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Biomedical subjects

G Michaels

Publications and source records attributed to G Michaels.

17 recordsLinked to original sources
PubMed1999

The identification of markers segregating with resistance to Schistosoma mansoni infection in the snail Biomphalaria glabrata.

Both snail and parasite genes determine the susceptibility of the snail Biomphalaria glabrata to infection with the trematode Schistosoma mansoni. To identify molecular markers associated with resistance to the parasite in the snail host, we performed genetic crosses between parasite-resistant and -susceptible isogenic snails. Because resistance to infection in adult snails is controlled by a single locus, DNA samples from individual F2 and F1 backcross progeny, segregating for either the resistant or susceptible phenotypes, were pooled (bulked segregant). Genotypes for both parents were determined with 205 arbitrary decamer primers by random amplified polymorphic DNA-PCR. Of the 205 primers, 144 were informative, and the relative allele frequencies between the pools for these primers were determined. Two primers, OPM-04 and OPZ-11, produced fragments in the resistant parent of one cross that were inherited in a dominant fashion in the resistant F2 and backcross-bulked segregant progeny. Subsequent typing of DNA samples of individual progeny snails showed that the 1.2-kb marker amplified by primer OPM-04 and the 1.0-kb marker produced by primer OPZ-11 segregated in the same dominant fashion with the resistant phenotype. Sequence analysis of the 1.2-kb marker showed that it corresponds to a repetitive sequence in the snail genome with no homology to existing DNA sequences in the public databases. Analysis of the 1. 0-kb marker showed that it also corresponds to a repetitive sequence in the B. glabrata genome that contains an imperfect ORF, with homology to retrovirus-related group-specific antigens (gag) polyprotein.

Animals↗
PubMed1995

Intraphagosomal oxygen in stimulated macrophages.

A new electron paramagnetic resonance (EPR)-based method was developed to obtain selective information on pO2 in a specific intracellular compartment (phagosomes). This method did not require the use of a broadening agent thereby eliminating one of the potential sources of experimental error with EPR oximetry. An oxygen-sensitive probe (4-(Trimethylammonium) 2,2,6,6-tetramethylpiperidine-d17-1-oxyl iodide (d-Cat1)) which has a net positive charge, was incorporated selectively into the phagosomes of macrophages stimulated with zymosan. Extracellular oxygen was measured by addition of a neutral nitroxide (4-oxo-2,2,6,6-tetramethylpiperidine-d16-1-oxyl (15N PDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 11.2 +/- 3.4 microM lower than that measured from the extracellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondrial consumption or by increasing the oxygen concentration in the extracellular compartment. These experiments provide what we believe to be the first reported measurements of the oxygen concentration in a specific intracellular location (intraphagosomal) and its comparison with the oxygen concentration in the extracellular space. The observed gradient cannot be explained in terms of known coefficients of diffusion, and these results are consistent with previous reports that a gradient in oxygen concentration can occur between the average intracellular and extracellular concentration of oxygen.

Animals↗
PubMed1987

Cloning, nucleotide sequence and expression of the pyrBI operon of Salmonella typhimurium LT2.

The pyrB-pyrI region of the Salmonella typhimurium LT2 chromosome has been cloned and sequenced. The two genes were found to constitute an operon, with pyrI being the distal gene and separated from pyrB by a 15-bp intercistronic region. Sequence analysis revealed the presence of two potential promoters; transcription initiated from the promoter proximal to pyrB would produce a transcript which could direct the synthesis of a 33-amino-acid leader peptide. The leader sequence possesses the requisite features of a rho-independent transcriptional terminator (attenuator) which is positioned 22 bp upstream from the pyrB structural gene. A regulatory mutation imparting a 30-fold elevated expression of pyrBI was identified as a two-base-pair deletion in the track of A X T base pairs located on the 3' side of the region of dyad symmetry of the attenuator. The leader sequence also has an additional region of dyad symmetry (putative transcriptional pause site) located 33 nucleotides upstream from the start of the proposed attenuator. The intervening sequence between the putative pause site and the indicated attenuator is characterized by encoding a high content of uracil residues in the transcript. Construction and analysis of transcriptional and translational fusions provided evidence that the leader region has the necessary features to mediate polypeptide synthesis in vivo, the removal of the region corresponding to the pause site and attenuator results in constitutive expression and the more distant potential promoter does not appear to facilitate significant transcriptional activity. Strong homology exists with the pyrBI operon from Escherichia coli K-12 but notable differences are observed.

Aspartate Carbamoyltransferase↗
PubMed1986

20 years cogeneration at a hospital.

Virtually all of the recoverable heat energy from the engine generator sets--jacket cooling, flue gas, and oil cooler--can be used to meet space cooling, space heating, and process loads at a 300 bed hospital.

Conservation of Energy Resources↗
PubMed1983

Construction and use of pyr::lac fusion strains to study regulation of pyrimidine biosynthesis in Salmonella typhimurium.

The technique developed by Rosenfeld and Brenchley [J Bacteriol 144, 848-851 (1980)] has been used to introduce Mu d1 (Apr lac) into Salmonella typhimurium for purposes of constructing pyr::lac fusion strains. A stable pyrB::lac fusion mutant was subsequently derived and used for the genetic characterization of the pyrB gene. The direction of transcription of pyrB was determined to be counterclockwise on the S. typhimurium linkage map and argI was shown to be located clockwise of pyrB. Mutants altered in the regulation of expression of pyrB were isolated and two of the isolates chosen for further study were tentatively categorized as promoter or operator mutants.

Aspartate Carbamoyltransferase↗
PubMed1976

Isolation of Klebsiella pneumoniae from lake water.

The question of the importance of aquatic borne Klebsiella pneumoniae to public health has been argued as the organism has appeared often in both polluted effluents and oligotrophic systems. Using a selective medium, double violet agar, which also differentiates K. pneumoniae from other organisms found in aquatic systems, we sampled water from three ponds and a large man-made lake, over a 3-month period. Results indicate that K. pneumoniae can be isolated consistently and in high numbers from eutrophic waters even when environmental stress reduces total numbers, but that isolations from cleaner waters are erratic. We conclude that mere isolation of the organism as an occasional contaminant probably does not present a hazard to public health.

Escherichia coli↗
PubMed1975

Magnetic resonance and kinetic studies of pyruvate, phosphate dikinase. Interaction of oxalate with the phosphorylated form of the enzyme.

Pyruvate, orthophosphate dikinase (EC 2.7.9.1) carries out its catalytic function in three successive partial reactions, the final step being the reaction of pyruvate with a stable phosphoenzyme intermediate to give phosphoenolpyruvate and free enzyme (Evans, H.J., and Wood, H. G. (1968), Proc. Natl. Acad. Sci. U.S.A. 61, 1448). Interactions of oxalate, a structural analog of enolpyruvate, with the phosphorylated form of the enzyme have been investigated by kinetic inhibition measurements and by magnetic resonance studies of manganous ion complexes with the enzyme. Oxalate inhibits the reaction catalyzed by pyruvate, phosphate dikinase, and the inhibition is linearly competitive with respect to pyruvate. The inhibitor constant for oxalate of 25 mu-M is fourfold lower than the Michaelis constant for pyruvate. The enhancement in the longitudinal relaxation rate of water protons (PRR) which occurs upon binding of Mn(II) to the enzyme has been used to monitor binding of oxalate to Mn(II)-enzyme complexes. PRR titrations indicate that the dissociation constant of oxalate from the Mn(II) complex of the free form of the enzyme is an order of magnitude weaker than the kinetically determined Ki. On the other hand, titrations of solutions which contain the phosphorylated form of the enzyme reveal a much stronger binding of oxalate. Moreover, the strength of oxalate binding to the phosphorylated enzyme is a function both of the species and of the concentration of monovalent cations in the solution. In the presence of Tl+, which has the most favorable activator constant for the final partial reaction, the dissociation constant for oxalate from its complex with the phosphorylated enzyme is less than 1 mu-M. Electron paramagnetic resonance (EPR) spectra for the enzyme-bound Mn(II) are sensitive to structural perturbations which occur upon binding of substrates or of oxalate to the enzyme. The EPR spectrum for the Mn(II)-phosphoenzyme-oxalate species is distinguished from spectra for other complexes of the enzyme by unusually narrow line widths and consequent resolution of fine structure from electronic quadrupole splitting. The narrow lines in the EPR spectrum are indicative of a rigid, pseudocrystalline environment for the bound Mn(II). The magnitude and frequency dependence of the PRR for the Mn(II)-phosphoenzyme-oxalate complex indicate that if any water molecules are bound to the Mn(II), their exchange with the bulk water is severely retarded. The kinetic and magnetic resonance studies support the hypothesis that oxalate mimics the reactive intermediate, enolpyruvate, in a complex with the phosphorylated enzyme which may resemble the structure of the transition state of the final partial reaction.

Bacillus↗