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Biomedical subjects

A W Smith

Publications and source records attributed to A W Smith.

At least 19 recordsLinked to original sources
PubMed1992

Analysis of promoter activity by transformation of Acremonium chrysogenum.

Promoter activity was examined in the beta-lactam-producing fungus, Acremonium chrysogenum, by assessment of the properties of transformant isolates. Transformation was achieved using plasmid constructs specifying hygromycin B resistance (HyR) linked to the promoter elements of gpdA (the glucose-6-phosphate dehydrogenase-encoding gene of Aspergillus nidulans), and pcbC [the gene encoding the isopenicillin N synthetase (IPNS) enzyme of A. chrysogenum]. Transformation frequency, HyR levels, and Hy phosphotransferase (HPT) levels suggested that the transformants of constructs using the gpdA promoter showed a higher level of expression of the HyR gene than in transformants obtained using the pcbC promoter. The patterns of integration of the transforming DNA also differed in that pcbC promoter construct transformants appeared to have tandem repeats. All integrations of plasmid DNA occurred on a single chromosome which was different in four out of five transformants studied. Multiple copy transformants of constructs using the pcbC promoter did not show the regulated pattern of expression of HPT activity observed with IPNS in untransformed strains.

Acremonium↗
PubMed1992

Siderophore-specific induction of iron uptake in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has two siderophore-based high-affinity iron-uptake systems utilizing pyoverdin and pyochelin. Using strain IA1, a mutant deficient in production of both siderophores, we have shown that addition of purified siderophore to the growth medium induces expression of specific iron-regulated outer-membrane proteins and increases 55Fe-siderophore transport. Addition of pyoverdin from the parent strain PAO1 or from a clinical strain 0:12 induced expression of an 85 kDa IROMP and increased the rate of 55Fe-pyoverdin transport. Transport rates for 55Fe-PAO1 pyoverdin increased from 1.27 to 3.57 pmol Fe min-1 per 10(9) cells. Addition of purified pyochelin induced expression of a 75 kDa IROMP accompanied with increased 55Fe-pyochelin uptake without affecting 55Fe-pyoverdin transport. 55Fe-pyochelin transport increased from 0.3 to 10.6 pmol min-1 per 10(9) cells. Addition of pyoverdin from the parent strain or a chromatographically distinct pyoverdin caused increased reactivity with an anti-85 kDa mAb in Western blotting, indicating that the same receptor is being induced. These results suggest that P. aeruginosa can respond specifically to the presence of siderophore and moreover that not only can the pyoverdin receptor transport its cognate ferri-pyoverdin but also different ferri-pyoverdins, albeit at a reduced rate.

Bacterial Outer Membrane Proteins↗
PubMed1992

The pyocin Sa receptor of Pseudomonas aeruginosa is associated with ferripyoverdin uptake.

We have used Tn5 mutagenesis to obtain a mutant resistant to pyocin Sa. When grown in iron-deficient succinate medium this mutant lacked an 85-kDa iron-regulated outer membrane protein (IROMP), and expression of a 75-kDa IROMP was increased compared with that in the parent strain. The mutant was deficient in pyoverdin biosynthesis and showed a 95% decrease in transport of ferripyoverdin purified from the parent strain, suggesting that the 85-kDa IROMP is the specific receptor for ferripyoverdin and pyocin Sa. The mutant compensated for the deficiency in pyoverdin biosynthesis and transport by exhibiting a fourfold increase in ferripyochelin transport. The low-level transport of ferripyoverdin in the Sa-resistant mutant, which extended to heterologous pyoverdins from other strains, suggests that Pseudomonas aeruginosa has a second ferripyoverdin uptake system of lower affinity and broader specificity.

Bacterial Outer Membrane Proteins↗
PubMed1992

Pinealitis coincident with recurrent uveitis: immunohistochemical studies.

Although experimental models of autoimmune uveitis predict pinealitis coincident with uveitis, there is no direct evidence of pineal pathology accompanying a human uveitis. Horses with naturally occurring uveitis are a potential source of eye and pineal tissues that are not available from human patients with active uveitis. We have observed pinealitis in a mare with equine recurrent uveitis. By immunohistochemistry we demonstrated immunoglobulin and MHC Class II antigen on infiltrating and resident cells of eye and pineal gland. These results support the relevance of the animal models and suggest that pinealitis may be coincident with some human uveitides.

Animals↗
PubMed1992

Metabolic oxidation and toxification of N-methylformamide catalyzed by the cytochrome P450 isoenzyme CYP2E1.

Alkylformamides, for example N-methylformamide, are hepatotoxic in rodents and humans. The mechanism by which N-methylformamide exerts its hepatotoxicity involves metabolic oxidation at the formyl moiety to yield a short-lived intermediate, perhaps methyl isocyanate, which reacts with glutathione to afford S-(N-methylcarbamoyl)glutathione. The hypothesis that the cytochrome P450 isozyme CYP2E1 catalyzes the metabolic toxification of N-methylformamide was tested. Hepatocytes obtained from mice that had received acetone, an inducer of CYP2E1, were incubated for up to 4 hr with N-methylformamide (5 and 10 mM). Whereas N-methylformamide caused cytotoxicity in these cells, as measured by release from the cells of lactate dehydrogenase, it was barely toxic, under these conditions, to cells from untreated mice. Coincubation of N-methylformamide with dimethylsulfoxide (10 mM), a CYP2E1 inhibitor, for 4 or 6 hr abolished the hepatocytotoxicity of N-methylformamide. Metabolism of N-methylformamide to S-(N-methylcarbamoyl) glutathione was measured in incubates with liver microsomes from rats, mice, or humans in the presence of glutathione. Pretreatment of rodents with acetone or ethanol induced the rate of metabolism of N-methylformamide and of p-nitrophenol, a known CYP2E1 substrate, but it did not increase aminopyrine N-demethylation. Metabolism of N-methylformamide and p-nitrophenol was elevated in microsomes from animals that had received acetone (1%) in their drinking water for 1 week to 230% and 200%, respectively, of control values in mouse microsomes and to 310% and 240%, respectively, of control values in rat microsomes. Pretreatment of animals with 4-methylpyrazole (200 mg/kg intraperitoneally, once daily for 3 days) increased metabolism of N-methylformamide to 410% of control values in rat liver microsomes but was without effect on murine microsomal metabolism of N-methylformamide. The metabolism of this compound was strongly inhibited by the CYP2E1 substrates or inhibitors dimethylsulfoxide (1-100 mM), p-nitrophenol (100 microM), and diethyldithiocarbamate (100 microM), which did not affect aminopyrine N-demethylation. A polyclonal antibody against rat CYP2E1 (10 mg of IgG/nmol of cytochrome P450) inhibited N-methylformamide metabolism in liver microsomes from rats and from a human by 75% and 80%, respectively. The rate of metabolism of N-methylformamide to S-(N-methylcarbamoyl) glutathione was determined in liver microsomes from six humans and correlated with extent of metabolic hydroxylation of chlorzoxazone, a CYP2E1 probe, and with amount of immunodetectable enzyme using an anti-rat CYP2E1 antibody (r = 0.81 and 0.80, respectively). The results suggest that CYP2E1 is the predominant, if not sole, cytochrome P450 isozyme responsible for the metabolic toxification of hepatotoxic N-alkylformamides.

Acetone↗
PubMed1991

Chromosome rearrangements in improved cephalosporin C-producing strains of Acremonium chrysogenum.

A number of A. chrysogenum strains from a lineage improved in cephalosporin C production were analysed by contoured-clamped homogeneous electric field gel electrophoresis (CHEF). Although antibiotic titre was increased across the lineage, chromosome rearrangements were only observed at two points in it. In one member of the lineage the chromosomal changes included those which altered the size of the chromosome on which the isopenicillin N synthetase gene (pcbC) was located. It is proposed that chromosome changes are a chance event in an industrial strain improvement programme.

Acremonium↗
PubMed1991

Production and characterization of monoclonal antibodies to outer membrane proteins of Pseudomonas aeruginosa grown in iron-depleted media.

The iron uptake systems of pathogenic bacteria provide potential targets for immunological intervention. We have partially purified the high molecular mass, iron-regulated outer membrane proteins (IROMPs) from Pseudomonas aeruginosa and used them to prepare a panel of monoclonal antibodies (mAbs). Five mAbs reacted with an 85 kDa IROMP separated by SDS-PAGE, but gave only low-level binding to whole cells by immunogold electron microscopy. However, iodination of whole cells indicated that the 85 kDa IROMP is surface-exposed. The mAbs were only cross-reactive with clinical isolates representing eight of the 17 International Antigenic Typing Scheme serotypes of P. aeruginosa, suggesting significant heterogeneity with respect to this IROMP.

Antibodies, Bacterial↗
PubMed1991

Immune response to Acinetobacter calcoaceticus infection in man.

After growth in an iron-depleted chemically-defined medium Acinetobacter calcoaceticus expressed four high mol. wt outer-membrane proteins (OMPs) which were repressed under iron supplementation or in a complex laboratory medium. Immunoblotting with serum from a septicaemic patient infected with A. calcoaceticus revealed antibody binding to these iron-repressible OMPs, indicating that they were expressed in vivo, and also to the 42- and 18-Kda OMPs. Although the antibody response to the OMPs did not vary significantly during convalescence, the response to the O-polysaccharide component of lipopolysaccharide decreased significantly. However, antibodies in serum from patients with A. calcoaceticus wound infections reacted with the iron repressible OMPs and a 54-Kda antigen suggesting a difference in immune recognition between local and systemic infection.

Acinetobacter↗
PubMed1991

Diversity of pilin of serologically distinct Bacteroides nodosus.

Pili from 11 distinct serotypes of Bacteroides nodosus were examined for diversity of pilin polypeptide subunits among serotypes and for purity of the pilin preparations. The pilin of all 11 samples was shown to be homogeneous. Mean +/- SD molecular weight of the pilin of 7 serotypes (A198, IV, V, VI, IX, XVII, and XVIII) was 18,500 +/- 100. The pilin of serotypes I, III, and VIII had molecular weight of 17,600, 19,400, and 19,000, respectively. Serotype XV differed greatly from the other 10 serotypes in that 2 distinct polypeptide bands with molecular weight of approximately 7,800 and 6,200 were detected. We suggest that these 2 low molecular weight bands resulted from proteolytic cleavage of the pilin protein.

Bacteroides↗
PubMed1991

Electron microscopic study of Bacteroides nodosus pili and associated structures.

Surface structures of Bacteroides nodosus were examined by electron microscopy. Collodion film and chrome shadowing were used for maximizing the visualization of B nodosus pili and ring structures. The existence of B nodosus pili in foot rot lesions was confirmed. Contrary to previous reports, it was found that B nodosus pili production can be retained through serial broth transfer under certain conditions. Capsule production by B nodosus was irregular in that it could be either lacking or variable in thickness. A bacteriophage capable of infecting B nodosus also was detected.

Animals↗
PubMed1991

Monoclonal antibodies against pili of serologically distinct Bacteroides nodosus.

Several monoclonal antibodies (McAbs) against pili of Bacteroides nodosus were examined to determine their reactivity with 11 different serotypes. One McAb was identified by enzyme-linked immunosorbent assay (ELISA) analysis that bound to nine of the 11 serotypes and another that bound to the remaining two serotypes tested. In addition, some McAbs demonstrated specificity for a single serotype, while others displayed specificities for up to five other serotypes. Comparison of immunoblot analysis with the ELISA revealed that the former method was not as sensitive in that all McAbs positive by the ELISA, were not positive by immunoblot. Possible explanations of these findings are discussed. There appear to be several antigenic determinants on B. nodosus pili and considerable sharing of these determinants between pili types. The 11 serotypes analyzed by the McAbs in this report are representative of all 20 US serotypes as well as the A-set and D-set categories of Australia. Therefore, the two epitopes recognized by two of the McAbs reported herein encompass all of the currently characterized B. nodosus serotypes and may provide a basis for bivalent vaccines efficacious for all types of B. nodosus induced footrot in sheep.

Animals↗
PubMed1990

Characterisation of a siderophore from Acinetobacter calcoaceticus.

The Gram-negative bacterium Acinetobacter calcoaceticus was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron chelator, 2,3-dihydroxybenzoic acid was identified in the culture supernatant by 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). A group of outer membrane proteins between 80 and 85 kDa were induced under iron restriction.

Acinetobacter↗
PubMed1990

Regulation of isopenicillin N synthetase (IPNS) gene expression in Acremonium chrysogenum.

Total RNA was extracted daily from the beta-lactam antibiotic producing fungus A. chrysogenum strain CO728 during a 7 day cephalosporin C fermentation. IPNS mRNA species, with a size of about 1.5 kb, were detected by Northern blotting at high levels between days 2 and 4. The rapid appearance of IPNS mRNA in mycelial extracts up to day 2 suggests that IPNS is regulated at the transcriptional level. Primer extension and S1 endonuclease mapping studies indicate the existence of two major and at least two minor transcription initiation start sites. There was no change in the relative levels of the four transcripts during the period they could be detected. A region upstream of the IPNS structural gene (pcbC) has been sequenced and the transcription initiation sites appear as major and minor pairs on either side of one of the pyrimidine-rich blocks that punctuate the promoter sequence.

Acremonium↗
PubMed1990

New marine calicivirus serotype infective for swine.

A new serotype of calicivirus was isolated from California sea lions (Zalophus californianus) with severe vesicular disease. Neutralizing antibodies were found in 27 of 82 (32.9%) serum samples from California sea lions and in 15 of 146 (10.3%) serum samples from Steller sea lions (Eumetopias jubatus) tested. The seropositive animals were widely dispersed along the margins of the eastern Pacific basin, from the Bering Sea to the Santa Barbara Channel. Seropositive samples were found from as early as 1976 through the present time. This new calicivirus serotype, San Miguel sea lion virus type 13, was inoculated into weaned pigs, resulting in induction of severe vesicular disease, which spread to all pigs, including uninoculated pen contacts. Virus was continually shed by most of the pigs throughout the 2-week duration of the experiment.

Animals↗
PubMed1990

Sultamicillin (sulbactam/ampicillin) versus amoxycillin in the treatment of acute otitis media in children.

A comparative study of sultamicillin (an orally active dimer of sulbactam and ampicillin) and amoxycillin for the treatment of acute otitis media was carried out in 86 children with acute otitis media. After 10 days' treatment, of those with traditional middle ear pathogens, 35/36 (97%) 50 mg/kg.day or 500 mg/day sultamicillin-treated children were cured with eight relapses after a further 18 days without treatment compared with 12/13 (92%) 40 mg/kg.day or 250 mg/day amoxycillin-treated children and three subsequent relapses. All six beta-lactamase-producing pathogens were in the sultamicillin treatment group: four were Haemophilus influenzae/H. parainfluenzae that were resistant to amoxycillin, and all were cured although two then relapsed. No significant clinical or laboratory side-effects were noted in either amoxycillin- or sultamicillin-treated patients. It is concluded that sultamicillin was comparable to amoxycillin in the treatment of acute otitis media.

Acute Disease↗
PubMed1989

Iron-deficiency anaemia and its response to oral iron: report of a study in rural Gambian children treated at home by their mothers.

Haematological and iron parameters, measured in 907 children aged from 6 months to 5 years in rural Gambia at the start of the rainy season, differed from those in American reference populations as follows: mean haemoglobin levels were much lower at ages 1 and 2 years and mean levels of mean corpuscular volume (MCV) were lower at all ages (at age 1 year mean haemoglobin was 11.2 g/dl and mean MCV 68.2 fl); in a sample of 249 children randomly selected from the whole study population, mean serum iron levels were similar but mean transferrin saturation and mean serum ferritin levels were lower, especially at ages 1-3 years (at age 1 year mean serum iron was 11.1 mumol/l, mean transferrin saturation 16.9%, and geometric mean serum ferritin 8.8 ng/ml. A total of 213 children (23%) whose haemoglobin and mean corpuscular volume were both less than the 3rd percentile of the reference population received oral iron or placebo from their mothers during the rainy season when malaria transmission is maximal. Mean levels of haemoglobin, mean corpuscular volume, serum iron, transferrin saturation and serum ferritin rose in the iron-treated group and fell in the placebo group at all ages, except under 1 year for serum ferritin, to produce significant differences between the groups by the end of the study. Total iron-binding capacity showed no significant changes during the study. We concluded that oral iron given by the mother during the rainy season can be used to treat iron-deficiency anaemia in Gambian children who would otherwise become more anaemic.

Administration, Oral↗
PubMed1989

The effects on malaria of treatment of iron-deficiency anaemia with oral iron in Gambian children.

In order to determine whether giving iron to iron-deficient children increases their susceptibility to malaria, 213 Gambian children aged between 6 months and 5 years with iron-deficiency anaemia were randomized to receive either oral iron or placebo during the rainy season when malaria transmission is maximal. Haematological and iron measurements improved significantly in the group given iron. Regular morbidity surveys showed that fever associated with parasitaemia occurred more frequently in the iron-treated group than in the placebo group. This difference was not significant for all parasitaemias grouped together, but became significant and progressively larger for parasitaemias of ten or more positive fields per 100 high power fields (P less than 0.025), and for parasitaemias of 50 or more positive fields per 100 high power fields (P less than 0.01). Three children in the iron-treated group but none in the placebo group had more than one episode of fever and parasitaemia. Splenomegaly rates rose appreciably during the study in both groups, but in children at age 2 years the splenomegaly rate at the end of the study was significantly greater in the iron-treated group. We concluded that there is a significantly increased risk of fever associated with severe malarial parasitaemia for children with iron-deficiency anaemia given iron during the season of maximal malaria transmission in this part of The Gambia.

Administration, Oral↗