Search PubMed⌕ Search

Biomedical subjects

R J Collier

Publications and source records attributed to R J Collier.

At least 181 records · Page 10Linked to original sources
PubMed1989

Temporal sequence of changes to the gray squirrel retina after near-UV exposure.

Monocularly aphakic gray squirrels (Sciurus carolinensis) were exposed for 10 min to monochromatic near-ultraviolet radiation (lambda = 366 nm, radiant exposure = 4.3 J/cm2) to determine if their yellow pigmented lens protected retinal tissue from photochemical damage. Eyes were examined from 1 to 30 days after exposure to determine the temporal sequence of retinal damage and the extent of recovery from such exposures. Light microscopy of exposed aphakic retinas revealed irreversible lesions to the photoreceptors. Swelling of inner segments, accumulation of heavy pigment deposits in the PE, presence of macrophages in the subretinal space, and pyknosis of photoreceptor nuclei were observed in the exposed region of the aphakic eye. Eyes exposed to ultraviolet radiation with their lenses intact were devoid of significant retinal lesions. This study represents a model system for studying the potential damaging effects of near-UV radiation to the aphakic eyes of humans.

Animals↗
PubMed1988

Pseudomonas aeruginosa exotoxin A: effects of mutating tyrosine-470 and tyrosine-481 to phenylalanine.

Directed mutagenesis was used to probe the functions of Tyr-470 and Tyr-481 of Pseudomonas aeruginosa exotoxin A (ETA) with respect to cytotoxicity, ADP-ribosylation of elongation factor 2 (EF-2), and NAD-glycohydrolase activity. Both of these residues lie in the active site cleft, close to Glu-553, a residue believed to play a direct role in catalysis of ADP-ribosylation of EF-2. Substitution of Tyr-470 with Phe caused no change in any of these activities, thus eliminating the possibility that the phenolic hydroxyl group of Tyr-470 might be directly involved in catalysis. Mutation of Tyr-481 to Phe caused an approximately 10-fold reduction in NAD:EF-2 ADP-ribosyltransferase activity and cytotoxicity but no change in NAD-glycohydrolase activity. The latter mutation did not alter the KM of NAD in the NAD-glycohydrolase reaction, which suggests that the phenolic hydroxyl of Tyr-481 does not participate in NAD binding. We hypothesize that the phenolic hydroxyl of Tyr-481 may be involved in the interaction of the toxin with substrate EF-2.

ADP Ribose Transferases↗
PubMed1988

Restoration of enzymic activity and cytotoxicity of mutant, E553C, Pseudomonas aeruginosa exotoxin A by reaction with iodoacetic acid.

Pseudomonas aeruginosa exotoxin A (ETA) is inactivated greater than 1,000-fold when an active site glutamic acid, E553, is mutated to aspartic acid (Douglas, C.M., and Collier, R. J. (1987) J. Bacteriol. 169, 4967-4971). To test the effect of creating a carboxyl-containing side chain at position 553 longer than that of glutamic acid, we first replaced Glu-553 with cysteine by site-directed mutagenesis of cloned ETA and then carboxymethylated the cysteine side chain with iodoacetic acid. The E553C mutation reduced ADP-ribosyltransferase and cytotoxic activities greater than 10,000-fold. Reaction of the mutant with iodoacetic acid enhanced enzymic activity 2,500-fold, to a level approximately one-sixth that of wild type toxin, and restored cytotoxicity to a slightly lesser extent. Iodoacetamide did not activate the mutant, and neither iodoacetic acid nor iodoacetamide affected the activity of wild type toxin. These results show that the carboxyl group of Glu-553 is important for ADP-ribosylation activity and imply flexibility in the enzyme-substrate complex in accommodating the slightly longer S-carboxymethylcysteine side chain. This general approach may have applications in protein engineering as well as in studying carboxyl side chain functions in enzymes.

ADP Ribose Transferases↗
PubMed1988

Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I.

Escherichia coli Shiga-like toxin I, a close relative of Shiga toxin and a distant relative of the ricin family of plant toxins, inhibits eukaryotic protein synthesis by catalyzing the depurination of adenosine 4324 in 28S rRNA. By comparing the crystallographic structure of ricin with amino acids conserved between the Shiga and ricin toxin families, we identified seven potential active-site residues of Shiga-like toxin I. The structural gene encoding Shiga-like toxin I A chain (Slt-IA), the enzymatically active subunit, was engineered for high expression in E. coli. Oligonucleotide-directed mutagenesis of the gene for Slt-IA was used to change glutamic acid 167 to aspartic acid. As measured by an in vitro assay for inhibition of protein synthesis, the specific activity of mutant Slt-IA was decreased by a factor of 1000 compared to wild-type Slt-IA. Immunoblots showed that mutant and wild-type Slt-IA were synthesized as full-length proteins and were processed correctly by signal peptidase. Both proteins were equally susceptible to trypsin digestion, suggesting that the amino acid substitution did not produce a major alteration in Slt-IA conformation. We conclude that glutamic acid 167 is critical for activity of the Shiga-like toxin I A chain and may be located at the active site.

Bacterial Toxins↗
PubMed1988

Amino acid sequence homology between the enzymic domains of diphtheria toxin and Pseudomonas aeruginosa exotoxin A.

Despite similarities in their enzymic properties, diphtheria toxin (DT) and exotoxin A (ETA) of Pseudomonas aeruginosa have major differences in structure and action: consequently, the question of possible evolutionary relatedness of these two proteins remains unanswered. Here we report the existence of significant amino acid sequence homology between the enzymic domain of DT and that of ETA. Major segments of sequence may be aligned with high percentages of identity and of conservative substitutions. The homologous stretches in ETA form much of the active-site cleft in the X-ray crystallographic structure. This evidence implies that these domains, at least, have diverged from a common ancestral protein and that active-site residues have been strongly conserved.

ADP Ribose Transferases↗
PubMed1988

Toxoid of Pseudomonas aeruginosa exotoxin A generated by deletion of an active-site residue.

Glutamic acid-553 of Pseudomonas aeruginosa exotoxin A (ETA), identified previously as an active-site residue, was deleted by oligonucleotide-directed mutagenesis of the cloned toxin gene in Escherichia coli. The purified mutant toxin was stable, fully immunoreactive, and capable of blocking toxin receptors. ADP-ribosyltransferase and cytotoxic activities were at least 10(6)-fold lower than those of wild-type ETA, and injection of mice with 50 micrograms (equivalent to 400 lethal doses of ETA) produced no ill effects. The mutant toxin elicited high levels of neutralizing anti-ETA antibodies in mice, which protected against a challenge with 100 micrograms of authentic ETA (greater than 600 lethal doses). The mutant protein has the attributes of a toxoid and may be useful as a component of vaccines for individuals at risk for infection by P. aeruginosa.

ADP Ribose Transferases↗
PubMed1988

Both type I and II insulin-like growth factor receptor binding increase during lactogenesis in bovine mammary tissue.

Bovine GH is a potent stimulant of lactation, and the insulin-like growth factors I and II (IGF-I and -II) are believed to mediate GH's growth-promoting actions. Since all of IGF's known actions are mediated through its receptor subtypes, we analyzed the distribution of IGF receptor subtypes in lactating and nonlactating bovine mammary tissue. Analysis of competition curves showed that IGF-I had greater potency than IGF-II in competing with [125I]IGF-I for binding to membranes prepared from both lactating and nonlactating animals. An insulin concentration of 4 micrograms/ml displaced less than 40% of the [125I]IGF-I bound to membranes prepared from both lactating and nonlactating animals, indicating that a high percentage of [125I]IGF-I was bound to the type II receptor. Lactation was associated with an increase in the total amount of [125I]IGF-I bound, and this change was due to an increase in binding to both receptor subtypes. Specifically, membranes prepared from lactating animals had a 3-fold increase in binding competed for by insulin and a 2-fold increase in binding not competed for by insulin. Affinity cross-linking of [125I]IGF-I to membranes prepared from both lactating and nonlactating animals, followed by polyacrylamide gel electrophoresis (PAGE) and autoradiography, showed that 260K and 135K bands were present. Competition experiments indicated that unlabeled IGF-I effectively competed for binding to the 260K band, whereas insulin did not. Binding to the 135K band could be inhibited by both IGF-I and insulin. The intensity of the labeled bands showed that type II receptors were relatively more abundant than type I receptors in membranes from both lactating and nonlactating animals. Membranes prepared from lactating animals showed both 135K and 127K species of the type I receptor, whereas nonlactating animals showed only the 135K band. We conclude that type I and II receptors are present in bovine mammary tissue, and type II predominate. Lactation is associated with increases in the concentration of both receptor subtypes, especially type I receptors. Lactation may be associated with structural changes in the type I receptor. These changes in receptor distribution could play a role in modulating the physiological effects of the IGFs on mammary tissue.

Animals↗
PubMed1988

Effects of thyroxine and growth hormone treatment of dairy cows on milk yield, cardiac output and mammary blood flow.

Four cows received thyroxine injections (T4; 20 mg/d) and three cows received growth hormone injections (GH; 44 mg/d) for 4 d during successive 16-d experimental periods. Measurement was made of milk yield, protein yield, mammary tyrosine and phenylalanine uptake, blood plasma hormone concentrations, mammary blood flow and cardiac output. Milk yield increased by 25% with T4 and 21% with GH treatment. Milk protein content tended to decline during T4 treatment and increase following GH treatment. Cardiac output increased by 8.9 liter/min (20%) and 4.6 liter/min (10%) with T4 and GH injection. Mammary blood flow (half-udder) increased from 3.6 to 4.9 liter/min (35%) and from 3.3 to 4.4 liter/min (33%) with T4 and GH treatment, respectively. These increases calculated on a whole-udder basis, accounted for 28% (T4) and 48% (GH) of the increases in cardiac output. The proportion of cardiac output perfusing the (whole) udder increased to 19.1% (T4) and 18.7% (GH), increases of 17 and 30%, respectively. Heart rate increased with T4 (but not GH treatment) from 80 to 115/min. Ratio of blood flow to milk yield was not changed by either treatment. The proportion of cardiac output perfusing the udder likely plays a major role in facilitating the partitioning of nutrients for milk synthesis.

Animals↗
PubMed1988

Effects of thyroxine and growth hormone treatment of dairy cows on mammary uptake of glucose, oxygen and other milk fat precursors.

Four cows received thyroxine injections (T4; 20 mg/d) and three cows received growth hormone injections (GH, 44 mg/d) for 4 d during successive 16-d experimental periods. Milk fat, lactose output, mammary uptake of glucose, oxygen and milk fat precursors were determined with each treatment. Injection of T4 increased lactose yield by 25% and fat yield 42%. The injection of GH increased fat and lactose yields by 24%. Both GH and T4 increased mammary glucose uptake by 35% and 45%, respectively, while T4 administration was associated with an increase in plasma glucose concentration from 67 to 84%. Thyroxine, but not GH, increased the ratio of mammary glucose uptake to lactose output from 1.24 to 1.58. Blood plasma acetate concentration declined following GH and T4 treatment by 17%. Mammary acetate uptake increased in response to GH injection in two of three cows but did not change with T4 injection. The injection of GH had no effect on plasma propionate concentration or mammary uptake. Thyroxine reduced plasma propionate content and mammary uptake. Neither T4 nor GH changed plasma free fatty acid concentration or mammary uptake. Thyroxine had no effect on plasma triglyceride concentration or mammary uptake, whereas GH increased mammary triglyceride uptake to the end of the experimental period. Mammary oxygen uptake was increased by GH as milk production increased. Increased mammary oxygen uptake following T4 treatment was transient. Change in mammary metabolism with T4 treatment permitted increased milk output without change in mammary oxygen consumption. Such a change may involve increased mammary utilization of pre-formed long-chain fatty acid and increased metabolism of glucose via glycolysis.

Animals↗
PubMed1988

Interactions of climatic factors affecting milk yield and composition.

Objectives were to evaluate effects of interactions of maximum temperature, minimum relative humidity, and solar radiation on milk yield and constituent traits. Effects of climate variables and their interactions were significant but small in most cases. Second order regression models were developed for several variables. Six were examined in detail: Holstein and Jersey milk yields, Holstein fat and Feulgen-DNA reflectance percent, and Jersey protein percent and yield. Maximum temperature had greatest influence on each response, followed by minimum relative humidity and solar radiation. Optimum conditions for milk production were at maximum temperatures below 19.4 degrees C, increasing solar radiation, and minimum relative humidity between 33.4 and 78.2% (cool sunny days, moderate humidity). Maximum Holstein fat percent of 3.5% was predicted for maximum temperatures below 30.8 degrees C, minimum relative humidity below 89%, and solar radiation below 109 Langleys; actual mean Holstein fat percent was 3.35%. Optimum climatic conditions for Jersey protein percent were at maximum temperature of 10.6 degrees C with solar radiation at 300 Langleys and relative humidity at 16% (cool sunny days, low humidity). Because noteworthy interactions existed between climate effects, response surface methodology was suitable for determining optimum climatic conditions for milk production.

Animals↗
PubMed1988

Climatic effects on occurrence of clinical mastitis.

Clinical mastitis records for 6.5 yr from a large north Florida dairy and corresponding daily weather data were analyzed. Monthly incidence of clinical mastitis was expressed as percent of cow-days in milk and graphed against monthly average daily maximum temperature humidity index values and monthly total rainfall. No trends were evident with rainfall. In 3 of 6 yr, monthly incidence of clinical mastitis increased more than 50% above annual incidence, and this followed high monthly temperature-humidity values. Least squares was used to estimate regression coefficients of temperature-humidity index categories based on 999,969 Holstein records. A temperature humidity index category represented the number of days used to calculate average daily maximum temperature-humidity index value. Sources of variation in observed occurrence of clinical mastitis were cow, parity, month, year, interaction of parity by month, and continuous effects of temperature-humidity index categories 2, 6, 15, 30, 60, each to third order and 60 by parity interaction. In all temperature-humidity index categories as the temperature-humidity index value increased, occurrence of clinical mastitis increased. When values rose from 55 to 80, twice as many for 2 d cows showed signs of clinical mastitis.

Animals↗
PubMed1987

Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin.

Photoaffinity labeling with native NAD, a method employed earlier with diphtheria toxin (DT), was used to identify an active site residue of Pseudomonas aeruginosa exotoxin A (ETA). An enzymically active fragment (Mr 27,000), derived by partial digestion of ETA with thermolysin, was irradiated with ultraviolet light (254 nm) in the presence of various radiolabeled preparations of NAD. Label from the nicotinamide moiety was efficiently transferred to the protein (maximally 0.79 mol/mol), and the label was exclusively located at position 553. This position, like that photolabeled in DT (position 148), corresponds to glutamic acid in the native protein. Chromatographically identical photo-products were generated at these positions in the two toxins. Glu-553 lies in a cleft in domain III that is believed to represent the active site of ETA, and other evidence supports the notion that Glu-553 of ETA and Glu-148 of DT are directly involved in catalysis. When Glu-553 of ETA was aligned with Glu-148 of DT, we found similarities of local primary structure not detected earlier. These results suggest that the catalytically active domains of ETA and DT may be evolutionarily related, and they provide information that should prove useful for preparing vaccines against ETA by recombinant DNA methods.

ADP Ribose Transferases↗
PubMed1987

Non-invasive method of measuring resonant frequency of a human tibia in vivo. Part 1.

We describe a method for measuring resonant frequency of a human tibia in vivo. Although the main reason for this measurement is the assessment of a fractured tibia, we emphasize the special instrumentation required; in particular the novel techniques which are used to overcome the difficulty of obtaining results through skin and soft tissue.

Acoustics↗
PubMed1987

Covalent coupling of bovine growth hormone to its receptor in bovine liver membranes.

The structure of bovine somatotropin receptor was examined following covalent coupling of iodinated recombinant bovine growth hormone ([125I]rbGH) to bovine liver membrane receptors using ethylene glycol bis(succinimidyl succinate). Iodinated rbGH was incorporated into a complex of estimated Mr of 140,000 under reducing conditions. Excess unlabeled rbGH, but not bovine prolactin (bPRL), inhibited completely the incorporation of [125I]rbGH into the Mr = 140,000 species. In dairy bulls, the Mr = 140,000 complex was undetectable soon after birth but became predominant at 6 months of age. No evidence was found to support presence of bPRL receptors in steer liver membranes. Assuming a 1:1 stoichiometry of hormone binding to receptor, it appears that bGH binds to a major receptor subunit of Mr = 119,000 which does not recognize bPRL.

Animals↗