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Biomedical subjects

R A Hart

Publications and source records attributed to R A Hart.

30 records · Page 2Linked to original sources
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↗
PubMed1981

Indirect solid-phase microradioimmunoassay for detection of Ascaris suum antibodies in swine sera.

Antibodies to Ascaris suum have been detected and quantitated in sera of swine orally inoculated with eggs of A suum, using an indirect solid-phase microradioimmunoassay. Antibody titer of 1:640 to 1:10,240 were quantitated, using purified and crude ascarid antigens in the assays. Swine sera from 71 of 72 farm herds located in 37 counties in Nebraska had at least 1 serum of the 7 tested with a titer equal to or greater than 1:20. A total of 437 sera were positive and 67 negative (ie, less than 1:20 titer). Six positive sera selected at random had titers of 1:640 to 1:5,120.

Animals↗
PubMed1980

Specific fluorescein-labeled antibodies to bovine viral diarrhea virus prepared from sera of rabbits immunized with purified virus.

Specific fluorescein-labeled antibody conjugates to three strains of bovine virus diarrhea virus were prepared from hyperimmune rabbit sera. Viruses used to hyperimmunize the rabbits were purified by four different procedures. Conjugates were comparable in quality and specificity to conjugates prepared from serum of a calf hyperimmunized to bovine virus diarrhea virus in our laboratory. The latter conjugate was tested by Biologics Laboratories, National Veterinary Services, U.S.D.A., Ames, Iowa.

Animals↗