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Biomedical subjects

J John

Publications and source records attributed to J John.

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

Kinetics of interaction of nucleotides with nucleotide-free H-ras p21.

A method is described for the convenient preparation of substantial quantities of nucleotide-free p21 or of 1:1 complexes with nucleotides other than GDP. The nucleotide-free protein has been used for kinetic studies of the binding of GDP and GTP, making use of the fluorescent analogues 3'-(methylanthraniloyl)-2'-deoxy-GDP and -GTP. Stopped-flow studies have led to the formulation of a two-step binding mechanism for both GDP and GTP, involving initial rapid but weak binding of the nucleotide followed by a relatively slow (10-20 s-1 at 25 degrees C; 3-5 s-1 at 5 degrees C) quasi-irreversible isomerization reaction. By use of a nonequilibrium competition method, guanosine and GMP have been shown to interact weakly but significantly with p21 (dissociation constants of 153 and 29 microM, respectively). The presence of guanosine or GMP at the active site of p21 leads to a marked stabilization of p21 against spontaneous denaturation when compared with the nucleotide- and nucleoside-free protein.

Guanosine↗
PubMed1990

Proton NMR studies of transforming and nontransforming H-ras p21 mutants.

One- and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and site-directed mutagenesis were used to study the influence of mutations on the conformation of the H-ras oncogene product p21. No severe structural differences between the different mutants, whether they were transforming or nontransforming, could be detected. Initially, selective incorporation of 3,5-deuterated tyrosyl residues into p21 and 2D NMR were used to identify the resonances representing the spin systems of the imidazole rings of the three histidyl residues in the protein, of six of the nine tyrosyl rings, and of four of the five phenylalanyl rings. The spin systems of the phenyl rings of Phe28, Phe78, and Phe82 could be assigned by using mutant proteins, since no severe structure-induced spectral changes in the aromatic part of the spectra of the mutant proteins were detected. Sequence-specific assignments of the histidine imidazole resonances could be obtained by comparison of the distance information obtained by nuclear Overhauser enhancement spectroscopy (NOESY) experiments with the crystal structure. The change in the chemical shift values of the Hl' proton and the alpha-phosphate of the bound GDP in the NMR spectra of the p21(F28L) mutant and the 28-fold increase in the GDP dissociation rate constants of this mutant suggest a strong interaction between Phe28 and the p21-bound nucleotide. In solution, the p21-bound GDP.Mg2+ has an anti conformation, and the phenyl ring of Phe28 is close to the ribose of the bound GDP.Mg2+.

Base Sequence↗
PubMed1990

Bactericidal effectiveness of modulated UV light.

Studies were designed to evaluate the effectiveness of pulsed modulated UV light waveforms for killing bacteria. Exposure of five strains of bacteria to the modulated information encoded in the light decreased the colony population from a confluent lawn to less than 20 colonies. However, approximately 2,000 colonies survived treatment with the same intensity and time of exposure to UV light lacking the modulated information.

Bacteria↗
PubMed1990

Interrelationships between serologic markers of immune activation and T lymphocyte subsets in HIV infection.

Relationships among four serologic activation markers and T cell subsets were measured in HIV-seropositive former blood donors (N = 64) and seronegative controls (N = 61). Significant correlations were observed for the HIV group in pairwise comparisons of soluble IL-2 receptor (sIL-2R), beta 2-microglobulin (beta 2M), neopterin (NEOP), and soluble CD8 (sCD8). CD4 cell levels (number/microliter) in the HIV group showed significant negative correlation with all four serologic markers; CD8 cell levels, in contrast, showed no significant correlation with any serologic activation marker measured. Significant correlations were observed, however, among various cell surface activation markers and serologic activation markers. Specifically, the proportion of CD8 cells expressing CD45RA showed significant negative correlations with NEOP and B2M levels, whereas the proportion of CD8 cells expressing HLA-DR showed significant positive correlations with B2M and sIL-2R levels. Further, the proportion of CD8 cells expressing CD38 showed significant positive correlations with all four serologic activation markers. These findings indicate that sIL-2R, B2M, NEOP, and sCD8 show similar quantitative changes and correlational relationships to CD4 cell destruction in HIV infection; they differ, however, in their relationships to proportional changes in activated CD8 cell subsets.

Antigens, CD↗
PubMed1990

[Intensive care aspects in eclampsia with multiple organ failure].

Because of a good prenatal care eclampsia has been very seldom. But it is still a severe disease. By means of a case report vital symptoms and possible etiologic mechanisms are discussed. The meaning of an interdisciplinary intensive care and a subtle monitoring has been emphasized.

Adult↗
PubMed1989

Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation.

The crystal structure of the guanine-nucleotide-binding domain of p21 (amino acids 1-166) complexed to the guanosine triphosphate analogue guanosine-5'-(beta, gamma-imido)triphosphate (GppNp) has been determined at a resolution of 2.6 A. The topological order of secondary structure elements is the same as that of the guanine-nucleotide-binding domain of bacterial elongation factor EF-Tu. Many interactions between nucleotide and protein have been identified. The effects of point mutations and the conservation of amino-acid sequence in the guanine-nucleotide-binding proteins are discussed.

Binding Sites↗
PubMed1989

C-terminal truncation of p21H preserves crucial kinetic and structural properties.

The human c-Ha-ras protooncogene product p21C was truncated at the C terminus by 23 amino acids. The resulting G-binding domain, p21 (1-166) = p21C', can be crystallized as a complex with the slowly hydrolyzing GTP analogues guanosin-5'-[beta,gamma-imido]triphosphate, guanosin-5'-[beta,gamma-methylene]triphosphate, and guanosin-5'-O-(3-thiotriphosphate). We show here that this protein has biochemical properties very similar to those of the intact protein. Activating mutations in position 12 (Gly12----Val; Gly12----Arg) have the same effect on the properties of the truncated protein as on intact protein. Nuclear magnetic resonance (NMR) measurements show no apparent effect of the C-terminal deletion on the solution structure of p21. This suggests that neither the structure of the G-binding domain nor any of its biochemical properties are markedly influenced by the truncation.

Amino Acid Sequence↗
PubMed1989

Crystallization and preliminary X-ray analysis of the human c-H-ras-oncogene product p21 complexed with GTP analogues.

The catalytic domain (amino acid residues 1 to 166) of the human ras-oncogene product p21 complexed with the GTP analogues beta,gamma-imido-GTP (GMPPNP), beta,gamma-methylene-GTP (GMPPCP), and guanosine-5'-(gamma-thiotriphosphate) (GTP gamma S) have been been crystallized. Crystals of the GMPPNP and GMPPCP complexes are well suited for high resolution X-ray crystallography. They belong to space group P3(1)21 (or its enantiomorph P3(2)21) with unit cell axes a=b=40.3 A and c = 162.2 A.

Base Sequence↗
PubMed1989

ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons.

Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24-ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal root ganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha-ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24-ras and c-Ha-ras lacking the 23 C-terminal amino acids including the membrane-anchoring, palmityl-accepting cysteine. These results suggest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors.

Adrenal Gland Neoplasms↗
PubMed1989

Biochemical and crystallographic characterization of a complex of c-Ha-ras p21 and caged GTP with flash photolysis.

The GTP binding domain of the c-Ha-ras protooncogene product (p21'c) and the corresponding region from an oncogenic mutant form of the protein in which glycine at position 12 has been replaced by valine [p21'(G12V)] have been crystallized with P3-1-(2-nitro)phenylethylguanosine 5'-O-triphosphate (caged GTP) at their active sites. The crystals give x-ray diffraction patterns to a resolution of better than 0.3 nm. Photolysis can be achieved in the crystal, after which GTP hydrolysis takes place at the rate expected from solution studies. Complete x-ray data sets have been obtained for the starting caged-GTP state and the final GDP state after photolysis and hydrolysis, demonstrating the feasibility of time-resolved structural investigations of the process of GTP hydrolysis.

Binding Sites↗
PubMed1989

Optical urethrotomy--experience in Oman.

Over a 4-year period 170 men with urethral strictures were treated with optical urethrotomy; 80% required no further treatment during the 2-year follow-up; 2 patients with urethral fistulae associated with stricture were also treated successfully.

Follow-Up Studies↗
PubMed1989

Staphylococcus saprophyticus urease: characterization and contribution to uropathogenicity in unobstructed urinary tract infection of rats.

We studied the biochemical properties of the urease of Staphylococcus saprophyticus and the possible role of the urease in experimental urinary tract infections. For this purpose, the nonhemagglutinating and nonadherent strain 9325, which was isolated from a case of symptomatic urinary tract infection, was used. The urease was shown to have a Km of 6.64 mM urea and a Vmax of 4.59 mumol NH3.min-1.mg-1. The enzyme was inhibited by acetohydroxamic acid in a noncompetitive manner. By means of Sephacryl S-300 column chromatography, we determined a mean molecular weight (+/- standard error of the mean) of 420,000 +/- 16,000. To assess the contribution of S. saprophyticus urease to uropathogenicity, a urease-negative mutant was constructed by nitrosoguanidine mutagenesis. In the rat model of ascending unobstructed urinary tract infection, higher numbers of CFU.gram of tissue-1 and more-severe lesions were detected with the parent strain. Moreover, bladder stones were found in animals infected with the urease-positive strain only. Interestingly, the difference in mean bacterial counts of the bladders was found to be significant by the Wilcoxon two-sample test (P less than 0.05), whereas that between the kidney bacterial counts was not. Immunoblot studies revealed a faint antibody response in rats infected with the mutant strain, although bacteria could still be detected in the kidneys after 7 days. Sera of animals challenged with the parent strain reacted strongly with many antigens of S. saprophyticus. Our data indicate that urease is a major factor for invasiveness of S. saprophyticus, especially in the tissue of the bladder, whereas persistence in the urinary tract and nephropathogenicity of this organism are governed by factors other than urease.

Animals↗
PubMed1989

Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway.

We have selected mutations in genes encoding components of the signaling pathway for alpha interferon (IFN-alpha) by using a specially constructed cell line. The upstream region of the IFN-regulated human gene 6-16 was fused to the Escherichia coli guanine phosphoribosyltransferase (gpt) gene and transfected into hypoxanthine-guanine phosphoribosyltransferase-negative human cells. These cells express gpt only in the presence of IFN-alpha. They grow in medium containing hypoxanthine, aminopterin, and thymidine plus IFN and are killed by 6-thioguanine plus IFN. Two different types of mutants were obtained after treating the cells with mutagens. A recessive mutant, selected in 6-thioguanine plus IFN, was completely resistant to IFN-alpha but responded normally to IFN-gamma and, unexpectedly, partially to IFN-beta. A constitutive mutant, selected in hypoxanthine-aminopterin-thymidine alone, was abnormal in expressing endogenous genes in the absence of IFN. Both types revert infrequently, allowing selection for complementation of the defects by transfection.

Blotting, Northern↗
PubMed1989

Strategic planning for nursing homes: a market opportunity analysis perspective.

Market opportunity analysis is a framework for systematic strategic planning involving the analysis of demand, market segments, competition, industry, and channels of referrals. This approach enables nursing homes to select target markets and to design marketing programs to attract those target markets.

Costs and Cost Analysis↗
PubMed1989

[Cryoimmunotherapy of herpetic and metaherpetic keratitis].

In 11 patients with relapsing herpetic keratitis and metaherpetic ulcers cryoimmunotherapy, as recommended by Amoils, was applied. Under instillation anaesthesia three cryocycles were made using a temperature of -80 degrees on the entire area of the lesion with subsequent instillation of 60 drops (1 drop per minute) of serum obtained from 15 ml of the patient's blood mixed with 5 ml gamma globulin. In all 11 patients progression of the disease was arrested, though it could not be checked by previous conservative therapy. The cornea cleared and vision improved. The method is effective, simple and expands therapeutic procedures which can be used in these tiresome conditions.

Adult↗
PubMed1988

Biochemical properties of Ha-ras encoded p21 mutants and mechanism of the autophosphorylation reaction.

Kinetic studies performed on p21H guanine nucleotide complexes with and without Mg2+ show that point mutations at positions 12, 59, and 61 each have a different effect on the rate of nucleotide dissociation. Double mutants with a combination of these amino acid substitutions reveal that the effects of each mutation on these kinetics are interactive (nonadditive) for positions 12 and 59 and approximately additive for the positions 12 and 61. The magnitude and direction of the effects seen are dependent on the nature of the nucleotide and whether or not the complexes contain Mg2+. All the mutants have reduced GTPase activity. It is also shown that the autophosphorylation reaction velocity is of first order with respect to the protein concentration and that this reaction is an intramolecular one, which takes place as a side reaction of the GTPase reaction. The autophosphorylation is not reversible under the experimental conditions. The covalently bound phosphate does not decrease the nucleotide-binding ability of the protein nor does it change the relative affinity of the protein for GTP versus GDP. The results are discussed in terms of the structural model and function of p21H.

Escherichia coli↗
PubMed1987

Evaluation of disposable cartridges for trace enrichment from aqueous solutions.

Selected aromatic hydrocarbons, phthalate esters, and food, drug and cosmetic (FD&C) dyes are isolated from aqueous solutions with Chrom-Prep PRP-1 and Sep-Pak C18 cartridges. The Chrom-Prep cartridges demonstrate greater adsorption capacity for model aromatic hydrocarbons and phthalate esters than Sep-Pak C18 cartridges; however, the latter exhibits greater capacity for the FD&C dyes, which have breakthrough volumes that are a function of pH. Both cartridge types show a small amount of analyte leakage during the trace enrichment step, which is measured by monitoring the cartridge effluents for the presence of 14C-labeled solute. Two PRP-1 cartridges connected in tandem reduce the amount of activity in the effluent by approximately one half.

Benzo(a)pyrene↗