M. tuberculosis pantothenate kinase are remarkable and novel.

Stereochemical and steric control of enzymatic glucuronidation : A rational approach for the design of novel Inhibitors for the human UDP-glucuronosyltransferase 2B7

The UDP-glucuronosyltransferases (UGTs) are enzymes of the phase II metabolic system. These enzymes catalyze the transfer of α-D-glucuronic acid from UDP-glucuronic acid to aglycones bearing nucleophilic groups affording exclusively their corresponding β-D-glucuronides to render lipophilic endobiotics and xenobiotics more water soluble. This detoxification pathway aids in the urinary and biliary excretion of lipophilic compounds thus preventing their accumulation to harmful levels. The aim of this study was to investigate the effect of stereochemical and steric features of substrates on the glucuronidation catalyzed by UGTs 2B7 and 2B17. Furthermore, this study relates to the design and synthesis of novel, selective inhibitors that display high affinity for the key enzyme involved in drug glucuronidation, UGT2B7. The starting point for the development of inhibitors was to assess the influence of the stereochemistry of substrates on the UGT-catalyzed glucuronidation reaction. A set of 28 enantiomerically pure alcohols was subjected to glucuronidation assays employing the human UGT isoforms 2B7 and 2B17. Both UGT enzymes displayed high stereoselectivity, favoring the glucuronidation of the (R)-enantiomers over their respective mirror-image compounds. The spatial arrangement of the hydroxy group of the substrate determined the rate of the UGT-catalyzed reaction. However, the affinity of the enantiomeric substrates to the enzymes was not significantly influenced by the spatial orientation of the nucleophilic hydroxy group. Based on these results, a rational approach for the design of inhibitors was developed by addressing the stereochemical features of substrate molecules. Further studies showed that the rate of the enzymatic glucuronidation of substrates was also highly dependent on the steric demand in vicinity of the nucleophilic hydroxy group. These findings provided a rational approach to turn high-affinity substrates into true UGT inhibitors by addressing stereochemical and steric features of substrate molecules. The tricyclic sesquiterpenols longifolol and isolongifolol were identified as high-affinity substrates which displayed high selectivity for the UGT isoform 2B7. These compounds served therefore as lead structures for the design of potent and selective inhibitors for UGT2B7. Selective and potent inhibitors were prepared by synthetically modifying the lead compounds longifolol and isolongifolol taking stereochemical and steric features into account. The best inhibitor of UGT2B7, β-phenyllongifolol, displayed an inhibition constant of 0.91 nM.

Exocrine pancreatic dysfunction in malnourished Australian Aboriginal children

Pancreatic exocrine dysfunction has been frequently recorded in protein-energy malnutrition in underdeveloped countries. In addition, the pancreas requires optimal nutrition for enzyme synthesis and potentially correctable pancreatic enzyme insufficiency may play a role in the continuation of protein-energy malnutrition. This problem has not been previously evaluated in Australian Aborigines. We have applied a screening test for pancreatic dysfunction (human immunoreactive trypsinogen [IRT] assay) to the study of 398 infants (6-36 months) admitted to the Alice Springs Hospital over a 20-month period. All infants were assessed by anthropometric measures and were assigned to to three nutritional groups (normal, moderate or severely malnourished) and two growth groups (stunted or not stunted). Of the 198 infants who had at least a single serum cationic trypsinogen measurement taken, normal values for serum IRT (with confidence limits) were obtained from 57 children, who were normally nourished. IRT levels were significantly correlated with the degree of underweight but there was no correlation with the degree of stunting or age. Mean IRT levels for the moderate and severely underweight groups were significantly greater than the mean for the normal group (P < 0.01). Seventeen children (8.6%) had trypsinogen levels in excess of the 95th percentile for the normally nourished group, reflecting acinar cell damage or ductal obstruction. We conclude that pancreatic dysfunction may be a common and important overlooked factor contributing to ongoing malnutrition and diseases in malnourished Australian Aboriginal children.

Inhibition of Peroxidase-Catalyzed Iodination by Cephalosporins: Metallo-beta-Lactamase-Induced Antithyroid Activity of Antibiotics

Broad-spectrum antibiotics with heterocyclic side chains strongly inhibit peroxidase-catalyzed iodination in the presence of metallo--lactamase. This suggests that antibiotic resistance due to hydrolysis of the -lactam ring in antibiotics would have negative effects on thyroid activity.

Paracrine action of sFLT-1 secreted by stably-transfected Ehrlich ascites tumor cells and therapy using sFLT-1 inhibits ascites tumor growth in vivo

Background Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis. A soluble form of Flt-1, a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidence suggests the applicability of sFlt-1 in tumor suppression. In the present study, we have developed and tested strategies targeted specifically to VEGF for the treatment of ascites formation.Methods As an initial strategy, we produced recombinant sFLT-1 in the baculovirus expression system and used it as a trap to sequester VEGF in the murine ascites carcinoma model. The effect of the treatment on the weight of the animal, cell number, ascites volume and proliferating endothelial cells was studied. The second strategy involved, producing Ehrlich ascites tumor (EAT) cells stably transfected with vectors carrying cDNA encoding truncated form of Flt-1 and using these cells to inhibit ascites tumors in a nude mouse model. Results The sFLT-1 produced by the baculovirus system showed potent antiangiogenic activity as assessed by rat cornea and tube formation assay. sFLT-1 treatment resulted in reduced peritoneal angiogenesis with a concomitant decrease in tumor cell number, volume of ascites, amount of free VEGF and the number of invasive tumor cells as assayed by CD31 staining. EAT cells stably transfected with truncated form of Flt-1 also effectively reduced the tumor burden in nude mice transplanted with these cells, and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Conclusions The inhibition of peritoneal angiogenesis and tumor growth by sequestering VEGF with either sFlt-1 gene expression by recombinant EAT cells or by direct sFLT-1 protein therapy is shown to comprise a potential therapy. Copyright (C) 2009 John Wiley & Sons, Ltd.

A Genetic Analysis of Lysosomal Enzyme Activities in Brahman Cattle

Analyses of variance and co variance were carried out on the activities of three lysosomal enzymes in mononuclear blood cells from Brahman cattle. These were hexosaminidase (HEX), beta-D-galacto-sidase (GAL) and acid alpha-glucosidase (GLU) which had been measured in blood mononuclear cells from 1752 cattle from 6 herds in a Pompe's disease control programme. Herd of origin and date of bleeding significantly affected the level of activity of all enzymes. In addition, HEX and GAL were affected by age and HEX by the sex of the animal bled. Estimates of heritability from sire variances were 0.29:t 0.09 for HEX, 0.31 :t 0.09 for GAL and 0.44:t 0.09 for GLU. Genetic correlations between all enzymes were positive. The data indicate the existence of a major gene causing Pompe's disease and responsible for 16% of the genetic variation in GLU. One standard deviation of selection differential for high GLU should almost eliminate Pompe's disease from the population. The effi-ciency of selection would be aided by estimating the breeding value for GLU using measurements of HEX and GLU and taking account of an animal's sex, age, date of bleeding and herd of origin.

Enzymic Conversion of Agmatine to Putrescine inL athyrms satiuus seedlings - Purification and properties of a multifunctional enzyme (putrescine synthase)

The participation of a multifunctional enzy(am sein - gle polypeptide with multiple catalytic activities (14)) has been demonstrated in the conversion of agmatine to putrescine in Lathyrus sativus seedlings. This enzyme (putrescine synthase) with inherent activities of agmatine iminohydrolase, putrescine transcarbamylase, ornithine transcarbamylase, and carbamate has been purified to homogeneity anhda s M, = 55,000.

The use of a synthetic prostaglandin e1 analogue (misoprostol) as an adjunct to pancreatic enzyme replacement in cystic fibrosis

Eleven cystic fibrosis children (mean age, 9.6 years) were chosen at random to participate in a study to observe the effects of concurrently stimulating gastric/duodenal bicarbonate secretion and inhibiting gastric acid secretion, using a methylated prostaglandin E1 analogue in patients with pancreatic insufficiency and taking pancreatic enzymes. Percentage fat absorption in 3-day stool collections were calculated before and after commencing therapy with misoprostol, 400 μg/day in divided doses. We found a significant reduction in fat output (14.7 ± 11.7 versus 7.5 ± 3.5 g/day, p < 0.05) in the study group as a whole and a significant reduction in steatorrhoeic level as a percentage of fat intake in all of the patients with abnormal base-line collections (23.1% versus 9.2% p < 0.002). We conclude that misoprostol should be considered in cystic fibrosis patients with steatorrhoea as a means of improving nutrient absorption. © 1988 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

First records of the papaya strain of Papaya ringspot virus (PRSV-P) in French Polynesia and the Cook Islands

The papaya strain of Papaya ringspot virus (PRSV-P), the cause of papaya ringspot disease, was confirmed in French Polynesia and the Cook Islands by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). In French Polynesia, the virus has probably been on the islands of Tahiti and Moorea for several years, but appears not to have spread to eight other islands. In contrast, PRSV-P has only recently appeared in the Cook Islands and is now the subject of an eradication campaign.