The present investigation was carried out to elucidate the possible involvement of the dopaminergic neurotransmitter system in the development of dichlorvos induced delayed neurotoxicity in the rat and to assess the protective efficacy of nimodipine against OPIDN (Organophosphate induced delayed neurotoxicity). Single subcutaneous dose of dichlorvos (200 mg/kg body weight) resulted in marked changes in the dopaminergic neurotransmitter system in terms of increased levels of both dopamine and norepinephrine along with significant increase in the activity of both the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase. This increase was accompanied with a concomitant decrease in the activity of the major degradative enzyme, monoamine oxidase. Scatchard plot analysis revealed a significant decrease in both K(d) and B(max) for dopamine D2 receptors. Administration of nimodipine, a centrally acting calcium channel blocker, along with dichlorvos restricted all these alterations to within control values and could also ameliorate certain behavioural deficits by maintaining the dopaminergic neurotransmitter system. The study underlines the importance of alterations in the dopamine system as a possible causative mechanism behind the behavioural and functional changes associated with delayed neurotoxicity.
Genes for the cell-counting factors in Dictyostelium discoideum, countin and countin2, are considered to control the size of the multicellular structure of this organism. A novel gene, countin3, that is homologous to countin and countin2 genes (49 and 39% identity in amino acid sequence, respectively) was identified in the D. discoideum genome. The expression of countin3 was observed in the vegetatively growing cells, decreased in the aggregating stage, increased in the mid-developmental stage and decreased again in subsequent stages. This expression pattern is different from that of countin and countin2. The distinct expression kinetics of three genes suggests that they would have unique roles in size control of D. discoideum.
The erythrocytes are widely used as model cells for studies of sodium-potassium pump (Na(+)-K(+) pump) in health and disease. Hence, to explore the possible role of the Na(+) transport across the cell membrane in the pathogenesis of pregnancy-induced hypertension (PIH), the present study was conducted to assess the Na(+)-K(+) pump functions in relation to its intrinsic kinetic properties using erythrocytes (RBC). Erythrocyte sodium concentration in pregnancy-induced hypertensive women was significantly (p<0.01) lower in comparison to normotensive pregnant women. On the contrary erythrocyte potassium was significantly higher (p<0.01) in PIH women as compared to normotensive pregnant women. Observed alterations in Na(+) and K(+) concentrations in erythrocytes were associated with significantly (p<001) increased Ouabain-sensitive sodium efflux rate and rate constants in erythrocytes from PIH women. Further, kinetic studies revealed that increased Ouabain-sensitive efflux rate constant in RBC from PIH women was accompanied by increased maximal velocity (V(max)) of Na(+)-K(+) pump. However, the affinity constant (K(m)) was unaltered in both the groups. Therefore, these findings suggest that increased Na(+)-K(+) pump activity in RBC of PIH women could be due to either increased numbers of Na(+)-K(+) pump units of increased numbers of active subunits of Na(+)-K(+) pump possibly due to specific plasma factors in PIH women.
Simple extraction of prolamins from cereal flour using 70% aqueous ethanol leads to co-extraction of lipids and other secondary products. Treatment of the crude extract with an excess of pure ethanol resulted in the removal of the majority of these compounds. Prolamin extracts obtained following ethanol precipitation showed little difference to products of more complex, multi stage, selective extractions, when compared using MALDI-TOF mass spectrometry. Toxicity tests on enzymic digests of ethanol-precipitated prolamins from coeliac-toxic cereals, coeliac-non-toxic cereals and oats were carried out using a rat liver lysosome assay. The prolamins from the ethanol-precipitated extracts showed greater activity than those extracted using only 70% ethanol for extraction. As the ethanol precipitation method is simple and provides a prolamin extract of sufficient purity for further evaluation, this procedure has been adopted as an alternative to more tedious procedures for preparation of cereal prolamins.
Cereal prolamins of wheat, rye and barley are the major proteins that have been implicated in toxicity in patients with coeliac disease. The gliadins of wheat are the best characterised with the identification of toxic peptides from rye and barley not as well advanced. This study has employed extended motifs, based on the known toxic motifs are derived from the sequence of A-gliadin, to search protein databases for matches with coeliac-toxic cereals. The results obtained have provided pointers to specific regions in rye and barley prolamins, which have received little attention in in vitro and in vivo studies of toxicity in coeliac disease. The results obtained in this study indicate that the size of the extended motif is critical when searching for coeliac-toxic cereals using protein databases. Extended motifs that are common to all three coeliac-toxic cereals and found in active wheat gliadin peptides are QQPYP, PQQPY and QQQPFP.
VCP (Valosin-Containing Protein), a member of the AAA (ATPases Associated to a variety of cellular Activities) family of proteins, possesses a duplicated highly conserved ATPase domain. An expressed sequence tag (EST), representing a clone from the Eimeria tenella merozoite cDNA library, was found to have high similarity to VCP genes from other organisms. A complete sequence derived from the corresponding clone (designated eth060) shows amino acid identity of 42-62% with other members of the VCP subfamily. Sequence analysis identified a putative ATPase domain in the eth060 sequence. This domain was PCR-amplified using gene-specific primers and cloned into a pBAD/Thio-TOPO expression vector. Expression in Escherichia coli demonstrated that the putative ATPase domain, which consists of 414 amino acid residues, produced a fusion protein of approximately 60 kDa in size.
Functional reagents known to bring about the formation of a distinct membrane molecular complex of the subunits of cytochrome b(558) (gp 91(phox) and p22(phox)) were investigated for their influence on the O2- generating capability of liposome incorporated cytochrome b(558) preparations. One, ethyleneglycolbis[sulfo-succinimidylsuccinate], (sulfo-EGS) was found to inhibit O2- generation at concentrations which are known to result in cross-linking the two subunits of cytochrome b(558). Sulfosuccinimidyl [4-azidophenyldithio] propionate, (sulfo-SADP) on the other hand, was found to be a powerful inhibitor of the cytochrome b(558) dependent O2- production at concentrations not able to result in cross linking of the two subunits. Sulfo-SADP inhibits the cytochrome b(558) O2- production 50% at 25 microM, while sulfo-EGS requires 400 microM. For these reagents, the succinimidyl group of sulfo-SADP and sulfo-EGS is the reactive group, which inhibit irreversibly, cytochrome b(558) generation of O2-. Both sulfo-SADP and sulfo-EGS have similar linker arms of 13.9 and 16.1 A, respectively. The difference, accounting for the strong inhibitory profile for sulfo-SADP as compared with sulfo-EGS, resides in the aryl group associated with the sulfo-SADP. The aryl group of sulfo-SADP has been found to be important in directing the specificity of the probe in its inhibition of O2- generation. When the disulfide bond linking the aromatic portion of the probe to the succinimidyl ring is cleaved by DTT (dithiothreitol), the product loses its specificity and has an inhibitory activity with respect to O2- generation comparable to that of sulfo-EGS. The partial protection against the inhibitory influence of sulfo-SADP by NADP(+) indicates that the reagent may interact at the pyridine nucleotide-binding domain of cytochrome b(558). Its low inhibitory titer and its water solubility suggest that sulfo-SADP reacts with a specific amine (the primary reactant for the succinimidyl group) on cytochrome b(558).
It is no long a question whether the process of evolution is affected by chance, but is to what extent. The random analysis can throw light on the underlying reasoning for the primary structure of proteins. The study on the Bcl-2 family shows that the rank per amino acid increases 93% in bovine than in chicken and rat, which indicates that the effect of chance has more impact on the evolutionary process of chicken and rat Bcl-2 and less impact on bovine Bcl-2. About one fourth of types of amino acids distribute in the highest probabilistic way in the Bcl-2 family. These phenomena are striking in four BH regions where vast majority of amino acids occur with the probabilistically simplest distribution. Mutations and variants can lead to the increased and/or decreased distribution probabilities of amino acids. Mutations 1, 2 and variant 3 target the rank increased in both affected amino acids. By contrast variants 1 and 2 lead to the rank decreased in both affected amino acids. In mutations 3 and 4, the affected amino acids change their ranks to opposite directions, i.e. the rank is lower in replaced amino acid and higher in replacing one. Using our random approaches as quantitative tools, we can measure, compare and explain the primary structures of proteins.
Leucine zipper-bearing kinase (LZK) is a new member of the mixed lineage protein kinase family. We previously cloned a cDNA encoding LZK from a human cerebellum cDNA library. The following studies indicated that LZK serves as a MAPKKK in the JNK/SAPK pathway in cells, and a scaffold protein, JIP-1, enhances LZK-induced JNK/SAPK pathway activation via physical association. Here we report characterization of the gene structure and fine chromosomal mapping of the human LZK gene. Polymerase chain-reaction (PCR) studies indicated that the human LZK coding sequence is composed of 13 exons, and that all the splice acceptor and donor sequences obey the GT-AG rule. Chromosomal localization studies involving FISH mapping demonstrated that the human LZK gene is located at 3q27.
Urease from seeds of pigeonpea showed a time-dependent and irreversible inactivation at very low concentrations of heavy metal ions. Concentration of Cu(2+), Hg(2+) and Ag(+) required for 50% inactivation, on 10 min of incubation, were found to be 2.2 x 10(-6), 2.9 x 10(-8) and 6.3 x 10(-12) M, respectively. The kinetics of inactivation with each of these metal ions was found to be biphasic, with half of the activity being lost in a fast phase and remaining in a slow phase. Acetohydroxamate (AHA) inhibits pigeonpea urease competitively and reversibly with a K(i) of 0.041 mM at pH 7.3. This inhibition was found to be pH dependent. A reversible and time-dependent inhibition was observed with AHA. AHA inhibition revealed biphasic kinetics as observed with the heavy metal ions. Pigeonpea urease was also inhibited by fluoride ions competitively with a K(i) value of 1.23 mM. These inhibition studies suggest the possible interaction of these inhibitors with active site thiol groups and Ni (II) ion. A mechanism has been proposed for each of these inhibitors and compared with inhibition studies reported for other ureases.
Ty1-copia-like retrotransposons have been identified and investigated in several plant species. Here, the internal region of the reverse transcriptase (RT) gene of Ty1-copia-like retrotransposons was amplified by PCR from total genomic DNA of 10 varieties of banana. Two to four clones from each variety were sequenced. Extreme heterogeneity in the sequences of Ty1-copia-like retrotransposons from all the varieties was revealed following sequence analysis of the reverse transcriptase (RT) fragments. The size of the individual RT gene fragments varied between 213 and 309 bp. Southern blots of genomic DNA digested from Musa acuminata and other banana varieties probed with W8 clone from M. acuminata and A4 clone from Pisang Abu Nipah showed similar strong, multiple restriction fragments together with other faint hybridization band patterns with variable intensities indicating the presence of many copies of the Ty1-copia-like retrotransposons in the genomes. There was no correlation between retroelement sequence and the banana species (with A or B genomes) from which it arose, suggesting that the probes are not useful for tracking genomes through breeding populations.
The gene of nitrous oxide reductase (nosZ) and entire region of nos gene cluster from Pseudomonas sp. strain MT-1, which is isolated from the sediment of Mariana Trench, was identified. Gene organization in nos gene cluster is similar to that of other bacteria, and nosZ was highly homologous to nosZ of other Pseudomonads. The remarkable property was the existence of a gap from Arg-302 to Ser-309 (numbered by NosZ of P. aeruginosa), which might be a feature of marine bacterial NosZ. It is interesting that nosZ of the strain MT-1 is not related so closely to P. stutzeri, although 16S rDNA of the strain MT-1 is highly homologous to that of P. stutzeri. The existence of nosZ gene in the strain MT-1 indicated the presence of a complete denitrification (nitrate to N(2)) in the Mariana Trench. This is the first report on genetic analysis of denitrification in the deep sea.
BACKGROUND:There is considerable evidence indicating that gall stone patients have altered gall bladder functions with respect to secretion and absorption and inflammation in mucus membrane when compared with gall stone free subjects. Increased levels of accelerated generation of reactive oxygen species and toxic degradative products of lipid peroxidation have been reported in the plasma of individuals with gall stones. The purpose of this study is to find out whether oxidative stress in mucosa plays any role in the pathogenesis of gall stone diseases. Levels of lipid peroxides, lipid hydroperoxides (LPH) and conjugated dienes were assessed in gall bladder mucosal scrapings obtained from 30 gall stone patients undergone cholecystectomy. Antioxidant enzymes such as catalase (EC 1.11.1.6), superoxidedismutase (EC 1.15.1.1), glutathione peroxidase (EC 1.11.1.9), glutathione transferase (EC 2.5.1.18) and glutathione reductase (EC 1.6.4.2) were assessed. The activity levels of alkaline phosphatase (EC 3.1.3.1), adenosine triphosphatase and protease (EC 3.4.24.11) were also assessed and data compared with identical data collected from 15 gall stone free subjects and 10 post-mortem cases. Gall stone patients had significantly higher levels of conjugated dienes, LPH and thiobarbituric acid reacting substances in their gall bladder mucosa. Lower levels of glutathione and glutathione related enzymes, catalase and superoxide dismutase were observed in those patients when compared to gall stone free subjects. The activities of functional enzymes in mucosa such as alkaline phosphatase, Na(+)-K+ (EC 3.6.1.3) and Ca2+ (EC 3.6.1.2) adenosine triphosphatase showed significant decreases. Histopathological observation showed lipid accumulation, dilated blood vessels, necrotic and fibrotic changes and inflammation in the gall bladder mucosa of gall stone patients. The data show that gall stone patients have a high level of oxidative stress in the gall bladder mucosa, a finding that may be related to a decreased activity of functional enzymes in mucosal cells. Such a condition might result in an altered gall bladder absorption and secretion of bile components such as mucins and glycoproteins. The resultant increased risk of bile saturation would further contribute to the progress of gall stone formation.
We have used the phagemid pComb3H to construct recombinant phages displaying the single chain variable fragment (ScFv) towards exotoxin of Burkholderia pseudomallei. Variable heavy and light chain fragments were amplified from the hybridoma 6E6A8F3B line, with a wide spectrum of primers specific to mouse antibody genes. Through overlapping extension polymerase chain reaction, the heavy and light chain fragments were linked to form the ScFv which was subsequently cloned into the phage display vector and transformed into ER2537 cells to yield a complexity of 10(8) clones. The transformants were screened by four rounds of biopanning against the exotoxin and resulted in selective enrichment of exotoxin-binding antibodies by 301 fold. The phage pool from the final round of selection displayed antibodies of high-affinity to the exotoxin as demonstrated by ELISA. Several clones were selected randomly from this pool and analysed by restriction enzyme digestion, fingerprinting and sequencing. Restriction analysis confirmed that all clones carried a 700-800 bp insert whose sequences, in general, corresponded to that of mouse IgG. Fingerprinting profiles delineated the antibodies into two families with different CDR sequences.
Aminobutyraldehyde dehydrogenase was purified to essentially homogeneity from putrescine-grown cells of Arthrobacter sp. TMP-1. The molecular weights of the enzyme and its subunit were 201,000 and 51,000, respectively, suggesting that the enzyme is a tetramer of identical subunits. The apparent Michaelis constants (K(m)) for 4-aminobutyraldehyde, 3-aminopropionaldehyde and 4-guanidinobutyraldehyde were approximately 65, 150, and 85 microM, respectively. Linear fatty aldehydes also tested were less active as a substrate, while the tested succinate-semialdehyde and branched fatty aldehydes were inert. The enzyme utilized both NAD(+) and NADP(+) as coenzymes. The optimum pH was 8.0. The enzyme lost 64% of its activity when held at 40 degrees C for 10 min.
We have used human cell lines, namely, K562 and HeLa cells as model systems in understanding the mechanism of lead toxicity and heat shock, that may be mediated by the heme-regulated eIF-2alpha kinase which is also called the heme-regulated inhibitor (HRI). RT-PCR analysis using HRI-specific primers indicated a two- to three-fold increase in HRI expression in K562 and HeLa cells exposed to lead acetate and heat shock, respectively. Further, in vitro eIF-2alpha kinase assay indicated a two- to three-fold increase in HRI kinase activity during lead toxicity in K562 cells. This increase in HRI expression and its activity was accompanied by a significant decrease in cell proliferation and cell viability. This is therefore, the first report indicating that both heavy metal exposure and heat shock cause inhibition of protein synthesis not by activation of HRI alone but by its over-expression as well as activation. Our data indicate further that lead-induced inhibition of cell proliferation may be caused due to inhibition of protein synthesis resulted due to induced expression and activity of HRI.
N-carbamoyl-beta-D-glucopyranosylamine (NCG) is a compound, which can chemically be synthesized by the condensation of glucose and urea by heating under acidic condition. In this study, we isolated and identified NCG and its anomer from human serum. This is the first study showing the occurrence and isolation of NCG from a natural source. The NCG level in human serum was estimated to be 71+/-33 microM using a glucose-3-dehydrogenase-based assay. Because NCG is not commercially used in foods or drugs, we conclude that the NCG isolated from human serum is synthesized from blood glucose and urea in vivo.
Ornithine decarboxylase (ODC) is an enzyme of one of the two pathways of putrescine biosynthesis in plants. The genes encoding ODC have previously been cloned from Datura stramonium and human. Using differential screening, we isolated ODC cDNA clone from a cDNA library of ripening Capsicum annuum fruit. The cDNA clone designated CUKM10 contains an insert of 1523 bp. The longest open reading frame potentially encodes a peptide of 345 amino acids with an estimated molecular mass of 47 kDa and exhibit striking similarity to other ODCs. Expression analysis showed that the capODC hybridised to a single transcript with a size of 1.7 kb. The capODC transcript was first observed in early ripening and increased steadily until it reached fully ripening stage. From the observation it is suggested that capODC is developmentally regulated especially during later stage of ripening.
The implication of oxidative stress in the pathology of insulin resistance has been shown recently. We investigated the effect of an insulin sensitizer, metformin, on the plasma lipid peroxidation and antioxidant defense system in the erythrocytes of high fructose fed rats which form an animal model of insulin resistance. The experimental animals were divided into two batches of 12 animals each. The control batch received the control diet, containing starch; the fructose group was given the high fructose diet. At the end of second week these were subdivided into two groups; one was given metformin (50 mg/kg/day in water) by gastric intubation and other group was left untreated. The rats were continued on the same dietary regimen for the next two weeks. Fructose-fed rats showed hyperglycemia, hyperinsulinemia and hypertriglyceridemia at the end of four weeks. Enhanced plasma lipid peroxidation and inadequate cellular antioxidant defense system were observed in them. Administration of metformin was associated with significant normalization of circulating insulin, glucose and triglyceride concentrations. The abnormal triglyceride distribution in the lipoprotein fractions was also ameliorated by metformin therapy. The imbalance between peroxidation and antioxidant defense system was mitigated when fructose-fed rats were treated with metformin. In the control rats, metformin did not affect the parameters studied. Significant positive correlation was obtained between insulin, triglycerides and glucose concentrations with lipid hydroperoxides suggesting that these metabolic variables could influence the lipid peroxide levels in plasma.
Eight selective nitrogen-sulfur donor ligands have been synthesized from the condensation of S-methyldithiocarbazate (SMDTC) with aldehydes and ketones with a view to evaluating their antimicrobial and cytotoxic activities, and also to correlate the biological properties with the structure of the ligands. The compounds were all characterized by elemental analyses and other physicochemical techniques. SMDTC and the Schiff bases were screened for antimicrobial and cytotoxic activities. SMDTC showed very large inhibition zones (24-44 mm) against bacteria and fungi with a minimum inhibitory concentration (MIC) of 390-25,000 and 1562-6250 microg ml(-1), against different bacteria and fungi, respectively. Streptomycin and nystatin were used as the internal standards against bacteria and fungi, respectively. SMDTC along with its Schiff bases with pyridine-2-carboxaldehyde, acetylacetone and 2,3-butanedione were strongly antifungal and the MIC values were comparable to nystatin. Most of the Schiff bases were strongly cytotoxic. In particular, those with pyridine-2-carboxaldehyde and 2,3-butanedione have CD(50) values of 5.5, 1.9-2.0 microg ml(-1), respectively, against leukemic cells, while against colon cancer cells, the values were 3.7 and 2.0 microg ml(-1), respectively. The glyoxal Schiff base was strongly active only against leukemic cell with CD(50) value of 4.0 microg ml(-1). The present findings have been compared with standard drugs.
