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Biomedical subjects

A W Johnson

Publications and source records attributed to A W Johnson.

At least 19 recordsLinked to original sources
PubMed1992

The association of household pollutants and socio-economic risk factors with the short-term outcome of acute lower respiratory infections in hospitalized pre-school Nigerian children.

In a 9-month study of acute lower respiratory infections (ALRI), the short-term prognostic implications of socio-economic and household risk factors were examined in 103 hospitalized pre-school Nigerian children. Seventy-nine (77%) subjects were potentially exposed to the combustion products of kerosene stoves, 16 (16%) to wood smoke and five (5%) to the products of cooking gas combustion. Only 17 subjects (17%) were exposed to household cigarette smoke. A highly significant association (p < 0.005) was shown between household cooking fuel and the outcome of hospitalization: five (63%) of the eight who died were potentially exposed to wood smoke. The duration of hospitalization was only significantly associated with paternal income (p < 0.05). None of the other domestic risk factors was significantly related to outcome or duration of admission. These findings suggest an association between an adverse outcome of ALRI and domestic exposure to wood smoke. While the hospital-based source will not allow definite conclusions, the present findings underscore the need for community-based studies. The implications for future strategies of ARI control are discussed, with suggestions.

Acute Disease↗
PubMed1992

Chloramphenicol-resistant Haemophilus influenzae meningitis in young urban Nigerian children.

In a developing country like Nigeria, the unusual emergence of Haemophilus influenzae type b, resistant to cost-effective antimicrobials, is of serious concern. We report three cases of H. influenzae type b meningitis in young Nigerian children in whom clinical and bacteriological features of resistance to chloramphenicol were identified. One of the cases had concomitant resistance to ampicillin (multiple-drug resistance). Significant anaemia was an associated feature in two cases, one of whom had a recent measles infection. All three cases were malnourished. The possible mechanisms of antimicrobial resistance in H. influenzae infections are highlighted while the need for periodic surveillance of antibiotic resistance profiles in resource-poor countries is emphasized. The potential value of prophylactic measures like H. influenzae type b conjugate immunization is discussed.

Anti-Bacterial Agents↗
PubMed1991

Strand exchange protein 1 from Saccharomyces cerevisiae. A novel multifunctional protein that contains DNA strand exchange and exonuclease activities.

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA molecules from single-stranded circles and homologous linear duplex DNA in vitro. Previously, Sep1 was purified as a 132,000-Da species; however, DNA sequence analysis indicates that the SEP1 gene is capable of encoding a 175,000-Da protein (Tishkoff, D.X., Johnson, A.W., and Kolodner, R.D. (1991) Mol. Cell. Biol. 11, 2593-2608). The SEP1 gene was cloned into a GAL10 expression vector and expressed in a protease-deficient yeast strain. Intact Sep1, which migrated as a Mr-160,000 polypeptide during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to apparent homogeneity and shown to have activities similar to those of the originally purified Mr = 132,000 fragment. We report here that, in addition to strand exchange activity, Sep1 contains an intrinsic exonuclease that is active on single- and double-stranded DNA with a severalfold preference for single-stranded DNA. The nuclease was induced in crude extracts upon induction with galactose, it co-purified with the strand exchange activity of Sep1, and the nuclease and strand exchange activities of Sep1 showed the same kinetics of heat inactivation. Sep1 nuclease, which requires Mg2+, can be functionally separated from the strand exchange activity by the substitution of Ca2+ for Mg2+. Under these conditions, the nuclease is inactive, and strand exchange activity is dependent on prior resection of the DNA ends by an exogenous exonuclease. Thus, the nuclease is necessary for synapsis but not strand exchange. Electron microscopic analysis revealed that true strand exchange products, alpha molecules and nicked double-stranded circular molecules, were formed. In addition, strand transfer proceeded to similar extents on 5'-resected and 3'-resected DNA. This result suggests that the polarity of strand transfer by Sep1 is determined by the polarity of its intrinsic nuclease.

Amino Acid Sequence↗
PubMed1991

Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1.

Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.

Amino Acid Sequence↗
PubMed1990

Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV.

DNA damage generated by oxygen radicals includes base-free apurinic/apyrimidinic (AP) sites and strand breaks that bear deoxyribose fragments. The yeast Saccharomyces cerevisiae repairs such DNA lesions by using a single major enzyme. We have cloned the yeast structural gene (APN1) encoding this AP endonuclease/3'-repair diesterase by immunological screening of a yeast genomic DNA expression library in lambda gt11. Gene disruption experiments confirm that the Apn1 protein accounts for greater than or equal to 97% of both AP endonuclease and DNA 3'-repair diesterase activities in yeast cell-free extracts. The DNA and predicted amino acid sequences for the APN1 gene are homologous to those for the nfo gene encoding DNA endonuclease IV of Escherichia coli. This conservation of structure between a eukaryotic enzyme and its prokaryotic counterpart underscores the fundamental nature of their roles in DNA repair.

Amino Acid Sequence↗
PubMed1989

Ascorbic acid: a factor concentrated in human gastric juice.

1. Concentrations of ascorbic acid (ascorbic and dehydro-ascorbic; A + D; measured by the 2,4-dinitrophenylhydrazine method) of nearly three times those of plasma are present in gastric juice samples from patients with normal gastric histology. 2. A significant reduction in gastric juice ascorbic acid (A + D) was observed in patients with chronic gastritis. This reduction in concentration was independent of the grade of gastritis. 3. Concentrations of ascorbic acid (A + D) in gastric biopsy specimens were consistently higher in the antrum than in the body of the stomach. 4. These data demonstrate that considerable quantities of ascorbic acid (A + D) are normally 'secreted' into the stomach. 5. Ascorbic acid (ascorbic only; A; measured by h.p.l.c.) was present predominantly in its biologically active form in the patients with normal gastric histology. However, in patients with gastritis, independent of grade, ascorbic acid was present predominantly in its oxidized, biologically inactive form.

Adult↗
PubMed1988

Yeast DNA diesterase for 3'-fragments of deoxyribose: purification and physical properties of a repair enzyme for oxidative DNA damage.

The DNA strand breaks resulting from exposure to the free radicals generated by ionizing radiation or oxidizing agents are refractory to DNA repair synthesis because of deoxyribose fragments that block their 3' termini. The restoration of normal 3'-OH nucleotide primers is the essential first step in the excision repair of these radical-induced strand breaks. We have used a synthetic DNA substrate containing 3'-phosphoglycolaldehyde esters to identify and purify to physical homogeneity the major yeast diesterase that removes such nucleotide fragments. Yeast 3'-phosphoglycolaldehyde diesterase had Mr = 40,500 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular weight estimate from gel filtration indicated that the active species is a nearly globular monomer. Purification of the enzyme removed a tightly bound metal, but the activity of the purified enzyme could be restored by the addition of Co2+, Mn2+, Ni2+, or Zn2+, with Co2+ the most effective cofactor. Even 3 microM Co2+ stimulated near-maximal activity, and this metal also conferred significant thermal stability on the purified protein. This is a novel enzyme, whose N-terminal amino acid sequence does not show any significant similarity to published sequences, and which is not the product of any gene in the RAD52 epistasis group.

Cations, Divalent↗
PubMed1988

Yeast DNA 3'-repair diesterase is the major cellular apurinic/apyrimidinic endonuclease: substrate specificity and kinetics.

DNA strand breaks with damaged 3' termini are potentially toxic lesions caused by free radicals. The purified yeast diesterase that removes small nucleotide fragments from such 3' termini in oxidized DNA has been further characterized with respect to its substrate specificity. In addition to the 3'-phosphoglycolaldehyde esters used to monitor the activity during purification, the enzyme efficiently hydrolyzed a variety of other 3'-esters in DNA. These included 3'-phosphates, 3'-(2,3-didehydro-2,3-dideoxyribose phosphates), and the 3'-blocking damages formed in vivo in Escherichia coli by H2O2 or in vitro by DNA treatment with bleomycin. This same transition metal-dependent enzyme also constitutes the major yeast endonuclease for apurinic/apyrimidinic sites in DNA, hydrolyzing these damages to yield normal 3'-hydroxyl nucleotides and 5'-phosphoryl base-free sugar termini (a Type II apurinic/apyrimidinic endonuclease). Yeast 3'-phosphoglycolaldehyde diesterase therefore appears to be involved in two distinct pathways of DNA repair: initiation of the repair of oxidative strand breaks in DNA and the restoration of sites of base loss caused by many types of DNA-damaging agents.

Cations, Divalent↗
PubMed1988

Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA.

Agents that act via oxygen-derived free radicals form DNA strand breaks with fragmented sugar residues that block DNA repair synthesis. Using a synthetic DNA substrate with a single type of sugar fragment, 3'-phosphoglycolaldehyde esters, we show that in Escherichia coli extracts the only EDTA-resistant diesterase for these damages depends on the bacterial nfo (endonuclease IV) gene. Endonuclease IV was purified to physical homogeneity (Mr = 31,000) from an E. coli strain carrying the cloned nfo gene and in which the enzyme had been induced with paraquat. Although heat-stable and routinely assayed in the presence of EDTA, endonuclease IV was inactivated in the absence of substrate at 23-50 degrees C by either EDTA or 1,10-phenanthroline, suggesting the presence of an essential metal tightly bound to the protein. Purified endonuclease IV released phosphoglycolaldehyde, phosphate, and intact deoxyribose 5-phosphate from the 3'-end of DNA, all with apparent Km of 5-10 nM. The optimal KCl or NaCl concentration for 3'-phosphoglycolaldehyde release was 50-100 mM. The purified enzyme had endonuclease activity against partially depurinated DNA but lacked significant nonspecific nuclease activities. Endonuclease IV also activated H2O2-damaged DNA for repair synthesis by DNA polymerase I. Thus, endonuclease IV can act on a variety of oxidative damages in DNA, consistent with a role for the enzyme in combating free-radical toxicity.

DNA Damage↗
PubMed1988

Measurement of the transfer of the nitrogen moiety of intestinal lumen glutamic acid in man after oral ingestion of l-[15N]glutamic acid.

1. The measurement of the intestinal metabolism of the nitrogen moiety of glutamic acid has been investigated by oral ingestion of l-[15N]glutamic acid and sampling of arterialized blood. 2. Measurements have been made in six normal adults weighing an average of 72.8 kg ingesting 100 mg of l-[15N]glutamic acid after an overnight fast. 3. Measurement of the enrichment of arterial glutamic acid, glutamine and alanine was by gas chromatography-mass spectrometry. Isotopic enrichment of the amino acids was followed for 150 min after the ingestion of the amino acid. 4. Arterialized venous blood amino acid concentrations, measured by h.p.l.c., demonstrated no significant changes during the course of the experiment. 5. From the observed appearance of label in arterialized glutamic acid, alanine and glutamine, little luminal glutamic acid reaches the extracellular pool. The majority of the administered nitrogen label appears in the arterial alanine and glutamine components.

Administration, Oral↗
PubMed1988

In vivo and in vitro studies of microsphere pancreatic supplements.

Over the past 5 years, the Leeds Regional Cystic Fibrosis (CF) Unit has provided comprehensive annual assessments of CF patients that include dietary assessments and fat absorption studies. Enteric-coated microsphere pancreatic enzyme preparations (microsphere preparations) were compared to conventional enzymes as therapeutic agents for these patients. Presently in the U.K., two microsphere preparations are licensed for use and a further two products are likely to receive licenses in the near future. The success of these preparations is dependent on the ability of the microsphere coating to resist dissolution until the pH exceeds approximately 5.5 and thus prevent inactivation of lipase in the acid environment of the stomach. A study comparing Pancrex V Forte, a conventional enzyme preparation, to three microsphere preparations, Pancrease, Creon, and pancreatin Merck, confirmed the superiority of Pancrease and Creon over Pancrex V Forte and pancreatin Merck with regard to control of symptoms, and nitrogen and fat absorption. Because of differences in the physical characteristics of various microsphere preparations, the dissolution rates of Pancrease, Creon, and pancreatin Merck were compared in vitro. In aqueous buffers, striking differences among the preparations were seen at pH 5.5; whereas only 25% of available lipase was released from Creon, both Pancrease and pancreatin Merck show almost complete dissolution at this pH. Only at pH 6.5 and above do all three preparations show complete dissolution. In duodenal juice, as in aqueous buffers, lipase release from Creon takes place at a lower rate than with the other two preparations until pH 6.0 or higher is attained.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1985

Elevated rates of whole body protein metabolism in patients with disseminated malignancy in the immediate postoperative period.

Protein metabolism after surgery in the presence of disseminated malignancy has been investigated and compared with results obtained from patients undergoing similar surgical procedures for benign disease and localized malignancy. Whole body nitrogen turnover, measured by primed continuous infusion of 15N glycine, was highest with disseminated malignancy. Similarly rates of whole body protein synthesis and breakdown, calculated from turnover and nitrogen excretion (nitrogen intake being zero), were elevated in the presence of disseminated malignant disease. The increased rates of protein metabolism may represent adaptation to the demands of inevitably growing malignant tissue.

Aged↗