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Biomedical subjects

S M Payne

Publications and source records attributed to S M Payne.

17 recordsLinked to original sources
PubMed1992

New methods for evaluating utilization management programs.

Blue Cross and Blue Shield of Massachusetts, Inc (BCBS), has developed two new methods for measuring the effect of utilization management (UM) in reducing unnecessary hospital use. The "program component" method measures the separate effect of preadmission review, concurrent review, and discharge planning. The "savable days" method produces a composite measure of the effectiveness of the program as a whole. The use of these two methods is illustrated with five years of utilization review data from the BCBS nongroup insurance product. The results can be used by operations managers and policymakers to measure the performance of individual UM components and the program as a whole, to establish goals and monitor program performance, to modify the program in response to changing utilization patterns, to assist in developing premiums, to establish risk-sharing agreements with employers or providers, and to demonstrate the effectiveness of the program for use in marketing.

Blue Cross Blue Shield Insurance Plans↗
PubMed1992

Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins.

IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.

Amino Acid Sequence↗
PubMed1992

Identification of the vibriobactin receptor of Vibrio cholerae.

Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.

Bacterial Outer Membrane Proteins↗
PubMed1992

Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae.

A 74-kDa iron-regulated outer membrane protein of Vibrio cholerae acts as the receptor for the V. cholerae iron-siderophore complex, ferric vibriobactin. MBG14, a mutant of V. cholerae 0395 containing a TnphoA insertion in a gene designated viuA, lacks this 74-kDa outer membrane protein and is unable to bind or utilize exogenous ferric vibriobactin. Introduction of a plasmid containing the complete viuA coding sequence and 513 bp of upstream DNA into MBG14 restored ferric vibriobactin utilization to the mutant. The DNA insert in this plasmid was sequenced, revealing a single open reading frame of 2,061 bp, encoding a deduced protein of 687 amino acids with a predicted molecular mass of 76,417 Da and a predicted initial signal sequence of 37 amino acids. ViuA showed only weak homology to two iron-regulated outer membrane proteins in Escherichia coli, IutA and FecA. Construction of viuA::TnphoA gene fusions allowed study of the regulation of viuA expression by iron. This regulation in E. coli was dependent on the fur gene. Northern (RNA) blot analysis of RNA from wild-type V. cholerae grown in high- and low-iron media revealed a monocistronic viuA message that was negatively regulated by iron at the transcriptional level. Primer extension analysis identified a single transcriptional start site, located 243 bp above the translational start site. The promoter region of viuA contained two interrupted dyad symmetric nucleotide sequences, overlapping the -10 and -35 boxes, each similar to the E. coli Fur binding consensus sequence. Another iron-regulated gene in V. cholerae that is negatively regulated by fur, irgA, requires a positive transcriptional activator (irgB) for expression. However, a strain of V. cholerae mutant in irgB was unaffected in viuA expression. These studies suggest that there is conserved, global coordinate iron regulation in V. cholerae by fur; additional regulatory factors, superimposed upon the fur system, may provide more precise control of individual iron-regulated genes.

Alkaline Phosphatase↗
PubMed1991

Synthesis and siderophore activity of albomycin-like peptides derived from N5-acetyl-N5-hydroxy-L-ornithine.

N5-Acetyl-N5-hydroxy-L-ornithine (1), the key constituent of several microbial siderophores, has been synthesized in 23% yield overall from N-Cbz-L-glutamic acid 1-tert-butyl ester (6) derived from L-glutamic acid. Reduction of 6 to 7 and treatment with N-[(trichloroethoxy)carbonyl]-O-benzylhydroxylamine (8), and diethyl azodicarboxylate and triphenylphosphine followed by deprotection produced the protected N5-acetyl-N5-hydroxy-L-ornithine derivatives 11 and 12 in large quantities (10-20 g). Following alpha-amino and alpha-carboxyl deprotections of 11 and 12, EEDQ [2-ethoxy-N-(ethoxycarbonyl)-1,2-dihydroquinoline] mediated peptide coupling and final deprotection provided amino acid 1 and six albomycin-like peptides (20, 23, 25, 28, 35, and 36). The growth-promoting ability of each was evaluated with the siderophore biosynthesis mutant Shigella flexneri SA240 (SA 100 iucD:Tn5). These results indicate that substantial modification of the framework of peptide-based siderophores can be tolerated by microbial iron-transport systems.

Anti-Bacterial Agents↗
PubMed1991

Synthesis and siderophore and antibacterial activity of N5-acetyl-N5-hydroxy-L-ornithine-derived siderophore-beta-lactam conjugates: iron-transport-mediated drug delivery.

N5-Acetyl-N5-hydroxy-L-ornithyl-N5-acetyl-N5-hydroxy-L-ornithyl-N5-acety l- N5-hydroxy-L-ornithine, the functionally instrumental component of the albomycins and ferrichromes, has been incorporated as a "carrier" substructure into both carbacephalosporin and oxamazin type beta-lactam antibiotics. The previously synthesized protected version of this tripeptide (14) was coupled with various beta-lactam analogues 17, 19, 24, and 25 to give protected conjugates 21, 22, 26, and 27. Final deprotection by hydrogenolysis provided the deprotected siderophore-beta-lactam antibiotic conjugates 1-4. The growth-promoting ability of each has been evaluated using either the siderophore-deficient mutant Shigella flexneri SA 100 or S. flexneri SA240 (SA 100 iucD:Tn5). Measurement of the growth-promoting activity using two isogenic Escherichia coli strains differing only in the presence or absence of fhuA (hydroxamate ferrichrome receptor) suggests uptake by the hydroxamate iron-transport system. The antibacterial activity of these conjugates has been investigated, and the potential for use of the ferrichrome iron-transport system as a means of drug delivery is discussed.

Anti-Bacterial Agents↗
PubMed1991

Iron uptake in Plesiomonas shigelloides: cloning of the genes for the heme-iron uptake system.

The iron uptake systems of Plesiomonas shigelloides strains were determined. Siderophore production was not detected by chemical or biological assays, and the strains tested were unable to use enterobactin, aerobactin, or vibriobactin for growth in low-iron media. Both hemin and hemoglobin supported full growth of the bacteria in media lacking other iron sources, but neither transferrin nor lactoferrin served as a source of iron. Hemolysin was detected, and the production of hemolysin was iron repressible. DNA sequences encoding hemolysin production and DNA sequences encoding the ability to use heme or hemoglobin as a sole source of iron were cloned from P. shigelloides and expressed in Escherichia coli. The abilities to use heme and hemoglobin as iron sources were closely linked, and the cloned sequences encoded the ability to transport the porphyrin, as well as iron, into the cells.

Biological Transport↗
PubMed1991

Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila.

Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete nucleotide sequence of amoA was determined. A putative iron-regulatory sequence resembling the Fur repressor protein-binding site overlapped a possible promoter region. A translational reading frame, beginning with valine and encoding 396 amino acids, was open for 1,188 bp. The C-terminal portion of the deduced amino acid sequence showed 58% identity and 79% similarity with the E. coli EntC protein (isochorismate synthetase), the first enzyme in the E. coli 2,3-DHB biosynthetic pathway, suggesting that amoA probably encodes a step in 2,3-DHB biosynthesis and is the A. hydrophila equivalent of the E. coli entC gene. An isogenic amonabactin-negative mutant, A. hydrophila SB22, was isolated after marker exchange mutagenesis with Tn5-inactivated amoA (amoA::Tn5). The mutant excreted neither 2,3-DHB nor amonabactin, was more sensitive than the wild-type to growth inhibition by iron restriction, and used amonabactin to overcome iron starvation.

Aeromonas↗
PubMed1991

Genetic analysis of the enterobactin gene cluster in Shigella flexneri.

The genes for transport and synthesis of the phenolate siderophore enterobactin are present on the chromosomes of both Ent+ and Ent- clinical isolates of Shigella flexneri. To determine why Ent- S. flexneri isolates fail to express a functional enterobactin system, the structure and expression of enterobactin genes were examined. Several alterations may be responsible for the inability of S. flexneri to express enterobactin. (i) The mRNA levels produced from the entC and fepB genes were not derepressed in low-iron media. (ii) DNA sequence analysis of the entC-fepB intergenic region revealed an 83-bp noncontiguous deletion in the putative fepB leader sequence. The deleted sequences are in a region which would be capable of forming extensive stem-and-loop structures. (iii) An amber codon in the 5' portion of the entC gene was also detected. (iv) An IS1 element, previously mapped to the Ent- S. flexneri enterobactin gene cluster, was found to lie within a potential transcriptional termination sequence in the entF-fepE intergenic region. (v) A mutation responsible for the inactivation of the entF gene was mapped to the entF coding region by using entF hybrid gene fusions. (vi) A comparison of outer membrane profiles from an E. coli strain harboring the cloned fepA gene from either an Ent+ or Ent- Shigella isolate revealed that the Ent- FepA protein is present in the outer membrane but at greatly reduced levels than that of the Ent+ FepA protein. This observation, along with additional studies, suggests that the Ent- FepA may be defective in translation and/or translocation.

Base Sequence↗
PubMed1978

Siderophore production by Vibrio cholerae.

Vibrio cholerae produces a phenolate-type siderophore that stimulates growth of the organism in low-iron medium. This compound is similar, but not identical, to enterochelin, the siderophore produced by Salmonella and Escherichia coli.

Chromatography, Paper↗
PubMed1978

Role of iron in disseminated gonococcal infections.

Strains of Neisseria gonorrhoeae isolated from disseminated gonococcal infections have enhanced ability to acquire iron in an experimental host when compared with urogenital gonococcal isolates from patients with uncomplicated infections.

Animals↗
PubMed1978

The critical role of iron in host-bacterial interactions.

The ability of potential pathogens to acquire iron in a host is an important determinant of both their virulence and the nature of the infection produced. Virulent gram-negative bacteria are capable of acquiring sufficient iron from the host because their virulence (for chick embryos) is unaffected by exogenous iron. Avirulent mutants which are apparently limited in their ability to acquire iron could be isolated from the virulent strains. The lethality of these mutants was significantly enhanced by exogenous iron. Reduction of the relatively high serum iron saturation of chick embryos (to levels more closely approximating those in man) by pretreatment with iron-binding proteins or endotoxin inhibits the lethality of some virulent bacteria. Those bacteria whose virulence was reduced include the Shigella, Vibrio cholerae and strains of Neisseria gonorrhoeae, all of which are nondisseminating pathogens in the normal human host. Pathogens which produce septicemic and disseminating infections such as Neisseria meningitidis, Haemophilus influenzae type B, Escherichia coli possessing K-1 antigen, Pseudomonas aeruginosa and Salmonella typhimurium and disseminating strains of N. gonorrhoeae were, in general, unaffected by reduced serum iron saturation. These disseminating bacteria appeared to produce greater quantities of compounds (siderophores) which stimulated microbial growth in low-iron media than did the nondisseminating pathogens. Thus, the gram-negative bacteria tested can be divided into four major classes according to their responses to modifications in iron levels in the chick embryo model and these results correlate with the nature of the infections which they typically produce in man.

Animals↗
PubMed1977

Detection and differentiation of iron-responsive avirulent mutants on Congo red agar.

Agar medium containing Congo red dye differentiates virulent and avirulent colonies of Shigella, Vibrio cholerae, Escherichia coli, and Neisseria meningitidis. Like virulent plague bacilli, wild-type cells of these species absorb the dye and produce red colonies. Mutants or colonial variants have been isolated that fail to absorb the dye and produce colorless colonies. These mutants exhibit reduced virulence in the chicken embryo model, but their virulence is enhanced by supplementation with iron. Of those species tested, only Neisseria gonorrhoeae isolates failed to grow in the presence of this dye. Inhibition of growth by Congo red may thus provide a simple means for differentiating gonococci from other Neisseria.

Bacteria↗
PubMed1977

Imferon agar: improved medium for isolation of pathogenic Neisseria.

Imferon, an iron-dextran complex, enhances the growth of Neisseria gonorrhoeae and N. meningitidis. The use of Imferon as a replacement for ferric nitrate, in a defined supplement for GC agar significantly increased the average colony sizes of both gonococci and meningococci. In comparison with Thayer-Martin medium, Imferon agar increased the speed and rate of isolation of gonococci from clinical specimens.

Agar↗
PubMed1975

Pathogenesis and immunology of experimental gonococcal infection: role of iron in virulence.

Addition of iron componds to inocula of the relatively avirulent T3 or T4 colony types of gonococci increased their lethality for chicken embryos after intravenous inoculation but had little or no effect on the highly virulent T1 or T2 types. The toxicity of nonviable inocula, killed cells or sonicates, was not significantly affected by ecogenous iron. Addition of the iron-binding protein conalbumin reduced or delayed the lethal effect of T1, but not T3, gonococci although growth of both colony types in the allantoic cavity of the embryo was inhibited by this protwin. This effect can be attributed specifically to deprivation of iron since the iron-complexed form of conalbumin had no apparent influence on growth or virulence. The results indicate that the ability to acquire iron in vivo is a significant factor in gonococcal virulence. The virulent colony types appear to have enhanced ability to compete with the host for iron and this may be related to the presence of pili, other surface components, or the synthesis of iron-chelating compounds.

Animals↗