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Biomedical subjects

M Knight

Publications and source records attributed to M Knight.

At least 37 records · Page 2Linked to original sources
PubMed1988

Delayed effects of doxorubicin on spermatogenesis and endocrine function in rats.

Doxorubicin was administered to adult male Wistar rats (1 mg/kg body weight, three times per week, for one, two, three, or four weeks) in order to examine testicular and reproductive endocrine toxicity 56 days after treatment. Doxorubicin treatment produced persistent dose-related reductions in testis, epididymis, and seminal vesicle weights, but did not alter ventral prostate weight. Testis and serum testosterone levels were not significantly affected by treatment, but serum LH was increased after treatment, and binding of iodinated hCG to testicular LH receptors was reduced. Serum FSH was elevated by the two lower total administered doses, but was not different from controls after treatment with the two higher total doses. There was clear histologic evidence of dose-dependent damage to the seminiferous tubules, which was reflected by decreased testicular and epididymal sperm content and by reductions in the stem-cell survival index. These results indicate that doxorubicin produces significant and persistent damage to the endocrine and spermatogenic compartments of the testis.

Animals↗
PubMed1987

Counter-current chromatographic purification of [D-Ala]peptide T amide.

A recently proposed octapeptide, [D-Ala]peptide T amide (D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH2), has been shown to bind to the CD4 receptor on human T-cell lymphocytes and block human immunodeficiency viral infectivity. This peptide may not only be itself a promising therapeutic for the disease of AIDS (acquired immune deficiency syndrome), but might open a new avenue of research towards more efficacious drugs. Accordingly, a counter-current chromatography system is here presented, utilizing the Ito flow-through coil planet centrifuge (solvent system, 1% trifluoroacetic acid-n-butanol (1:1), upper phase mobile) which allows facile preparative purification of [D-Ala]peptide T amide. The method may be applicable to purifying analogues of [D-Ala]peptide T amide as they are developed.

Chromatography, High Pressure Liquid↗
PubMed1987

Characterisation of the structure and expression of the gene encoding a major female specific polypeptide of Schistosoma mansoni.

A previously described cDNA clone, pSF10, of Schistosoma mansoni encoding the very dominant female specific polypeptide (FSP) has been used to characterize the gene and its expression. The gene is detectable in different isolates of S. mansoni and is estimated to be present in 3 copies per haploid genome. The gene is not sex linked and exhibits neither amplification nor rearrangement concomitant with expression. Expression of the gene by parasites maturing in hamsters is first detected after 5 weeks when the RNA is present at 1/10 the level of that of 6 week worms. Although the FSP gene is specifically and highly expressed by egg laying female worms a corresponding polypeptide produced by the cell-free translation of RNA is not detectable. It was confirmed, however, that pSF10 does indeed encode a mRNA by DNA sequence analysis. The sequence demonstrated a mRNA containing a poly(A) tail and two open reading frames. One reading contains no methionine but is very high (47%) in glycine. This amino acid composition could account for the inability to detect the gene product by cell-free translation in the presence of [35S]methionine.

Animals↗
PubMed1987

The purification and characterization of subunits alpha, beta, and gamma from the rabbit reticulocyte eukaryotic initiation factor 2.

Eukaryotic initiation factor 2 (eIF-2) contains three nonidentical subunits, alpha, beta, and gamma. The simultaneous purification of all three subunits was achieved by reverse-phase HPLC using a 0.1% trifluoroacetic acid-acetonitrile binary solvent system. The order of the eluted subunits, beta, alpha, and gamma, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After hydrolysis in 6 N HCl, picomole level amino acid composition analysis was achieved by the ninhydrin reaction on a Beckman 6300 system. Using second-derivative spectroscopic analysis, Trp was detected in all three subunits. All three subunits were subjected to amino-terminal sequence analysis. The amino-terminal of eIF-2 alpha from amino acid positions 1 to 23 inclusive was determined. The order of eight amino acids from the amino-terminal of eIF-2 gamma was also determined. This characterization and partial determination of the primary sequence of these subunits permit the utilization of molecular biology techniques in order to elucidate the complete primary structure. Additionally, the partial amino acid sequence data permitted the designation of synthetic gene probes as well as the identification of eIF-2 alpha and gamma cDNA and/or genomic clones.

Amino Acid Sequence↗
PubMed1987

Resistance to reinfection with Schistosoma haematobium in Gambian children: analysis of their immune responses.

The relationship between reinfection with Schistosoma haematobium and immunological parameters was studied in a group of Gambian children aged from 8 to 13 years. Each individual's exposure to infection was assessed from observations of water contact, cercarial densities and infected snail densities at water contact sites. Eosinophil counts were made and responses to egg antigen (SEA) and adult worm antigen (WWH) measured by ELISA. Low levels of reinfection were associated with a high eosinophil count, high levels of antibodies against WWH and SEA, increased age and low exposure. In a multiple regression analysis of the association of reinfection with eosinophil count, antibody levels, exposure, age and sex, the effects of eosinophil count and exposure were still very significant after allowing for all the other variables. The effects of the antibody levels were close to significance after allowance for exposure and eosinophil count (for WWH: P = 0.09; for SEA: P = 0.07), although the evidence was less clear after additional allowance was made for age and sex. The ability of sera from the children to recognize different parasite antigens was also examined by immunoprecipitation of labelled schistosomulum surface, WWH, SEA and S. haematobium adult worm mRNA in vitro translation products. Schistosomulum surface antigens were recognized by all the sera and there was little variation in this response. There was more variation in their responses to SEA and WWH and a marked heterogeneity in the response to in vitro translation products. However, the pattern of antigen recognition appeared unrelated to susceptibility to reinfection.

Adolescent↗
PubMed1987

Surface and species-specific antigens of Schistosoma haematobium.

Of the surface antigens identified by radio-iodination, two-dimensional gel analyses showed no similarities between those of Schistosoma haematobium and Schistosoma mansoni, thus providing a basis for the species specificity of these antigens described previously (Simpson, Knight, Hagan, Hodgson, Wilkins & Smithers (1985) Parasitology 90, 499-508). The surface antigens of S. haematobium were glycosylated and comprised an acidic polypeptide of Mr 17,000 as well as a complex set of polypeptides of approximate pI 6-7, which resolved in the Mr range 20,000-30,000. At least one of the lower Mr forms of this complex is also present in the adult worm. Limited cross-reaction was observed with S. mansoni infection sera and this may be due to a shared carbohydrate epitope. In contrast, extensive cross-reaction was observed using sera from mice immunized with S. bovis. This pattern parallels the species-specificity of vaccine-induced immunity. Extensive cross-reaction was also observed within cell-free translation products of m-RNA from adult worms of S. haematobium and S. mansoni by use of heterologous human infection sera. The few antigens which were species-specific may represent surface antigens.

Animals↗
PubMed1987

Comparison of the antioxidant properties of alpha-tocopherol and alpha-tocopheryl acetate in newborn rabbit lung.

The antioxidant properties of alpha-tocopherol and alpha-tocopheryl acetate in lung tissue were quantitated in vitro by measurement of thiobarbituric acid (TBA) reactants, pentane production and lipid peroxides following either an iron/ascorbate or a xanthine/xanthine oxidase oxidant stress. Lung homogenates were obtained from newborn rabbits treated intravenously with either 10 or 100 mg/kg of either alpha-tocopherol or alpha-tocopheryl acetate. The animals were killed 5 min after dosing to minimize the conversion of alpha-tocopheryl acetate to alpha-tocopherol. Lung homogenates from animals treated with alpha-tocopherol had decreased concentrations of TBA reactants and pentane production after incubation with either oxidant stress compared to lung homogenate from animals treated with alpha-tocopheryl acetate or vehicle alone. Lipid peroxide concentrations were no different in homogenates from alpha-tocopherol or alpha-tocopheryl acetate treated animals. Correlations between antioxidant activity and tissue concentrations of alpha-tocopherol indicate 50% inhibition is achieved with 30-100 micrograms/g lung tissue. In vitro addition of alpha-tocopherol required concentrations between 100 and 200 micrograms/g to reduce lipid peroxidation by 50%.

Animals↗
PubMed1986

The recognition of Schistosoma mansoni surface antigens by antibodies from patients infected with S. mansoni and S. haematobium.

Polypeptide surface antigens of Schistosoma mansoni recognized by schistosomiasis patients have been identified and their strain and species specificity investigated. Antibodies from individuals infected with S. mansoni were used in immunoprecipitation assays of 125I-labelled schistosomulum surface antigens. All individuals surveyed from St. Lucia strongly precipitated antigens of approximately Mr 38,000 to 32,000 and 20,000. These antigens were shown by two-dimensional gel electrophoresis to be the same as those recognized by experimentally immunized mice. Although individuals showed a highly heterogeneous response against total polypeptide antigens synthesized in vitro by cell-free translation of adult S. mansoni mRNA, all individuals recognized the same surface antigens. Immunoprecipitation with sera from patients infected with S. mansoni in many different parts of Africa resulted in generally the same antigens being precipitated, although a very high molecular weight antigen(s), not strongly recognized by the St. Lucian sera was also precipitated by most of the African patient sera. One serum from Ghana precipitated the high molecular weight antigen but not the low molecular weight antigens, raising the possibility of the existence of S. mansoni strain(s) exhibiting some diversity in surface antigens. The surface of S. mansoni schistosomula was found to bind strongly antibodies from individuals infected with S. haematobium, demonstrating that most surface antigens are cross-reactive. Immunoprecipitation demonstrated, however, that of the polypeptide surface antigens only the very high molecular weight antigen was recognized by anti-S. haematobium antibodies and that the 38,000 to 32,000 and 20,000 Mr antigens were species-specific. Immunoprecipitation of the polypeptide antigens derived from purified adult surface membranes demonstrated recognition of the same 32,000, 25,000 and 20,000 Mr antigens recognized by chronically infected mice. Again these antigens were found to be species-specific.

Adult↗
PubMed1986

Repetitive DNA as a tool for the identification and comparison of nematode variants: application to Trichinella isolates.

DNA prepared from four isolates of Trichinella was compared by genomic DNA cross-hybridisation, by electrophoresis following restriction endonuclease digestion and by hybridisation studies using a cloned repetitive DNA sequence from T. spiralis. The DNA from T. spiralis, T. nelsoni and T. pseudospiralis isolates was distinct and the interrelationships of these isolates were inferred. In contrast to previous work on T. nativa and T. spiralis, our work suggests that these two isolates are very similar.

Animals↗
PubMed1986

Adult schistosome cDNA libraries as a source of antigens for the study of experimental and human schistosomiasis.

Protective immunity has been demonstrated in experimental schistosomiasis and is also believed to occur in man. It can be mediated by antibodies from infected animals or animals immunized with attenuated organisms. Recombinant Escherichia coli synthesizing antigenic polypeptides from the three principal species of schistosome that infect man, Schistosoma mansoni, S. japonicum and S. haematobium, have been constructed. Libraries of adult worm cDNA were prepared from each species in the expression vector lambda gt 11 and directly screened with antibodies from animals experimentally immunized with S. mansoni and S. japonicum and from humans infected with S. haematobium. The S. mansoni clones have been analysed in greatest detail. At least four different types of clones were identified. All the detected recombinant polypeptide antigens were recognised by antibodies from chronically infected mice and most were also recognised by antibodies from mice immunized with attenuated cercariae and anti-surface membrane antibodies. Clones synthesizing species-specific antigens for both S. mansoni and S. japonicum were identified by simultaneous screening of both libraries. At least three types of S. haematobium clones were identified by screening with human infection serum, most of which were species-specific. All the antigens were in the form of fusion peptides with E. coli beta-galactosidase and their expression was induced by isopropylthiogalactopyranoside. Since known protective monoclonal antibodies recognise highly glycosylated membrane proteins which cannot be identified in the form of nascent polypeptides, the direct identification of polypeptide antigens defined by their reactivity, as reported here, is an essential step in producing reagents by recombinant DNA technology, suitable for vaccination and diagnosis.

Animals↗
PubMed1986

Cloning of a major developmentally regulated gene expressed in mature females of Schistosoma mansoni.

A cDNA library constructed from RNA isolated from adult Schistosoma mansoni has been screened by differential hybridization to identify clones corresponding to genes highly expressed by female worms. Several such cDNAs encoding the same highly abundant mRNA species were identified. Studies with one of these (pSF10) which contained a 500 base pair insert demonstrated that this gene was not expressed in immature females or eggs and encoded a polypeptide of approximately 35 000 daltons. Quantitation of the levels of RNA showed that 10% of the total RNA of female parasites was homologous to pSF10. A single gene corresponding to pSF10 was identified in Southern blotting experiments using adult worm DNA. The cloning of this gene will facilitate study of the molecular and genetic events controlling female schistosome maturation.

Animals↗
PubMed1986

Lipid peroxidation in newborn rabbits: effects of oxygen, lipid emulsion, and vitamin E.

The extent of in vivo lipid peroxidation and the in vivo antioxidant effects of alpha-tocopherol and alpha-tocopheryl acetate were studied in newborn rabbits exposed to one of two oxidant stresses: hyperoxia (FIO2 greater than 0.9) or parenteral lipid emulsion infusion. Lipid peroxidation was monitored by measurement of expired ethane and pentane, tissue thiobarbituric acid (TBA) reactants, and tissue lipid peroxides. Seventy-two h of hyperoxia did not increase any of the parameters of lipid peroxidation although mortality was higher in oxygen exposed animals. alpha-Tocopherol (100 mg/kg, intravenous) lowered expired hydrocarbons and tissue TBA reactants, but raised liver lipid peroxides in both air and hyperoxia exposed pups. Infusion of soybean oil emulsion increased production of ethane and pentane, liver TBA reactants, and lung lipid peroxides. Both alpha-tocopherol and alpha-tocopheryl acetate prevented the soybean oil emulsion induced increase in volatile hydrocarbons. alpha-Tocopherol (100 mg/kg, intravenous) administration also prevented the increase in liver TBA reactants and lung lipid peroxides. In identically treated animals, alpha-tocopheryl acetate administration decreased liver TBA reactants but had no effect on lung lipid peroxides. We conclude that alpha-tocopherol reduces lipid peroxidation in newborn rabbits including animals exposed to hyperoxia or infused with lipid emulsions. alpha-Tocopheryl acetate results in lower tissue alpha-tocopherol concentrations and is less effective as an antioxidant in lipid emulsion infused rabbits.

Animals↗
PubMed1985

A comparison of peripheral and central effects of CCK8 on water-reinforced operant responding.

Systemic administration of cholecystokinin-octapeptide (CCK8) suppresses operant responding in water deprived rats, but it is unclear whether this effect is centrally or peripherally mediated. Rats were trained to press a lever for water in an operant conditioning chamber under a variable-interval schedule of reinforcement. After response rate stabilized injections were administered, and response suppression was measured by comparing injection response rate to baseline rate. Intracerebroventricular injections of CCK8 reduced lever pressing at relatively high doses (20 and 50 micrograms/rat). But in a direct comparison, the same dose of CCK8 (20 micrograms/rat) given intraperitoneally reduced responding significantly more than when given into the lateral ventricle. The suppressive effects of CCK8 (30 and 300 micrograms/kg i.p.) were significantly reduced by complete abdominal vagotomy. The effects of CCK8 (30 micrograms/kg i.p.) were blocked by pretreatment with the specific competitive CCK8 antagonist dibutyryl cyclic GMP (70 and 140 mg/kg i.p.), but not by the acetylcholine antagonist atropine (0.1 to 10 mg/kg i.p.). These data suggest that suppression by CCK8 of operant lever pressing in water-deprived rats is primarily mediated by vagal afferent fibers.

Animals↗
PubMed1985

Products of cholecystokinin (CCK)-octapeptide proteolysis interact with central CCK receptors.

Peptidases present in central nervous system (CNS) synaptic membranes, hydrolyze the neuroactive peptide cholecystokinin-octapeptide (CCK-8; Asp-Tyr-SO3H-Met-Gly-Trp-Met-Asp-Phe-NH2). In order to determine the pathway of degradation, synthetic CCK-8 was incubated at 37 degrees C with purified synaptic membranes; at various intervals reaction samples were removed from the reaction mixture and analysed by high-performance liquid chromatography to identify and quantify the peptide fragments. The results indicate an initial endopeptidase cleavage at the Met-Gly bond producing CCK-5 (Gly-Trp-Met-Asp-Phe-NH2). The carboxyl-terminal pentapeptide is further proteolysed to CCK-4 (Trp-Met-Asp-Phe-NH2) by a puromycin-sensitive aminopeptidase and to CCK-3 (Met-Asp-Phe-NH2) and Gly-Trp by an endopeptidase action. CCK-3 and CCK-2 appear to be relatively stable end-products. Moreover, these proteolytic fragments are shown to bind to the CCK receptor in brain with varying potencies.

Animals↗