Search PubMed⌕ Search

Biomedical subjects

A W Johnson

Publications and source records attributed to A W Johnson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Regulation and intracellular localization of Saccharomyces cerevisiae strand exchange protein 1 (Sep1/Xrn1/Kem1), a multifunctional exonuclease.

The Saccharomyces cerevisiae strand exchange protein 1 (Sep1; also referred to as Xrn1, Kem1, Rar5, or Stp beta) catalyzes the formation of hybrid DNA from model substrates in vitro. The protein is also a 5'-to-3' exonuclease active on DNA and RNA. Multiple roles for the in vivo function of Sep1, ranging from DNA recombination and cytoskeleton to RNA turnover, have been proposed. We show that Sep1 is an abundant protein in vegetative S. cerevisiae cells, present at about 80,000 molecules per diploid cell. Protein levels were not changed during the cell cycle or in response to DNA-damaging agents but increased twofold during meiosis. Cell fractionation and indirect immunofluorescence studies indicated that > 90% of Sep1 was cytoplasmic in vegetative cells, and indirect immunofluorescence indicated a cytoplasmic localization in meiotic cells as well. The localization supports the proposal that Sep1 has a role in cytoplasmic RNA metabolism. Anti-Sep1 monoclonal antibodies detected cross-reacting antigens in the fission yeast Schizosccharomyces pombe, in Drosophila melanogaster embryos, in Xenopus laevis, and in a mouse pre-B-cell line.

Animals↗
PubMed1995

Microbial inciters of acute asthma in urban Nigerian children.

BACKGROUND: In tropical Africa the role of microbial agents of acute respiratory infections in acute exacerbations of bronchial asthma remains largely unexplored. However, empirical antibacterial therapy is frequently initiated in moderate to severe cases of acute asthma with symptoms of acute respiratory infection. A study was set up to determine how often acute respiratory infection is associated with acute asthma, to identify the associated pathogens, and to proffer appropriate therapeutic suggestions. METHODS: Over a 16 month period, 86 episodes of acute asthma were studied for clinical and laboratory features of acute respiratory infection at the University College Hospital (UCH), Ibadan. Virological diagnosis was based on immunofluorescence studies of nasopharyngeal aspirates and/or serological tests using the microtitre complement fixation technique. Throat swabs and blood were cultured for bacterial agents. RESULTS: Of the 64 cases who presented with rhinorrhoea, 51 (79.7%) were pyrexial (T > or = 37.6 degrees C). Inflammatory changes (frequently interstitial streakiness) were identified in 10 (19.6%) of the 51 chest radiographs; only two of these had lobar shadowing. Significant bacterial isolates were made in only three (3.5%) of the throat swabs and two (2.4%) of the blood cultures from the 86 cases; none had clinical septicaemia. On the other hand, 55 viral agents were identified from 39 (53%) of the 74 subjects studied; 16 (41.0%) had dual viral identifications. Respiratory syncytial virus (RSV) accounted for 20 (36.4%) identifications, parainfluenza virus (PIV) type 3 for 15 (27.3%), and influenza type A (Flu A) for 12 (21.8%). Viral identifications were significantly higher in infants and preschool subjects (< 5 years) and in those presenting with either rhinorrhoea or pyrexia. CONCLUSIONS: The results of this study underscore the importance of viral upper respiratory infections in asthma exacerbations in a tropical setting. The paucity of clinical and investigative features of bacterial acute respiratory infection suggests that there is little rationale for routine antibiotic cover in children with acute exacerbations of asthma in the tropics.

Acute Disease↗
PubMed1994

The activity of the Saccharomyces cerevisiae strand exchange protein 1 intrinsic exonuclease during joint molecule formation.

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA from single-stranded and homologous linear duplex DNA. The initial pairing reaction requires limited exonucleolytic digestion of the double-stranded DNA (dsDNA) by the intrinsic 5' to 3' exonuclease of Sep1 or by an exogenous exonuclease. Subsequent strand exchange proceeds without the need for exonuclease activity. Sep1 degrades linear dsDNA at a rate of 20 nucleotides/min with an average processivity of 45 nucleotides. During strand exchange reactions joint molecules are first observed after 1-2 min, suggesting that only limited digestion is necessary for pairing. The linear dsDNA was found to pair with single-stranded DNA (ssDNA) when it was resected by only 22 nucleotides, and linear dsDNA digested by more than 22 nucleotides was observed only in joint molecules. Approximately 20 nucleotides were also the minimum extent of digestion that could support pairing. In the absence of exonuclease activity, Sep1-promoted pairing requires dsDNA molecules with single-stranded tails homologous to the circular ssDNA. These results suggest that Sep1-promoted strand exchange requires a single-strand annealing event prior to the strand displacement phase of the reaction.

Bacteriophage M13↗
PubMed1993

Tuberculous laryngitis in a Nigerian child.

A 12.5-year-old Nigerian child presented with progressively worsening hoarseness of 9 months duration, without overt features of chronic ill health. Superficial discrete cervical adenitis, radiological evidence of miliary tuberculosis and hilar adenopathy were identified on admission. The laryngoscopic findings comprised fully mobile but 'beefy' red, oedematous vocal cords and interarytenoid region. The diagnosis of tuberculous laryngitis was made on the basis of the laryngoscopic, radiological and clinical improvement that followed antituberculous therapy. The possible pathogeneses of tuberculous laryngitis are discussed. We emphasize the importance of considering tuberculous laryngitis in a child with persistent hoarseness.

Child↗
PubMed1993

Bacterial aetiology of acute lower respiratory infections in pre-school Nigerian children and comparative predictive features of bacteraemic and non-bacteraemic illnesses.

In a 9-month surveillance of the microbial agents causing acute lower respiratory infections (ALRI) in hospitalized preschool Nigerian children, 24 bacterial isolates were made from 22 (33 per cent) out of 66 blood cultures, including seven (70 per cent) of the 10 cultures from subjects with pleural effusion. Of the 24 positive isolates, Staphylococcus aureus accounted for 14 (58 per cent), Klebsiella pneumoniae for four (17 per cent), Streptococcus pneumoniae and Staphylococcus albus for two (8 per cent) each, and Haemophilus influenzae for only one case. Seven subjects had a mixed aetiology, comprising one bacteraemic case with measles and six with concomitant viral identifications. Sixteen cases, 10 (63 per cent) of whom were malnourished, had two or more pathogens, including 10 with multiple viruses. Bacterial isolates from the throat, were not significantly predictive of the ALRI aetiology. Overt malnutrition, empyema, and anaemia, were significantly commoner in bacteraemic cases (P < 0.03, 0.01, and 0.05), while rhinorrhoea and crepitations were each associated with the non-bacteraemic ones. (P = 0.05 and < 0.05). A similar association was shown between rhinorrhoea, wheezing/rhonchi and the mixed aetiological group (P < 0.05 and 0.05, respectively). The case-fatality in bacteraemic subjects, was 9 per cent and was 2.1 times higher than that for non-bacteraemic cases, in whom a shorter mean duration of admission was recorded. In developing countries, the need for periodic local surveillance of ALRI pathogens, as a prerequisite for evolving rational antimicrobial policies, is emphasized. Our findings underscore the predictive importance of malnutrition as a risk-factor of severe bacteraemic ALRI, frequently associated with multiple pathogens. The relative usefulness of blood culture in identifying bacterial agents of ALRI is discussed.

Acute Disease↗
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↗