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Biomedical subjects

R J Collier

Publications and source records attributed to R J Collier.

At least 235 records · Page 13Linked to original sources
PubMed1983

Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage beta.

A 1,942-base-pair DNA segment encoding the structural gene for diphtheria toxin was sequenced, and the primary structure of the toxin was deduced. Restriction enzyme fragments corresponding to nontoxic or hypotoxic peptides of the toxin were isolated from corynebacteriophage beta and cloned into Escherichia coli on plasmid pBR322, and the sequence was determined. The mature toxin molecule deduced from the sequence has 535 amino acid residues and a molecular weight of 58,342. The deduced sequence for the fragment A moiety was the same as that determined at the protein level, except for a single serine residue, which had been mispositioned in the earlier study. Several differences were noted with respect to the partial sequence data available on the fragment B moiety, some or all of which may reflect genetic variations among populations of corynephages carrying the toxin gene. The DNA sequence predicts a 25-residue leader peptide preceding the mature protein, which is presumably involved in secretion of the toxin from lysogenized Corynebacterium diphtheriae. We infer that initiation of translation probably occurs at a GTG codon (codon -25). Cloned restriction fragments containing sequences for the amino-terminal region of toxin, together with 5' flanking regions, were expressed in E. coli. Toxin-related peptides were synthesized and secreted into the periplasmic space. These results provide a basis for applying recombinant DNA methods to the study of diphtheria toxin and for producing novel, genetically altered forms of the toxin suited to the construction of new classes of immunotoxins.

Amino Acid Sequence↗
PubMed1983

Molecular cloning and expression of gene fragments from corynebacteriophage beta encoding enzymatically active peptides of diphtheria toxin.

Two restriction fragments from corynebacteriophage beta vir tox+ that encode peptides similar to diphtheria toxin fragment A and the chain termination fragment, CRM45, have been cloned into Escherichia coli in plasmid pBR322. Clones containing the recombinant plasmids produced gene products that were active in catalyzing the ADP ribosylation of elongation factor 2 and were reactive with diphtheria toxin antiserum. Toxin-related peptides were found primarily in the periplasmic compartment and were degraded to nonimmunoreactive forms within 1 to 2 h of synthesis. The expression of both gene fragments appears to have originated from the diphtheria toxin promoter.

Bacteriophages↗
PubMed1983

Climatological and genetic effects on milk composition and yield.

Results confirm most other research on milk composition and yield. All responses were affected by climate, some considerably more than others, if percent of error variance is the criterion. Jersey yields were less sensitive to climate than were Holstein, but Jersey milk composition appeared more sensitive. Somatic cell count (REF), a measure of mastitis, was affected by climate but less than all other variables except protein/fat and LM%. Needed are estimates of interactions between climatic effects and response surface models to quantify possible improvement in performance following environmental modification. Genetic correlations between milk yield and REF and chloride % suggest that single trait selection for milk yield might increase incidence of mastitis although phenotypic correlations indicate that high yields and absence of mastitis are correlated.

Animals↗
PubMed1982

X-ray grade crystals of diphtheria toxin.

Diphtheria toxin, complexed with the endogenous dinucleotide ApUp, has been crystallized under novel conditions involving high concentrations of both polyethylene glycol and salt. Among four crystal forms obtained, one was found suitable for high resolution structural analysis by x-ray diffraction. This form is triclinic (space group P1) with 2 molecules per unit cell; unit cell parameters are a = 70.8 A, b = 70.7 A, c = 65.3 A, alpha = 95.2 degrees, beta = 91.3 degrees, gamma = 99.7 degrees.

Crystallization↗
PubMed1982

From Pap to ApUp.

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Adenosine Triphosphate↗
PubMed1982

The mechanical resonances of a human tibia: part I--in vitro.

This paper represents the beginning of a research programme whose aim is to develop a practical method of monitoring the healing progress of fractures. The objectives of the following are to identify the various resonances, and to show the types of modes which exist in long bones in vitro. A model has been evaluated to predict the tibial resonance frequencies. It is composed of a homogeneous, hollow beam with the constant cross section of an isosceles triangle. Such a beam can explain the two main types of fundamental transverse mode along with their triple resonances. The predicted resonances of the transverse waves are less than 5.8% in error in comparison to the measured values. This error increases to 23.8% for the torsional waves due to their different characteristics.

Biomechanical Phenomena↗
PubMed1982

Effects of heat stress during pregnancy on maternal hormone concentrations, calf birth weight and postpartum milk yield of Holstein cows.

For an evaluation of the effects of heat stress during pregnancy on calf birth weight and postpartum maternal milk yield, 21 cows and 10 heifers were assigned to shade (S; n = 16) or no shade (NS; n = 15) treatments during the last trimester of pregnancy in June 1978. At parturition, all cows were removed from treatment and uniformly managed in the milking herd. At 4-d intervals from d 199 of pregnancy to parturition, respiration rates, rectal temperature, heart rate and Black Globe temperature were recorded and blood samples obtained via tail venipuncture between 1300 and 1600 h. Weekly prepartum body weights of dam, birth weight of calf and subsequent daily milk yield were recorded. Black Globe temperature, rectal temperature and respiration rates were higher in cows given NS. Calf birth weight was lower in the NS group. Milk yield was correlated in a linear manner with calf birth weight, and cows in group NS exhibited reduced lactation performance after calving. Plasma progestin concentrations were higher in heat-stressed cows (6.0 vs 5.1 ng/ml). Estrone-sulfate concentrations were reduced in plasma of NS cows (2,505 vs 4,433 pg/ml). Thus, hormone concentrations of maternal and fetal origin were altered by environment. Plasma thyroxine concentrations were lower in NS cows (51.2 vs 66.4 ng/ml), while plasma triiodothyronine concentrations were elevated (1.8 vs 1.5 ng/ml), indicating altered thyroid hormone metabolism in heat-stressed cows. Heat stress altered endocrine dynamics during pregnancy and reduced calf birth weight and may have indirectly altered subsequent milk yield.

Animals↗
PubMed1982

Nucleic acid, metabolic and histological changes in gilt mammary tissue during pregnancy and lactogenesis.

Changes in mammary gland histology, dry weights, nucleic acids and in vitro rates of substrate oxidation in incorporation into lipid were measured in mammary biopsies of three gilts each on d 30, 45, 60, 75, 90, 105 and 112 of pregnancy, and d 1 and 4 of lactation. Histological changes noted were progressive duct growth early in pregnancy followed by rapid lobulo-alveolar development between d 75 and 90 to complete mammogenesis. Colostrum and lipid were evident by d 105 with marked distension of alveolar lumina on d 112. Complete differentiation of the secretory process was apparent on the day of parturition. Concentrtion of dry, fat-free tissue (DFFT) and DNA changed little before d 60 but increased fourfold between d 75 and 90. No further increases in DFFT or DNA were noted. RNA concentrations paralleled DNA through d 90, after which they steadily increased. Rates of acetate and glucose oxidation increased transiently during midpregnancy then declined and remained low until initiation of lactogenesis. Substrate incorporation into lipid increased slightly at midpregnancy and again at d 105, after which it increased markedly. Collectively, results indicate that mammogenesis is complete by d 90, after which lactogenesis is initiated in a two-stage process. Metabolic rates expressed on a DNA basis indicated considerable rates of oxidation, but not of lipogenesis by proliferating mammary tissue. Preferential metabolism of acetate vs glucose near parturition suggests coordination of metabolism between the mammary gland and other maternal tissues.

Acetates↗
PubMed1982

Influences of environment and its modification on dairy animal health and production.

Physiological state of dairy animals is a predisposing factor in environmental influences on animal health. Critical phases of life cycle include neonatal period, postpubertal reproduction, and lactation. Primary effect of environment in neonatal period is increased disease incidence associated with reduced immunoglobulin content in plasma of calves. Cold stress has little effect on reproduction; in contrast, heat stress reduces libido, fertility, and embryonic survival in cattle. Heat stress in late gestation reduces fetal growth and alters endocrine status of the dam. Carryover effects of heat stress during late gestation on postpartum lactation and reproduction also are detectable. Heat stress of lactating cattle results in dramatic reductions in roughage intake and rumination. Decreases in roughage intake contribute to decreased volatile fatty acid production and may contribute to alteration in ratio of acetate/propionate. Rumen pH also declines during thermal stress. Electrolyte concentrations, in particular sodium and potassium, also are reduced in rumen fluid of heat stressed cattle. The decrease in sodium and potassium are related to increases in loss of urinary sodium and loss of skin potassium as well as decline in plasma aldosterone and increase in plasma prolactin. Reduction in thyroxine, growth hormone, and glucocorticoid concentrations in chronically heat stressed cattle appear to be related to decreases in basal metabolism.

Adaptation, Physiological↗
PubMed1981

Characterization of hybrid molecules containing fragment A from diphtheria toxin linked to concanavalin A or the binding subunit of ricin toxin.

Earlier we described the synthesis of a toxic, disulfide-linked conjugate between Fragment A from diphtheria toxin (DTA) and the lectin concanavalin A (Con A). Here we report further characterization of this Con A-SS-DTA conjugate and of a nontoxic conjugate containing DTA linked through a disulfide bridge to the binding subunit of ricin toxin, RTB. The Con A-SS-DTA conjugate preparation was heterogeneous, and contained both divalent and tetravalent forms of Con A linked to DTA. The divalent form of the conjugate, which lacked hemagglutination activity, was as toxic as the tetravalent form for 3T3 cells. Consistent with this result, Con A-SS-DTA analogs prepared with the chemically modified dimeric derivatives of Con A, succinyl-Con A and acetyl-Con A, were as toxic as DTA disulfide linked to tetravalent Con A. Two analogs of Con A-SS-DTA containing nonreducible intermolecular linkages between DTA and Con A were at least 1000-fold less toxic than Con A-SS-DTA, although they were indistinguishable from unmodified Con A in binding to 3T3 cells. This suggests that reduction of the disulfide bridge between Con A and DTA may be necessary for conjugate toxicity. Con A-SS-DTA was equally toxic for a variety of cell lines varying in sensitivity to diphtheria toxin, including Vero, CHO, HeLa, and 3T3. The conjugate was also toxic for a diphtheria toxin-resistant variant of V79 cells (V79 Dtxr-3) that has lower affinity than wild type cells for diphtheria toxin. Chloroquine, colchicine, cytochalasin B, and ammonium chloride had no effect on Con A-SS-DTA toxicity for Vero cells, although ammonium chloride and chloroquine inhibited diphtheria toxin action.

Animals↗
PubMed1981

An endogenous dinucleotide bound to diphtheria toxin. Adenylyl-(3',5')-uridine 3'-monophosphate.

Diphtheria toxin has recently been fractionated into two forms, one of which contains tightly but noncovalently bound nucleotide-like material. Here we report identification of the major nucleotide constituent (comprising 80% of the total extractable ultraviolet-absorbing material) as adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). Upon incubation with ApUp, the nucleotide-free form of toxin bound approximately one molar equivalent of the dinucleotide and was converted to a form similar or identical with the native nucleotide-bound form.

Adenosine Monophosphate↗
PubMed1981

Hormonal responses of lactating dairy cattle to TRH and ACTH in a shade management system within a subtropical environment.

Lactating cows (64) were balanced by breed (54 Holstein and 10 Jersey) and assigned randomly to shade (S) or no shade (NS) management treatments for a continuous 20 wk trial beginning 5-5-76. A sub-sample of Holstein cows, five S and five NS, were fitted with jugular catheters 84 days after initiation of experiment. Thyrotropin Releasing Hormone (TRH; 100 mug) was administered intravenously at 1200 h to evaluate prolactin responses. Two days later each cow received intravenously 200 IU of ACTH at 1100 h to compare acute corticoid responses to ACTH. Mean prolactin response to TRH was greater for NS cows (291 vs 169 ng/ml; P < .01) as was peak plasma concentrations at 20 min (467 vs 267 ng/ml; P < .01). Mean corticoid response to ACTH injection was less for NS cows (52 vs 70 ng/ml; P < .10). Corticoid concentrations of plasma in both treatments had declined 65% by 7 h postinjection. These endocrine differences may be associated with thermoregulation and/or metabolic adjustments of cows exposed to different environmental systems of management during a seasonal period of thermal stress.

Journal Article↗
PubMed1981

Chromogenic detection of antigen in bacteriophage plaques: a microplaque method applicable to large-scale screening.

We have developed a simple and rapid (24 h) enzyme-linked immuno-detection method to screen for rare antigen-positive phage among large numbers of antigen-negative ones. Horse-radish peroxidase-antibody conjugate, incorporated into the soft agar layer of a plaque assay system, is precipitated locally by antigen produced during plaque formation, and is detected by standard chromogenic methods. The method has been used to screen plaques of bacteriophage beta tox+ for the presence of diphtheria toxin and related cross-reacting material. When phage were plated on very dense bacterial lawns, they formed minute plaques (microplaques). Because of the high local concentration of antigen generated by lysis of the dense lawn, the microplaques gave more intense chromogenic signals than larger plaques formed on less dense Corynebacterium diphtheriae lawns. Thus, antigen-positive microplaques could be easily recognized even in the presence of very large numbers of antigen-negatives. In a reconstruction experiment, small numbers of antigen-positive phage were detected with high efficiency (greater than 75%) against a background of 3.8 X 10(4) antigen-negatives/cm2 of agar surface (equivalent to 2.4 X 10(6) plaques/9 cm petri plate). This screening method should facilitate isolation of phage mutants affecting production of given antigens and may be of particular value in detecting specific genes cloned into phage vectors.

Antigens↗
PubMed1981

Appendix: a model of plaque formation.

Equations describing plaque formation in soft agar have been based on certain simplifying assumptions, for which data are presented. The derived equations permit one to calculate (i) average plaque size as a function of the initial density of indicator cells (Do), (ii) the number of cells lysed per plaque as a function of Do, and (iii) the cumulative number of cells lysed at various stages of plaque development. The calculated values agree well with those determined experimentally.

Bacteriophages↗
PubMed1981

Periparturient endocrine changes of conceptus and maternal units in Jersey cows bred for milk yield.

Control cows, sired by and bred to bulls with assumed zero estimated breeding values, and selected cows, sired by and bred to bulls of high predicted difference for milk yield, were used. Blood samples were collected via jugular venipuncture on alternate days from 35 to 14 days prepartum, from 14 to 28 days postpartum, and daily from 14 days prepartum to 14 days postpartum. We examined blood hematocrit, and concentrations in plasma of protein, estrone, estradiol, estrone sulfate, progestins, glucocorticoids, luteinizing hormone, and prolactin. Total plasma volume was measured on days -21, -7, and +11. Weekly body weights, prepartum and postpartum, were recorded. All data were analyzed by least squares analysis of variance with day and hematocrit as continuous independent variables. Hematocrit was higher for selected cows throughout the sampling period. Prepartum concentrations of progesterone were higher in selected cows, but concentrations of estrone, luteinizing hormone, and prolactin were lower than in control cows. Hormonal and physiological responses indicated that selection for milk yield influenced both the conceptus and maternal units as measured by prepartum endocrine function.

Animals↗
PubMed1981

Periparturient and postpartum endocrine changes of conceptus and maternal units in Jersey cows bred for milk yield.

Control cows, sired and bred by bulls of zero estimated breeding value, and selected cows, sired by and bred to bulls of high predicted differences for milk yield, were used to evaluate maternal endocrine changes from 14 days prepartum to 28 days postpartum. Examined were concentrations in plasma of 13, 14 dihydro-15 keto-prostaglandinF2 alpha, progesterone, estrone sulfate, estrone, luteinizing hormone, and prolactin. Ability of cows to release prolactin and luteinizing hormone on day 10 postpartum was evaluated after a simultaneous injection of thyrotropin releasing hormone (100 microgram) and gonadotropin releasing hormone (100 microgram). Changes in progesterone and estrogens prepartum lead to peak concentrations of prolactin and prostaglandin at parturition and 3 days postpartum, respectively. Higher basal concentrations of prolactin for control cows prepartum were associated with a higher prolactin release by thyrotropin releasing hormone at 10 days postpartum. Although release of luteinizing hormone in response to gonadotropin releasing hormone did not differ between groups on day 10 postpartum, a subsequent increase in progesterone to above 1 ng/ml was earlier and more precisely synchronized among control cows (16 +/- .43 versus 23 +/- 2.33 days). Within cow concentrations of F2 alpha 13, 14 dihydro-15-keto-prostaglandin-F2 alpha were correlated with size of previous gravid uterine horn (.67) and milk yield (-.39). Selection for milk yield influenced postpartum endocrine function.

Animals↗