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Biomedical subjects

R J Collier

Publications and source records attributed to R J Collier.

At least 271 records · Page 15Linked to original sources
PubMed1978

Chimeric toxins: toxic, disulfide-linked conjugate of concanavalin A with fragment A from diphtheria toxin.

A disulfide-linked conjugate of concanavalin A (Con A) and fragment A from diphtheria toxin has been synthesized and shown to be toxic for HeLa (human), Chinese hamster ovary (CHO), and SV3T3 (murine) cells. The conjugate was constructed by first coupling cystamine to Con A with a carbodiimide reagent and then reacting the modified Con A with reduced fragment A under conditions promoting disulfide interchange. The desired conjugate, obtained in nearly 50% yield relative to input of fragment A, was purified by affinity chromatography on Sephacryl S-200 and NAD-Sepharose; on analysis, it gave an average of 1.4 molecules of fragment A per tetrameric Con A molecule. The conjugate proved to be about equally active in inhibiting protein synthesis in HeLa, CHO, or SV3T3 cells in culture but was inactive relative to controls in a toxin-resistant strain of CHO cells containing altered elongation factor 2, the target protein of fragment A. With toxin-sensitive strains the conjugate was 100- to 1000-fold more active than controls, including fragment A, cystaminyl-Con A, and mixtures thereof, but was 1/50th to 1/500th as toxic as diphtheria toxin itself. Similar activity relative to controls was observed after intradermal inoculations in rabbits, and intravenous injections of the conjugate were lethal for mice. The activity of the conjugate in tissue culture was inhibited by Con A or alpha-methylmannoside but not by galactose. This and similar conjugates should be useful in studying mechanisms of entry of biologically active proteins into cells.

Cell Line↗
PubMed1978

Affinity filters, a new approach to the isolation of tox mutants of Vibrio cholerae.

We have devised a novel plate assay method for detecting mutants of Vibrio cholerae altered in the production of cholera toxin (tox mutants). Colonies replicated from a master plate are grown on the surface of a cellulose filter disc to which ganglioside-albumin conjugates have been attached. Toxin secreted by the colonies is tightly bound to the ganglioside filters. After removal of the cells by washing, the bound toxin may be detected by treating the filters with radioactively labeled antibodies against either whole toxin or one of its constituent polypeptide chains, followed by autoradiography. Colonies producing significantly greater of lesser amounts of toxin than the parental type are easily recognized and can be shown in liquid culture to have the corresponding hypertoxinogenic or hypotoxinogenic phenotype. This method, termed "the ganglioside filter assay," is applicable to screening large numbers of colonies and should facilitate isolation of various specific classes of mutants in cholera toxin production. In modified form the method will be applicable to various systems in which mutants of secreted proteins are sought.

Cholera Toxin↗
PubMed1978

Purification of cholera toxin and its subunits: new methods of preparation and the use of hypertoxinogenic mutants.

Cholera toxin was obtained in pure form by fractionation on two phosphocellulose columns successively. Cholera toxin and choleragenoid were quantitatively and selectively adsorbed to the first column in 10 mM phosphate buffer, pH 7.0, and were subsequently eluted with buffer of high ionic strength. The toxin was then separated from choleragenoid on the second column by chromatography at pH 8.3. The toxin obtained was highly active and pure as judged by electrophoresis, isoelectric focusing, and various immunological and chemical tests. Pure choleragenoid was by-product of the procedure. The A1 chain of the toxin was obtained in pure form by treating phosphocellulose-bound toxin with urea and a reducing agent. The anionic A1 peptide was thereby released, leaving a complex of the B and A2 chains (A25B) bound to the resin. The latter was then eluted and further purified to obtain nontoxic antigen. The overall yields of cholera toxin and choleragenoid were increased two- to threefold by the use of hypertoxinogenic mutants of Vibrio cholerae.

Biological Assay↗
PubMed1978

Regulation of cortisol uptake in mammary tissue of cows.

Mammary tissue explants from four nonlactating, nonpregnant cows were placed into culture with media containing various combinations of insulin, prolactin, growth hormone, 17beta-estradiol, dexamethasone, and progesterone. Combinations of insulin, prolactin, growth hormone, or 17beta-estradiol had no effect on cytoplasmic or nuclear uptake of tritiated cortisol compared with values at zero time. Combinations containing dexamethasone of progesterone reduced cytoplasmic and nuclear uptake of tritiated cortisol. To examine inhibition by progesterone of binding of tritiated cortisol, mammary tissue from each of four lactating, nonpregnant and four nonlactating, nonpregnant cows were placed in flasks containing tissue culture medium 199, tritiated cortisol (2 ng/ml), and progesterone at concentrations of 0, 10(-12), 10(-11), 10(-10), 10(-9), 10(-8), 10(-7), 10(-6), or 10(-5) X 6.4 M. Cytoplasmic uptake of tritiated cortisol into nonlactating tissue decreased linearly as progesterone increased, whereas tritiated cortisol uptake in lactating tissue did not decrease until progesterone exceeded 10(-7) M. We postulated progesterone is sequestered in milk fat of cytoplasm of lactating tissue whereas in nonlactating tissue progesterone is available to compete with cortisol at sites of cortisol binding.

Animals↗
PubMed1977

The mechanism of ADP-ribosylation of elongation factor 2 catalyzed by fragment A from diphtheria toxin.

Measurements of the initial rate of ADP-ribosylation of elongation factor 2 (EF-2) catalyzed by Fragment A from diphtheria toxin support a sequential mechanism and suggest that the reaction proceeds through a central ternary complex involving Fragment A and the substrates, EF-2 and NAD. The Michaelis constants for EF-2 and NAD are 0.15 and 1.4 muM, respectively. As determined by equilibrium gel permeation, EF-2 does not bind Fragment A significantly, alone or in the presence of adenine, ADPribose, nicotinamide or NADH. Based on these and earlier results, we propose an ordered sequential mechanism for the reaction; the sequence of binding of substrates is NAD, followed by EF-2.

Adenine↗
PubMed1977

Simple method for purifying choleragenoid, the natural toxoid of Vibrio cholerae.

Choleragenoid, a nontoxic aggregate of the B subunit of cholera toxin, has been purified from concentrated culture filtrates in a single step by ion-exchange chromatography on phosphocellulose or other cation-exchange resins. This procedure is far simpler than others currently used to isolate choleragenoid and yields a preparation essentially free from nucleic acid, lipopolysaccharide, toxin, and other proteins present in the crude culture filtrates. The purified choleragenoid retained the specific receptor-binding capacity of the toxin but exhibited no enterotoxic activity by either the ileal loop assay or the skin permeability assay. This purification methods may therefore be superior to others currently used for obtaining choleragenoid for immunization or other purposes.

Amino Acids↗
PubMed1977

Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa.

A nontoxic peptide (molecular weight, 26,000), which is active in catalyzing the adenosine diphosphate (ADP)-ribosylation of elongation factor 2, has been isolated from the culture supernatant of Pseudomonas aeruginosa strain 103 in stationary phase. Like fragment A from diphtheria toxin, the active peptide catalyzed the hydrolysis of nicotinamide adenine dinucleotide as well as the ADP-ribosylation of elongation factor 2 and showed similarities to fragment A in specific activity, kinetic constants, pH optimum, and ionic sensitivity. These results provide strong evidence for a high degree of homology in the structures of their active sites. That the peptide is not identical to fragment A is shown by the fact that it was not neutralized by fragment A-specific antiserum and was different in amino acid composition and pH and thermal labilities. Although definitive evidence is lacking, there are data suggesting that this peptide is a proteolytic fragment from the ADP-ribosylating toxin (exotoxin A; molecular weight, 66,000) produced by the same strain of P. aeruginosa.

Adenosine Diphosphate Sugars↗
PubMed1977

Lactogenesis in explant cultures of mammary tissue from pregnant cows.

The hormonal requirements for lactogenesis were investigated using explant cultures of mammary tissue obtained from cows at 30-40 days prepartum. Hormones used were insulin, hydrocortisone, and prolactin, and parameters examined were radioactive acetate incorporation into fatty acids, secretory response ratings, and histological and ultrastructural analysis. Data indicated that insulin was essential for mammary epithelial cell survival, but insulin alone did not result in the intiation of milk synthesis. The culture of explants in a medium containing insulin plus hydrocortisone resulted in alterations in the cytology of alveolar cells but no induction of milk synthesis. Biosynthesis results and secretory response ratings indicated that the initiation of milk synthesis occurred when explants were cultured in insulin and prolactin; however, synthesis was limited and alveolar integrity was not well maintained. Results from all parameters demonstrated that the maximal lactogenic response was obtained when the culture medium contained insulin, hydrocortisone and prolactin. After 48 h of culture in this medium, the rate of acetate incorporation into fatty acids had increased 3-fold and the markedly distended alveolar lumina contained many fat droplets and abundant eosinophilic staining secretion. However, an unusal amount of casein-like micelles and especially lipid also accumulated in the alveolar cells. The accumulation of milk components within the epithelial cells may be related indirectly to the accumulation of products in the lumina or perhaps related to a difference in the hormonal requirement between the initiation of milk synthesis and initiation of milk secretion.

Acetates↗
PubMed1976

Amino-acid sequence of fragment A, an enzymically active fragment from diphtheria toxin.

The amino-acid sequence of Fragment A from diphtheria toxin is reported. Fragment A (molecular weight, Mr, 21,145) is the major enzymically active fragment produced upon activation of the intact toxin (Mr about 60,000) by limited tryptic digestion and reduction. It, or a similar fragment, is believed responsible for the inhibition of protein synthesis in animal cells exposed to the toxin. Fragment A, which corresponds to the amino terminus of the toxin, is shown here to consist of three major forms (190, 192, and 193 residues) resulting from cleavage by trypsin adjacent to any of three closely spaced arginine residues. All three forms are enzymically active.

Amino Acid Sequence↗
PubMed1976

Effects of dietary caffeine on the testis of the domestic fowl, Gallus domesticus.

Roosters were fed 0-1% caffeine mixed by weight into a standard ration. With continued dietary caffeine administration, the average fertility of eggs collected for 2 weeks from untreated pullets inseminated with semen from the treated males at 0, 7 and 14 days after the start of treatment was 30-8, 33-5 and 3-3%, respectively. After 14 days of treatment fertility was significantly lower (P less than 0-001) than before (0 days) or 7 days after treatment. Semen output and sperm concentration were markedly reduced 17-21 days after treatment, and no semen could be collected from the roosters after they had received caffeine for 30 days. Removal of dietary caffeine resulted in resumption of semen production and a return of fertility to the control level. Testicular histology showed that spermatocyte divisions ceased and spermiogenesis was abnormal, although Leydig tissue and the response of the males to massage for semen collection was not affected. The effects on spermatogenesis and fertility were reversible after treatment for 30 days.

Animals↗
PubMed1976

Cellular studies of mammary tissue from cows hormonally induced into lactation: lactose and fatty acid synthesis.

Temporal changes in ability of mammary gland to synthesize lactose and fatty acids were identified during the treatment of cows hormonally induced to lactate, and animal differences were compared to subsequent milk production. Hormonal treatment involved 17 beta-estradiol + progesterone on days 1 to 7 and dexamethasone on days 17 to 19. Mammary tissue obtained by biopsy on days 0, 8, 16, and 26 of treatment was examined for biosynthetic capacity by tissue slice incubations. In terms of peak daily milk yield, one cow was very successful (greater than 30 kg), two were intermediate (9 to 10 kg), and one cow was unsuccessful (less than 3 kg). Differences between cows in the capability to synthesize lactose and fatty acids were evident as early as day 8 and were further magnified by day 16. In particular, the tissue from the successful cow was undergoing lactogenesis by day 8 while this was not evident until the day 16 biopsy sample in the less successful cows. In contrast to the other cows, tissues from the unsuccessful animal regressed in its ability to synthesize lactose and fatty acids between day 16 and 26. Relative differences between animals in measurements of metabolic capacity were consistent with subsequent milk production.

Acetates↗
PubMed1976

Cellular studies of mammary tissue from cows hormonally induced into lactation: histology and ultrastructure.

Cytological changes were investigated in mammary parenchyma of cows hormonally induced to lactate. Four cows were induced with injections of 17 beta-estradiol plus progesterone (days 1 to 7) and dexamethasone (days 17 to 19). Tissue biopsies were obtained on days 0, 8, 16, and 26; and milking commenced on day 18 or 21 of the treatment period. Mammary tissue of cow A, the most successful animal, showed marked cellular proliferation and differentiation by day 8. By day 16, the parenchyma of cow A had undergone lactogenesis and exhibited histological and ultrastructural characteristics associated with actively secreting mammary tissue. Mammary epithelia of cow D, the least successful animal, showed minimal development by day 8 and ultrastructurally resembled nondifferentiated mammary cells. Mammary parenchyma of cow D had not undergone differentiation and was involuting by day 16. Mammary tissues of cow B and C showed intermediate responses with only slight development by day 16 and only limited areas of secretory activity by day 26. Results suggest that animals successfully induced into lactation undergo critical periods of cellular proliferation and differentiation which unsuccessful animals do not experience. Cytological differences were first evident by day 8, further magnified by day 16 of the treatment period, and subsequently expressed in terms of milk production (peak milk yield equaled 30.5, 10.0, 9.2, and 2.4 kg/day for cows A, B, C, and D).

Animals↗