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Biomedical subjects

R A Hart

Publications and source records attributed to R A Hart.

At least 19 recordsLinked to original sources
PubMed1995

The spread of Corynebacterium pseudotuberculosis infection to unvaccinated and vaccinated sheep.

The decrease in the prevalence of Corynebacterium pseudotuberculosis after two generations of vaccination against the disease it causes, was used to estimate the rate of control of caseous lymphadenitis (CLA). Three groups of 150 sheep, of which 50 in each group were artificially infected with C pseudotuberculosis and 100 in each group were uninfected sheep, were run separately for 40 months and shorn 5 times to promote the spread of CLA. One lot of 50 infected sheep and 2 lots of 100 uninfected sheep were vaccinated against CLA. The rate of spread of CLA was recorded. Sheep vaccinated against CLA and naturally exposed to infection had a 74% lower infection rate than unvaccinated sheep. Sheep vaccinated against CLA and exposed to only vaccinated infected sheep had a 97% lower infection rate. Unvaccinated sheep had a 76% infection rate, with 77% of the transmission occurring at the 4th and 5th shearings, without any discharging CLA abscesses being observed. This study supports the view that in Australian wool producing flocks, CLA spreads mainly from sheep with discharging lung abscesses to sheep with shearing cuts. Vaccinated sheep infected with CLA have 96% fewer lung abscesses compared with unvaccinated infected sheep and are therefore less likely to spread this disease to other sheep.

Animals↗
PubMed1995

Use of multifactorial analysis to develop aqueous two-phase systems for isolation of non-native IGF-I.

A high yield procedure was developed to solubilize and extract IGF-I from recombinant E. coli by adding chaotrope and disulfide reductant to alkaline fermentation broth. To enhance centrifugation performance and recovery yield, a salt/polymer aqueous two-phase extraction procedure was developed whereby soluble non-native IGF-I and biomass solids are enriched in separate liquid phases. To develop this extraction system a multifactorial experimental approach was used to simultaneously map the phase diagram and identify conditions to suitably partition IGF-I and cell remnants. The presence of urea in these systems tended to disrupt two-phase formation and solids sedimentation. This, in turn, constrained the concentrations of phase forming solutes which could be effectively used. Systems containing low levels of salt (less than about 4% w/w) and polymer (less than about 10% w/w) did not form two phases. Systems containing high levels of salt (greater than about 7% w/w) and polymer (greater than about 18% w/w) formed two phases with floating solids. Intermediate levels of salt (between about 4% and 7% w/w) and polymer (between about 10% and 18% w/w) formed two phases in which solids were enriched in the heavy phase. Systems in this latter desired category were produced with a variety of different salts and polymers and all enriched non-native IGF-I in the light phase. Highest recovery yield (about 90%) was obtained with systems composed of 5% sodium sulfate and 14% PEG-8000.(ABSTRACT TRUNCATED AT 250 WORDS)

Centrifugation↗
PubMed1994

Large scale, in situ isolation of periplasmic IGF-I from E. coli.

Human insulin-like growth factor I (IGF-I) accumulates in both folded and aggregated forms in the fermentation medium and cellular periplasmic space when expressed in E. coli with an endogenous secretory signal sequence. Due to its heterogeneity in form and location, low yield of IGF-I was obtained using a typical refractile body recovery strategy. To enhance recovery yield, a new procedure was developed to solubilize and extract IGF-I from cells while in fermentation broth. This method, called in situ solubilization, involves addition of chaotrope and reductant to alkaline fermentation broth and provides recovery of about 90% of all IGF-I in an isolated supernatant. To further enhance recovery, a new aqueous two-phase extraction procedure was developed which partitions soluble non-native IGF-I and biomass solids into separate liquid phases. This two-phase extraction procedure involves addition of polymer and salt to the solubilization mixture and provides about 90% recovery of solubilized IGF-I in the light phase. The performance of the solubilization and aqueous extraction procedures is reproducible at scales ranging from 10 to 1000 liters and provides a 70% cumulative recovery yield of IGF-I in the isolated light phase. The procedure provides significant initial IGF-I purification since most host proteins remain cell associated during solubilization and are enriched in heavy phase. ELISA analysis for E. coli proteins indicates that 97% of the protein in the light phase is IGF-I. Together, the techniques of in situ solubilization and aqueous two-phase extraction provide a new, high yield approach for isolating recombinant protein which is accumulated in more than one form during fermentation.

Escherichia coli↗
PubMed1994

New infection with Corynebacterium pseudotuberculosis reduces wool production.

The effect of natural Corynebacterium pseudotuberculosis infection on wool production and quality in sheep was examined in light of evidence that artificial C pseudotuberculosis infection causes wool production loss. A toxin ELISA was used to identify sheep that had been infected with C pseudotuberculosis. Greasy and clean fleece weights and fibre diameter were compared in infected and uninfected sheep. C pseudotuberculosis infection caused a 3.8 to 4.8% decrease in greasy wool production and a 4.1 to 6.6% decrease in clean wool production. C pseudotuberculosis infection did not affect fibre diameter. The effects of caseous lymphadenitis (the disease caused by C pseudotuberculosis infection) cause an annual loss of about $17 million in wool production to the Australian wool industry.

Abattoirs↗
PubMed1994

Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli.

Vitreoscilla hemoglobin (VHb) is accumulated at high levels in both soluble and insoluble forms when expressed from its native promoter on a pUC19-derived plasmid in Escherichia coli. Examination by atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy revealed that the insoluble form uniformly lacks the heme prosthetic group (apoVHb). The purified soluble form contains heme (holoVHb) and is spectroscopically indistinguishable from holoVHb produced by Vitreoscilla cells. This observation suggested that a relationship may exist between the insolubility of apoVHb and biosynthesis of heme. To examine this possibility, a series of experiments were conducted to chemically and genetically manipulate the formation and conversion of 5-aminolevulinic acid (ALA), a key intermediate in heme biosynthesis. Chemical perturbations involved supplementing the growth medium with the intermediate ALA and the competitive inhibitor levulinic acid which freely cross the cell barrier. Genetic manipulations involved amplifying the gene dosage for the enzymes ALA synthase and ALA dehydratase. Results from both levulinic acid and ALA supplementations indicate that the level of soluble holoVHb correlates with the heme level but that the level of insoluble apoVHb does not. The ratio of soluble to insoluble VHb also does not correlate with the level of total VHb accumulated. The effect of amplifying ALA synthase and ALA dehydratase gene dosage is complex and may involve secondary factors. Results indicate that the rate-limiting step of heme biosynthesis in cells overproducing VHb does not lie at ALA synthesis, as it reportedly does in wild-type E. coli (S. Hino and A. Ishida, Enzyme 16:42-49, 1973).

Bacterial Proteins↗
PubMed1994

Effect of environment on insulin-like growth factor I refolding selectivity.

Recombinant human insulin-like growth factor I (IGF-I), a 70-amino-acid peptide containing three disulphide bonds, produces two monomeric and several multimeric species during refolding. To optimize production of correctly folded IGF-I, conditions which influence protein refolding, stability and solubility were systematically examined. Combinations of solution components and conditions were analysed to identify synergistic interactions which enhance or reduce refolding efficiency. IGF-I concentration had the largest effect on formation of correctly folded peptide, due to competing association reactions. Solution polarity had the next largest individual effect, both on the level of multimeric peptide and on the relative proportion of correctly folded to misfolded monomeric peptide. Salt type and concentration and chaotrope type and concentration also had large individual effects on the distribution of IGF-I forms produced. Solution polarity modulated the effects of many other conditions including chaotrope concentration, salt type and concentration, and osmolyte concentration. Simultaneously decreasing the solution polarity and increasing the salt concentration improved the yield of correctly folded IGF-I relative to either individual change. Optimum solution conditions for refolding were 2M urea, 1M NaCl and 20% (v/v) ethanol. A possible mechanism for the importance of solution polarity on IGF-I refolding is discussed.

Amino Acid Sequence↗
PubMed1992

Ultrastructural morphometry of anterior cruciate and medial collateral ligaments: an experimental study in rabbits.

This study presents morphometric analyses of collagen subfascicle area fraction and collagen fibril diameter distributions for the anterior cruciate (ACL) and medial collateral (MCL) knee ligaments from transmission electron micrographs of ligament cross sections of five mature, female New Zealand White rabbits. Statistically significant differences in subfascicular area fractions were found between the ACL and MCL (0.89 +/- 0.02, 0.97 +/- 0.01, respectively; p less than 0.001). Mean fibril diameters for the ACL and MCL were also significantly different (0.059 +/- 0.005, 0.085 +/- 0.011 microns, respectively; p less than 0.025). Fibril eccentricity (a measure of parallel alignment of collagen fibrils within the ligaments, defined as the ratio of minor to major axes of elliptical fibril outlines) was 0.89 +/- 0.03 and 0.85 +/- 0.08, respectively, for the ACL and MCL; these data were not significantly different (p greater than 0.1). The relative amount of variation in the pooled fibril diameter data due to variation between animals, ligaments, locations within ligaments, and among fibrils at individual locations are reported. The variation of fibril diameter distributions between the ACL and MCL was substantially greater than the variation between different locations within each ligament cross section as well as between different animals. The structural differences reported may help explain known differences in the biomechanical properties of the ACL and MCL.

Animals↗
PubMed1992

Epiphytic microflora on alfalfa and whole-plant corn.

Epiphytic microflora were identified and counted on four cuttings of alfalfa, each harvested at three stages of maturity, and on three whole-plant corn hybrids. Enterobacteriaceae were predominant on both crops. Yeasts and molds also were major epiphytic microorganisms on whole-plant corn. The group--including lactobacilli, pediococci, and leuconostocs, which are genera that produce lactic acid and thus are instrumental in silage preservation--constituted only a small proportion of the total population (less than .5%) on both crops. Lactate-fermenting clostridial spores were not detected on standing alfalfa, and occurrences of these spores on standing corn plants were due to soil contamination from rainfall prior to harvest. The numbers of epiphytic microorganisms, except for the lactobacilli, pediococci, and leuconostocs group, were higher on standing corn than on alfalfa. The epiphytic microflora on alfalfa increased with increasing temperature during the growing season. However, neither cutting number nor maturity affected the epiphytic microflora on standing alfalfa, and wilting following mowing had little effect on most populations. Higher temperatures during wilting increased yeast and mold counts but had no effect on other microbial counts. The chopping process tended to increase the epiphytic microflora populations compared with those on the standing crops, and the group containing lactobacilli, pediococci, and leuconostocs was most enhanced. Only yeast and mold counts on the chopped alfalfa increased with greater DM content and buffering capacity.

Animal Feed↗
PubMed1991

Purification and aqueous two-phase partitioning properties of recombinant Vitreoscilla hemoglobin.

Soluble recombinant Vitreoscilla hemoglobin was purified from E. coli lysate by sequential two-phase extraction techniques. Extraction of lysate containing VHb in PEG/dextran gave a 3.6-fold increase in VHb purity in the PEG-rich phase via a size exclusion mechanism. Further extraction of the recovered PEG phase in PEG/sodium sulfate gave an additional 2.0-fold increase in purity in the PEG-rich phase due to an electrostatic mechanism. Final extraction of the PEG phase in PEG/magnesium sulfate gave an additional 1.3-fold increase in VHb purity in the magnesium sulfate-rich phase. The final yield from the extractive purification was 47% with purity of VHb estimated to be greater than 95%. Yields from the sulfate salt extractions are essentially quantitative due to the extreme partitioning behavior of VHb in these systems. VHb partition coefficients as large as 46 in PEG/sodium sulfate and as small as 0.06 in PEG/magnesium sulfate were observed. Similar small partition coefficients were obtained with PEG/manganese sulfate extractions. This dramatic effect of divalent cation content on the partition coefficient of VHb in PEG/sulfate salt systems was investigated by pH and magnesium ion titration experiments. Results show the effect to be largest and nearly constant for pH values greater than 6.0 and diminished at lower pH values. A model based on magnesium ion binding to negatively charged amino acids is shown to correlate with the data well. Based on model formulation and the partitioning behavior of contaminant proteins, the observed effect is expected to be applicable to other proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

A finite helical axis as a landmark for kinematic reference of the knee.

Reference coordinates based on the finite helical axis for flexion of the knee from 0 to 90 deg are proposed. Six degree-of-freedom tracking allows the use of such a helical axis as a kinematic landmark for knee motion representation. Data from five human subjects in vivo are presented as a path of finite helical axes for flexion of the knee from 20 to 80 deg. The finite helical axis rotates by an average of 11.4 deg, the centrode translates an average of 19.8 mm, and the total axial translation averages 0.1 mm during flexion from 20 to 80 deg. Error due to the transducer was measured on a fixed-pivot pendulum and found to be 1.0 deg and 1.9 mm rms for the helical axis orientation and position, respectively, and 0.1 mm for the axial translation. Reproducibility and soft tissue effects on the measurements were repeatable to 4.0 deg and 2.7 mm rms in orientation and position, respectively, and 0.1 mm for the axial translations. Soft tissue errors averaged 4.9 deg and 3.6 mm in position and orientation, and 0.3 mm in the axial translations.

Adult↗
PubMed1990

Protein composition of Vitreoscilla hemoglobin inclusion bodies produced in Escherichia coli.

The protein composition of inclusion bodies produced in recombinant Escherichia coli overproducing Vitreoscilla hemoglobin (VHb) was analyzed by one-dimensional and two-dimensional electrophoresis techniques. Results indicate the presence of two types of cytoplasmic aggregates of differing morphology in single bacterial cells. These aggregates also differ in their relative content of VHb and pre-beta-lactamase and are separable by differential centrifugation. Results further suggest that the cytoplasmic protein elongation factor Tu is integrated into VHb inclusion bodies. The presence of the outer membrane proteins OmpA and OmpF in inclusion body preparations is attributed to cell envelope contamination rather than specific involvement in inclusion bodies. The specificity of in vivo protein aggregation is discussed.

Animals↗
PubMed1990

Lesions of hippocampal circuitry define synaptosomal-associated protein-25 (SNAP-25) as a novel presynaptic marker.

Synaptosomal-associated protein, 25 kD, (SNAP-25) is a novel protein containing a possible transition metal binding site and encoded by a neuronal-specific mRNA. We examined the distribution of SNAP-25 mRNA and protein in the hippocampal formation of the adult rat following kainic acid, colchicine, and entorhinal lesions. The results show that destruction of granule cells of the dentate gyrus and CA3 pyramidal cells did not diminish SNAP-25 immunoreactivity in the dendritic fields of these cells. In contrast, lesioned neurons exhibited an extensive loss of immunoreactivity at the site of their axonal projections. These results support the identification of SNAP-25 as a novel presynaptic protein. In addition, SNAP-25 immunoreactivity was increased in afferent fibers which project to areas adjacent to the deafferented region, and expression of SNAP-25 mRNA was increased in neurons deafferented by the lesion. Examination of SNAP-25 immunoreactivity and mRNA expression may provide a useful marker of major hippocampal pathways and of axonal plasticity in neurological disorders such as Alzheimer's disease and temporal lobe epilepsy.

Animals↗
PubMed1989

The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.

cDNA clones of a neuronal-specific mRNA encoding a novel 25-kD synaptosomal protein, SNAP-25, that is widely, but differentially expressed by diverse neuronal subpopulations of the mammalian nervous system have been isolated and characterized. The sequence of the SNAP-25 cDNA revealed a single open reading frame that encodes a primary translation product of 206 amino acids. Antisera elicited against a 12-amino acid peptide, corresponding to the carboxy-terminal residues of the predicted polypeptide sequence, recognized a single 25-kD protein that is associated with synaptosomal fractions of hippocampal preparations. The SNAP-25 polypeptide remains associated with synaptosomal membrane components after hypoosmotic lysis and is released by nonionic detergent but not high salt extraction. Although the SNAP-25 polypeptide lacks a hydrophobic stretch of residues compatible with a transmembrane region, the amino terminus may form an amphiphilic helix that may facilitate alignment with membranes. The predicted amino acid sequence also includes a cluster of four closely spaced cysteine residues, similar to the metal binding domains of some metalloproteins, suggesting that the SNAP-25 polypeptide may have the potential to coordinately bind metal ions. Consistent with the protein fractionation, light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus. The mRNA was found to be enriched within neurons of the neocortex, hippocampus, piriform cortex, anterior thalamic nuclei, pontine nuclei, and granule cells of the cerebellum. The distribution of the SNAP-25 mRNA and the association of the protein with presynaptic elements suggest that SNAP-25 may play an important role in the synaptic function of specific neuronal systems.

Amino Acid Sequence↗
PubMed1985

Serum crosslinked fibrin (XDP) and fibrinogen/fibrin degradation products (FDP) in disorders associated with activation of the coagulation or fibrinolytic systems.

Soluble crosslinked fibrin derivatives (XDP) in serum were determined by enzyme immunoassay utilizing monoclonal antibodies and compared with serum fibrinogen/fibrin degradation products (FDP) assayed by conventional techniques. In healthy subjects and patients with miscellaneous disorders not usually associated with activation of the haemostasis mechanism, mean XDP levels were 45 and 70 ng/ml respectively. However, elevated levels of XDP occurred in conditions commonly associated with intravascular and possibly extravascular activation of the coagulation system. Markedly raised mean XDP values (677-6900 ng/ml) occurred in treated pulmonary embolism, disseminated neoplasia, severe inflammatory disorders and complicated postoperative states, and lesser but significant elevation (mean 150-400 ng/ml) in treated venous thrombosis, uneventful postsurgical states, localized neoplasia, liver disease and symptomatic arterial disease. Levels during initial streptokinase therapy (mean 24 000 ng/ml) fell tenfold as treatment was continued. The degree of XDP elevation over normal values was significantly higher than that of FDP in conditions with a propensity for venous thrombosis (post-operative states, disseminated neoplasia and inflammatory diseases) than in liver disease, localized neoplasia or patients receiving heparin therapy for venous thromboembolism.

Adult↗
PubMed1985

Computerized nutrient analysis for foodservice.

The need for computerized nutrient analysis does differ depending on the type of foodservice institution. Variation in the menu cycles, type of funding, experience with computers, number and type of clientéle fed, and the limited number of respondents affiliated with various institutions make it difficult to recommend a standard nutrient analysis software system. Additionally, the researchers frequently noted two obstacles to computerization of foodservice and nutrition departments: (a) respondents believe their institutions or departments are too small for computers, and (b) there is limited knowledge about computers and nutrient analysis. It is hoped that foodservice personnel can use the information from this study to learn of features available in nutrient analysis programs and that professionals can use it in designing and marketing nutrient analysis software.

Attitude↗