Uptake and distribution of 203Hg by fish fingerlings, Cirrhina mrigala, exposed to linear alkyl benzene sulphonate.
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Biomedical subjects
Publications and source records attributed to V Misra.
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The major glycoproteins of bovine herpesvirus type 1 showed distinct temporal patterns of expression. The glycoproteins GVP 11 and GVP 6 as well as its cleavage products, GVP 11a and GVP 16, were expressed early in the infectious process, whereas GVP 9 was expressed late. Temperature-sensitive mutants were developed and characterized. Mutants belonging to two complementation groups were unable to synthesize DNA at 40 degrees C, the non-permissive temperature. In cells infected with these mutants the late glycoprotein GVP 9 was not synthesized at 40 degrees C, whereas the synthesis of the early glycoproteins GVP 11 and GVP 6 continued at wild-type levels. These studies suggest that the transition from early to late glycoprotein synthesis is linked to viral DNA synthesis.
Preruminant calves on several farms had diarrhea nonresponsive to treatment and were doing poorly, despite being fed a high quality calf milk replacer. Because these reconstituted milk replacers always had a sediment, they were suspected of containing insoluble nonmilk-derived proteins. Microscopic examination of the milk replacer, however, did not show any evidence of starch granules. We therefore analyzed the samples by SDS PAGE. We were able to identify and quantitate pea protein in calf milk replacers in which all the protein was supposedly milk-derived. We were also able to differentiate polypeptides derived from pea and soy. We concluded that PAGE is a sensitive technique for detecting nonmilk-derived proteins in calf milk replacers.
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The 130-kDa bovine herpesvirus-1 (BHV-1) glycoprotein GVP 6 was found to cross-react immunologically with the herpes simplex glycoprotein gB. Antibodies in polyclonal serum against gB immunoprecipitated GVP 6 and its cleavage products from a lysate of BHV-1-infected cells. Conversely, polyclonal serum against GVP 6 precipitated gB from HSV-1-infected cell lysates. Sera against the other glycoproteins did not demonstrate cross-reactivity. A 3.6-kb Kpnl-Hpal fragment of BHV-1 DNA that hybridized to the gene for gB was cloned and the nucleotide sequence of both strands was determined. The longest codon reading frame in the fragment coded for a protein that showed extensive homology with gB1 and related sequences from pseudorabies virus, varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus. The strongest homologies were observed in two segments of the ectodomain, the transmembrane domain, and sequences adjacent to the transmembrane domain.
The immunobiology of luteinizing hormone releasing hormone (LHRH) was explored, to provide a conceptual and practical basis for the use of LHRH in immunocastration. Cysteine substituted analogues of LHRH were synthesized including Cys1-LHRH (C1-LHRH), Cys6-LHRH (C6-LHRH) and Cys10-LHRH (C10-LHRH). These were reacted to carrier molecules using the heterobifunctional cross-linking reagent m-maleimidobenzoylsulfosuccinimide ester (SMBS), producing peptide-carrier conjugates of known peptide content and conjugation orientation. This reaction regime was found to be rapid, efficient and allowed for easy control of peptide to carrier ratios. Conjugates were used in active immunization trials in BALB/c mice to characterize the murine immune response against LHRH. BALB/c mice were shown to have the capacity to recognise all three cysteine substituted LHRH analogues and to produce antibodies cross-reactive with native LHRH. The specificity of LHRH antisera generated was found to be dependent on the site of conjugation of the peptide to carrier molecule. C1-LHRH generated carboxy terminal directed antibodies, C10-LHRH generated amino terminal directed antibodies, while C6-LHRH could generate amino terminal directed or carboxy terminal directed antibodies, or both within a given animal. No intrinsically immunodominant epitopes were seen within the LHRH molecule.
Cysteine substituted analogues of luteinizing hormone releasing hormone (LHRH) were coupled to carrier molecules, and the resulting conjugates used to characterize the immune response to native LHRH generated in BALB/c mice and to formulate vaccines in an effort to maximize titer development. In an active immunization trial designed to characterize temporal aspects of anti-LHRH titer development, titers could be detected 1 week after initial immunization. No differences were observed in response between male and female mice. Booster immunizations could enhance the titers against LHRH. The titers developed against the carrier molecule were uniformly higher than the corresponding anti-LHRH titers throughout the course of the trial. In separate trials, vaccines were formulated and tested in BALB/c mice for titer development against LHRH. Adjuvants, carrier molecules, dosage and peptide to carrier ratio were considered. Dosages of 50 micrograms conjugate per immunization per mouse, at conjugation ratios of 3-12 peptides per 10(5) Da carrier molecule, were found to produce immune responses. Adjuvants including Havlogen and dimethyldioctadecylammonium bromide (DDA), and carrier molecules including keyhole limpet hemocyanin (KLH), porcine thyroglobulin (TGB) and equine gamma globulin (EGG) were all found to be effective.
Pathomorphological changes in the skin was noticed under the scanning electron microscope in fish fingerlings (Cirrhina mrigala) exposed to 0.005 ppm (25% of the LC50) concentration to linear alkyl benzene sulfonate. The epithelial cells present in the epidermis of the skin were found to secrete more mucus with linear alkyl benzene sulfonate (LAS) than did controls. The presence or deposition of mucus on the surface of skin indicated likely molecular interaction between constituents of mucus and LAS.
An attempt has been made to study the biochemical properties of lung alkaline phosphatase so that it may serve as a model for measuring the toxicity of air pollutants, especially metals. Lung was shown to be a better source of this enzyme in comparison to other organs in rats. The optimum pH, metal requirements, substrate specificity, and affinity constants were characterized with different substrates, and the effect of different metals was studied. The involvement of zinc in the lung enzyme was not indicated as shown by Sephadex G-200 elution profile of postmitochondrial supernatant of 65Zn-treated animals.
Intratracheal injection of zinc oxide dust, a component of many cosmetic preparations caused elevation of alkaline phosphatase and lactate dehydrogenase in serum and lung of guinea pigs. Clearance of zinc oxide dust from lungs through zinc-carrying proteins was evident.
Fish fingerlings (Cirrhina mrigala) exposed to 0.005 ppm (25% of LC50) concentration to detergents (linear alkyl benzene sulfonate) showed marked behavioral changes and distorted appearance of primary and secondary lamellae along with damage to gill epithelium under scanning electron microscopy at various magnifications. Mucosal cells of gills were found to secrete mucus showing primary reactions for membrane damage leading to dysfunction in respiration and osmoregulation.
Mouse myeloma NS1-Ag4 cells were fused with spleen cells from a BALB/c mouse previously immunized with luteinizing hormone releasing hormone (LHRH) conjugated to serum bovine albumin (BSA). Fused cells were grown in HAT restrictive medium which was screened for LHRH binding ability via a primary binding assay employing [125I]LHRH and cold ethanol precipitation. One clone (hy-USASK/DSIL-LHRH-A1) was selected for further study. Cell culture fluid and ascites fluid bound 30% of [125I]LHRH at 1:4000 and 1:400,000 dilution respectively. A competitive inhibition assay using ascites fluid at 1:2,000,000 dilution and LHRH standards at 0.125-32.0 ng/ml was established. Initial studies using rabbit anti-mouse allotype sera in a horseradish peroxidase (HRP)-ELISA system indicate the antibody is IgG1. A dose of 0.5 ml ascites fluid containing LHRH antibody given intravenously (i.v.) on day 9 of gestation was effective in terminating pregnancy in rats. A 1 cm progesterone implant made of elastomer polymer and placed interperitoneally blocked this effect. Ascites fluid (4.5 ml) containing LHRH antibody, when infused i.v. into mature spayed female dogs induced a precipitous decline in mean luteinizing hormone (LH) levels and reduced LH pulsatility over 4 days. It was concluded that the mouse monoclonal antibody is specific for LHRH, and can interrupt reproductive events in vivo.
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Hybridoma cell lines producing monoclonal antibodies to bovine herpes virus type 1 (BHV-1) were established. The monoclonal antibodies were characterized with respect to their antigen specificities and biological activities. One group of eight monoclonal antibodies precipitated the glycoproteins GVP 3 (180K) and GVP 9 (91K), a second group of thirteen monoclonal antibodies precipitated GVP 6 (130K), GVP 11 (74K) and GVP 16 (55K), and one monoclone secreted antibodies specific for GVP 7 (105K). Analysis of the immune precipitates by electrophoresis under nonreducing conditions suggested that GVP 3 is a dimer of GVP 9. It also indicated that GVP 11 and GVP 16 are components of a disulfide-linked complex, GVP 6. The results, obtained by immunoprecipitation were confirmed by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA), using electrophoretically separated viral glycoproteins. In addition, these techniques demonstrated differential reactivities of the monoclonal antibodies with GVP 11 and GVP 16. The monoclonal antibodies were used to analyze the biological roles of these three sets of glycoproteins. Monoclonal antibodies directed against GVP 3/GVP 9 did not neutralize viral infectivity, but most of them mediated complement-dependent lysis of the infected cell. Individual monoclonal antibodies directed against GVP 6/GVP 11/GVP 16 could neutralize virus as well as participate in complement-mediated lysis. The only available monoclone against GVP 7 did not show any biological activity in the above two assays. Thus, GVP 6/GVP 11/GVP 16 may contain the attachment site of the virion.
A biostatic assay method involving 45Ca uptake into shells and tissues of snails (Lymnaea vulgaris) in 72 hr was developed to follow the effect of detergent-polluted water on ecosystems. There was a marked decrease in the 45Ca uptake by shells and tissues of linear alkyl benzene sulfonate-exposed animals as compared to controls. No change in 45Ca uptake was observed in dead shells, thereby excluding the possibility of passive exchange.
The water flea (Daphnia magna) was used as a sensitive indicator for assessing the toxicity due to synthetic detergents. Acute and chronic toxicity of detergents to the water flea was studied under laboratory conditions by following the median tolerance limit (TLM) at 48 hr and the rate of survival. A significant decrease in the rate of reproduction (number of hatching and neonates produced) were found at 21 days. During acute toxicity studies behavioural changes were also noticed.
A possible mechanism of biological effects of silicate dusts is the interaction between silicic acid and tissue constituents, and this has been studied in vitro. On incubating silicic acid with lung homogenate, appreciable amounts of silicon were recovered in the trichloroacetic acid precipitate in both organic solvent extract and residual protein. Polyvinyl pyrrolidone inhibited this interaction as well as reducing the dissolution of silicic acid from asbestos dust. The capacity of serum albumin to bind silicic acid was also observed. Chemical interaction between macromolecules and silicic acid could be one of the factors responsible for the biological effects of silicon-containing dusts.
In an effort to determine whether distinct types of bovine herpesvirus 1 are responsible for causing specific syndromes, the polypeptides and DNA of 93 BHV 1 isolates from the province of Alberta as well as vaccine strains and numerous other Canadian, U.S. and European isolates were analyzed by PAGE and restriction endonuclease fingerprinting, respectively. The polypeptide patterns showed only slight variations: only six isolates contained polypeptides that varied from the norm in their molecular weight, or were absent. Although on the basis of endonuclease patterns the isolates could be categorized into three "strains" and nine "sub-strains", we were unable to associate any of the strains with specific clinical signs. This suggests that the type of disease caused may be determined more by the route of infection and animal management practices than by the inherent properties of certain types of BHV 1. Most of the Albertan isolates were of one BHV 1 type and sub-type.