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Biomedical subjects

J Field

Publications and source records attributed to J Field.

At least 109 records · Page 6Linked to original sources
PubMed1990

Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein.

Adenylyl cyclase from S. cerevisiae contains at least two subunits, a 200 kd catalytic subunit and a subunit with an apparent molecular size of 70 kd, which we now call CAP (cyclase-associated protein). We cloned a cDNA encoding CAP by screening a yeast cDNA expression library in E. coli with antisera raised against the purified protein. The cDNA contained an open reading frame capable of encoding a 526 amino acid protein that is not homologous to any sequences in the current data bases. Adenylyl cyclase activity in membranes from cells that lacked CAP was not stimulated by RAS2 proteins in vitro. These results suggest that CAP is required for at least some aspects of the RAS-responsive signaling system. Mutants lacking CAP had four additional phenotypes that appear to be unrelated to effects of the RAS/adenylyl cyclase pathway: the inability to grow on rich medium (YPD), temperature sensitivity on minimal medium, sensitivity to nitrogen starvation, and a swollen cell morphology.

Adenylyl Cyclases↗
PubMed1990

Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae.

The interaction between RAS proteins and adenylyl cyclase was studied by using dominant interfering mutations of adenylyl cyclase from the yeast Saccharomyces cerevisiae. RAS proteins activate adenylyl cyclase in this organism. A plasmid expressing a catalytically inactive adenylyl cyclase was found to interfere dominantly with this activation. The interfering region mapped to the leucine-rich repeat region of adenylyl cyclase, which is homologous to domains present in several other proteins and is thought to participate in protein-protein interactions.

Adenylyl Cyclases↗
PubMed1990

High-field localized in vivo proton spectroscopy on micro volumes.

A water-suppressed volume-selected in vivo 1H spectrum of 0.2 ml of a rat brain has been obtained at 200 MHz using the SPACE localization method. Good signal-to-noise and spectral resolution were obtained by averaging 256 acquisitions. The spectrum shows little T2 weighting effect.

Animals↗
PubMed1990

Application of volume-selected, two-dimensional multiple-quantum editing in vivo to observe cerebral metabolites.

The volume selection technique SPACE has been combined with a two-dimensional multiple-quantum editing sequence to uniquely detect certain J-coupled cerebral metabolites. In vivo results demonstrating edited glutamate/glutamine and lactate from 0.4 ml of a rat's brain at 4.7 T are presented. The sequence was optimized to balance multiple-quantum generation and signal loss due to T2 relaxation. Without due regard to T2 relaxation little signal is observed.

Animals↗
PubMed1990

Signal-to-noise ratio improvements in in vivo high resolution micro-volume selected spectroscopy.

A system capable of in vivo volume selected 1H NMR spectroscopy of voxels as small as 0.2 cm3 is described. Signal-to-noise ratio improvements with probe design and a novel signal steering device are detailed. A high-resolution, image-directed proton spectrum from 0.2 cm3 of a rat's brain at 200 MHz obtained using the SUBMERGE/SPACE pulse sequence is presented. Single-scan voxel shimming was implemented to improve spectral resolution.

Animals↗
PubMed1990

Mutational mapping of RAS-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase.

Large deletion and small insertion mutations in the adenylyl cyclase gene of Saccharomyces cerevisiae were used to map regions required for activation by RAS protein in vitro. The amino-terminal 605 amino acids were found to be dispensable for responsiveness to RAS protein. All other deletions in adenylyl cyclase destroyed its ability to respond to RAS. Small insertion mutations within the leucine-rich repeat region also prevented RAS responsiveness, while other insertions did not.

Adenylyl Cyclases↗
PubMed1989

Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae.

We report a class of interfering mutants of the human H-ras gene capable of inhibiting phenotypes arising from the expression of the activated RAS2 gene, RAS2val19, in the yeast Saccharomyces cerevisiae. All these mutants encode unprocessed H-ras proteins that remain in the cytoplasm. One of the mutants, H-rasarg186, was examined in detail. H-rasarg186 protein is a competitive inhibitor of RAS2val19 protein. It does not interfere with processing and membrane localization of RAS2val19, nor does it appear to compete with RAS protein for its proposed regulator, the CDC25 protein. By several criteria the RAS2val19 adenylate cyclase interaction is unaffected by H-rasarg186. We infer from our results that H-rasarg186 protein interferes with an alternative function of RAS2val19.

Adenylyl Cyclases↗
PubMed1989

Water suppression with B0 field gradient homospoil pulses in high-resolution NMR spectroscopy.

The combination of a frequency nonselective excitation suppression method (1331 sequence) with selective excitation followed by gradient-induced dephasing of water transverse magnetization yielded suppression ratios of greater than 10,000:1. The need for gradient preemphasis and correction of B0 field shifts is discussed. The suppression efficiency of this method compared favorably to results obtained using the CPMG spin-echo technique to observe metabolite resonances in a urine sample.

Equipment Design↗
PubMed1989

In vivo high-resolution volume-selected proton spectroscopy and T1 measurements in the dog brain.

Successful in vivo NMR spectroscopy requires a combination of techniques to address the problems of volume selection, water suppression, and resolution. All this needs to be done in the very heterogeneous environment found in living organisms. Previously published techniques are used to obtain 1H spectra from a dog brain, observing metabolites with concentrations below 1 mM. Measurements of spin-lattice relaxation times (T1) are also presented. The 1H relaxation times are long (T1 greater than 1.0 s) yielding information about the fluidity of the molecular environment. Comments are made concerning the achievable linewidth in vivo and the deficiencies that phase-encoding spectroscopic methods may have in obtaining high-resolution 1H spectra.

Animals↗
PubMed1989

The adenylyl cyclase gene from Schizosaccharomyces pombe.

We cloned the adenylyl cyclase gene from the fission yeast Schizosaccharomyces pombe using low-stringency hybridization to the Saccharomyces cerevisiae adenylyl cyclase gene. The Sc. pombe gene encodes a 1692-amino acid-residue protein. The identity of this gene was confirmed by studies of its expression in Sa. cerevisiae. Expression of the carboxyl-terminal region of the Sc. pombe adenylyl cyclase protein will suppress a temperature-sensitive mutation in the Sa. cerevisiae adenylyl cyclase gene. Furthermore, Sa. cerevisiae that lack their endogenous adenylyl cyclase gene and express the carboxyl-terminal region of the Sc. pombe adenylyl cyclase protein have measurable adenylyl cyclase activity. The carboxyl-terminal region of this protein has strong homology with the catalytic domain of the Sa. cerevisiae adenylyl cyclase. Also, Sc. pombe adenylyl cyclase, like Sa. cerevisiae adenylyl cyclase, contains a tandemly repeated motif rich in leucine. Neither yeast protein is particularly homologous to the recently cloned Gs-responsive mammalian adenylyl cyclase [Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R. & Gilman, A. G. (1989) Science 244, 1558-1564].

Adenylyl Cyclases↗
PubMed1989

How do doctors and patients react to the introduction of a practice formulary?

A formulary covering 10 drug groups and over 50% of prescribing was devised in a general practice and doctors' attitudes to the idea to a formulary were assessed before and after the study. The prescribing of formulary drugs rose from 72% to 81% over two years, and the general practitioners were significantly more positive towards formularies in the practice which devised the formulary but not in three control practices. A group of patients receiving repeat prescriptions was interviewed in three practices in three consecutive years, and there was no difference in satisfaction with drugs between the formulary practice and the two control practices. However, an association between changing a drug and dissatisfaction was noted, and there was a trend of decreasing satisfaction with prescribing and with information given about drugs over the three years in all the practices. Overall 51% of patients felt that they had either too little or no information given to them about their drugs. Improving information given to both doctors and patients about drugs may be important in improving prescribing without causing discontentment.

Attitude of Health Personnel↗
PubMed1988

Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.

We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be a multisubunit complex consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.

Adenylyl Cyclases↗