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S I Miller

Publications and source records attributed to S I Miller.

At least 19 recordsLinked to original sources
PubMed1992

Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes.

Survival of Salmonella typhimurium within macrophage phagosomes requires the coordinate expression of bacterial gene products. This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S. typhimurium virulence regulators PhoP and PhoQ. Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold. In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage phagosomes. The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments. Measurements of pH documented that S. typhimurium delayed and attenuated acidification of its intracellular compartment. Phagosomes containing S. typhimurium required 4-5 hr to reach pH < 5.0. In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5. The eventual acidification of phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression. These observations implicate phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate phagosome acidification.

Ammonium Chloride↗
PubMed1992

Purification and primary structure of murine cryptdin-1, a Paneth cell defensin.

We have purified and determined the amino acid sequence of cryptdin-1, a murine Paneth cell defensin. The peptide corresponds to a previously characterized mRNA that accumulates to high abundance during postnatal ontogeny of the small bowel. Acid-extracted intestinal protein was fractionated by cation-exchange chromatography and fractions were assayed for antimicrobial activity. One peak of anti-Salmonella activity contained a putative defensin, based on its predicted electrophoretic migration in acid-urea PAGE. The peptide was purified to homogeneity by RP-HPLC and sequenced. These studies demonstrate defensin expression in non-myeloid tissue. The N-terminal extension of cryptdin-1 is a unique structural feature of this novel epithelial defensin.

Amino Acid Sequence↗
PubMed1992

Enteric defensins: antibiotic peptide components of intestinal host defense.

Five intestinal defensins, termed cryptdins 1-5, have been purified from mouse small bowel, sequenced, and localized to the epithelium by immunohistochemistry. Although identified as members of the defensin peptide family by peptide sequencing, enteric defensins are novel in that four cryptdins have amino termini which are three to six residues longer than those of leukocyte-derived defensins. A fifth cryptdin is the first defensin to diverge from the previously invariant spacing of cysteines in the peptide structure. The most abundant enteric defensin, cryptdin-1, had antimicrobial activity against an attenuated phoP mutant of Salmonella typhimurium but was not active against the virulent wild-type parent. Immunohistochemical localization demonstrated that cryptdin-1, and probably cryptdins 2 and 3, occur exclusively in Paneth cells, where the peptides appear to be associated with cytoplasmic granules. Biochemical and immunologic analysis of the luminal contents of the small intestine suggest that cryptdin peptides are secreted into the lumen, similar to Paneth cell secretion of lysozyme. The presence of several enteric defensins in the intestinal epithelium, evidence of their presence in the lumen, and the antibacterial activity of cryptdin-1 suggest that these peptides contribute to the antimicrobial barrier function of the small bowel mucosa.

Amino Acid Sequence↗
PubMed1992

An unusual pagC::TnphoA mutation leads to an invasion- and virulence-defective phenotype in Salmonellae.

Two phenotypes believed to contribute to the pathogenesis of Salmonella infections are macrophage survival and invasion of epithelial cells. It was recently observed that the Salmonella macrophage survival factor PagC has significant amino acid similarity to the Yersinia invasion factor Ail. This observation raised the possibilities that macrophage survival is in part determined by the pathway of entry and that PagC confers an entry mechanism that does not trigger the microbicidal activities of the macrophage. Thus, we sought to investigate the role of PagC in invasion by examining (i) the invasion phenotype of pagC mutants and (ii) the invasion phenotype of Escherichia coli carrying pagC. A previously identified invasion-defective TnphoA insertion mutant of Salmonella enteritidis was found to have TnphoA inserted into the signal sequence-encoding region of pagC; the pagC allele from this mutant, SM5T, was designated pagC64. In contrast, Salmonella typhimurium carrying the pagC1 allele (a TnphoA insertion mutation, downstream of the region encoding the signal sequence) was not defective for invasion. Further analysis of these two pagC alleles suggested that the invasion-defective phenotype associated with pagC64 is not due to the loss of PagC function but rather is due to the synthesis of a hybrid PagC-alkaline phosphatase protein that is aberrantly localized, most likely to the inner membrane, and thus may prevent proper localization or function of a factor(s) required for efficient invasion. The observation that pagC did not confer an invasive phenotype to E. coli further suggests that PagC is not an invasion factor. A cloned pagC gene complemented the macrophage survival defect of S. typhimurium pagC1 mutants, but the cloned ail gene did not. Together these results suggest that the structural similarity between PagC and Ail may not extend to a similarity in function. Interestingly, S. enteritidis carrying the pagC64 allele that results in both an invasion defect and a macrophage survival defect was less virulent for mice infected intragastrically or intraperitoneally than was S. enteritidis carrying the pagC1 allele that results only in a macrophage survival defect.

Alkaline Phosphatase↗
PubMed1991

PhoP/PhoQ: macrophage-specific modulators of Salmonella virulence?

The regulation of gene expression by the two-component regulatory system PhoP/PhoQ is necessary for Salmonella typhimurium survival within macrophages, defensin resistance, acid resistance, and murine typhoid fever pathogenesis. Salmonella experience multiple environments during mammalian infection and survival requires tightly regulated gene expression. After phagocytosis by macrophages, signal transduction by PhoQ results in the transcription of phoP-activated genes (pags) encoding proteins essential to bacterial survival and virulence. One such gene, pagC, encodes an envelope protein with amino acid similarity to an epithelial cell invasion protein of Yersina enterocolitica, Ail, and a bacteriophage lambda outer membrane protein, Lom. The PhoP and PhoQ proteins can also repress the synthesis of proteins, encoded by phoP repressed genes (prgs), when pags are maximally expressed. If prgs encode receptors for toxic compounds, prg repression may protect the cell within macrophages when pag expression is most necessary. At least one prg locus, prgH, is required for full S. typhimurium mouse virulence. Within the macrophage, different environments may stimulate a switch from pag to prg expression that is necessary to Salmonella survival. prg expression may also be necessary for surviving nonmacrophage environments. Study of the PhoP regulon should lead to the discovery of new virulence factors, increase knowledge of how gene regulation is essential to bacterial virulence, and perhaps lead to the development of better vaccines for typhoid fever.

Animals↗
PubMed1991

A Salmonella typhimurium virulence protein is similar to a Yersinia enterocolitica invasion protein and a bacteriophage lambda outer membrane protein.

The phoP-phoQ-regulated pagC locus is essential for full virulence and survival within macrophages of Salmonella typhimurium. The protein product, DNA sequence, and transcript of pagC were determined. The pagC locus encodes a single 188-amino-acid membrane protein that is similar to the ail-encoded eucaryotic cell invasion protein of Yersinia enterocolitica and the lom-encoded protein of bacteriophage lambda. The similarity of PagC and Ail to Lom leads us to hypothesize that Lom is a virulence protein and that bacteriophage gene transfer and lysogeny could have led to the development of proteins essential to survival within macrophages and eucaryotic cell invasion.

Adhesins, Bacterial↗
PubMed1990

Characterization of defensin resistance phenotypes associated with mutations in the phoP virulence regulon of Salmonella typhimurium.

The defensin sensitivities of Salmonella typhimurium strains with mutations in the phoP/phoQ two-component virulence regulon were tested by using purified defensins NP-1 and NP-2. Strains with mutations in either gene of the regulatory pair (phoP [transcriptional activator] or phoQ [membrane sensor kinase]) had increased sensitivities to defensin. The predicted periplasmic domain of the PhoQ protein contained a markedly anionic domain that could interact with cationic proteins and that could be responsible for resistance to defensin. Because insertion mutations in phoP are polar on phoQ, we constructed strains that expressed the PhoQ protein in the absence of PhoP to test whether resistance to defensin requires only the phoQ gene product. We found that resistance to defensin requires the function of both components of this regulatory system, because strains expressing PhoQ without PhoP were still markedly sensitive to defensins. This implied that a pag (phoP-activated gene) product is responsible for defensin resistance. We also tested for the ability of defensins NP-1, NP-5, and HNP-1 to activate pag expression and found that these peptides have no effect. Defensin resistance is not the only virulence characteristic controlled by the PhoP-PhoQ regulon because mutations in pagC, as well as ones in the phoP locus that resulted in constitutive pag activation (phenotype PhoPc), had no effect on defensin resistance, even though they rendered the organism avirulent and deficient in survival within macrophages. The virulence defect conferred by mutations in the phoP-phoQ two-component regulatory system is not completely explained by alterations in resistance to cationic proteins and involves the control of other proteins necessary for S. typhimurium survival within macrophages.

Animals↗
PubMed1990

Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages.

The phoP genetic locus is a two-component regulatory system (phoP-phoQ) that controls the expression of genes essential for Salmonella typhimurium virulence and survival within macrophages. Strains with a phoP constitutive mutation (phenotype PhoPC) showed up to 10-fold greater expression of phoP-activated genes (pag loci) than did strains with a wild-type phoP locus (phenotype PhoP+). While the phoP constitutive mutation resulted in increased expression of pag loci, it also dramatically reduced the expression of other protein species. Comparison of the protein content of PhoP+ and PhoPC strains by two-dimensional protein gel electrophoresis demonstrated that at least 40 separate protein species were changed in expression as a result of this mutation. The PhoPC S. typhimurium were found to be attenuated for virulence and survival within macrophages. This finding suggests that a balanced PhoP-PhoQ regulatory response, which allows expression of phoP-repressed as well as -activated genes, is required for full virulence of S. typhimurium. We have further shown that small numbers of PhoPC bacteria can be used as a live attenuated vaccine to protect against mouse typhoid. As few as 15 PhoPC bacteria protected mice against challenge with 10(5) 50% lethal doses of wild-type organisms, suggesting that important protective antigens are regulated by the PhoP-PhoQ virulence regulon.

Animals↗
PubMed1990

Use of commercially available rapid chloramphenicol acetyltransferase test to detect resistance in Salmonella species.

Chloramphenicol resistance among Salmonella spp. has important public health and clinical implications, especially in areas of the world where these strains are endemic. The availability of rapid and sensitive screening methods for detection of antibiotic resistance is important. Therefore, we tested 33 strains of Salmonella for chloramphenicol acetyltransferase (CAT) activity using two rapid techniques. Evaluation of a 1-h tube method and a 30-min commercial disk procedure demonstrated that they are as accurate as standardized susceptibility techniques. Both the 1-h tube and 30-min disk methods detected CAT enzymatic activity produced by one CAT gene copy per cell.

Chloramphenicol O-Acetyltransferase↗
PubMed1979

Cupro-zinc superoxide dismutase: a possible biologic marker for alcoholism (studies in black patients).

Cupro-zinc superoxide dismutase (SOD-1) was measured by competition radioimmune assay of erythrocyte lysates of alcoholic and control subjects. Normal amounts of SOD-1 were found in the extract from control subjects (815 ng/mg Hb) and from white alcoholics (874 ng/mg Hb). However, the level of SOD-1 in lysates from black alcoholics (1033 ng/mg Hb) was significantly increased (p = 0.0001, rank sum test) compared with black and white controls. This increase was not related to any other observed hematologic disorder or to any historical, demographic, or laboratory value examined.

Adult↗
PubMed1978

Are divided doses of tricyclic antidepressants necessary?

A double blind control comparison on the effects of 50 mg of clomipramine given three times a day and 150 mg of clomipramine in a single bedtime dosage revealed no significant differences in therapeutic efficacy. Patients reported more lightheadedness and dizziness but no differences on other side effects. The theoretical basis was the long biological half-life of such tricyclic antidepressants. Advantages included ease of administration, patient compliance, and timing of sedative effect.

Adjustment Disorders↗
PubMed1978

Social class and psychiatric diagnosis: differential findings in a lower-class sample.

Social class and the American Psychiatric Association Diagnostic and Statistical Manual II Diagnosis were examined for all patients contracting the Department of Psychiatry over a year at a large county hospital. This sample was primarily lower class -50 per cent Class V and 33 per cent in Class IV. All diagnoses showed average class between IV and V. Organic brain syndrome, both psychiatric and non-psychotic, and mental retardation had lower average social class than other diagnoses. A small sample of the diagnosis behaviour disorder of childhood and adolescence was also lower than other diagnoses. Class V patients with mental malfunction may present differently than patients from other social classes. The absence of differences on neurosis and functional psychosis may have been due to the predominance of lower class patients, diagnosticians more familiar with lower class patients, or the allowable choice of diagnoses.

Adolescent↗
PubMed1977

Logic as a tool for clinical training in social work.

The paper is an attempt to illustrate the usefulness of logic as a technique in the clinical training of mental health professionals. The specific concepts that are examined include the nature of deductive and inductive reasoning, hypothesis testing, necessary and sufficient conditions, "if-then" propositions, and the nature of clinical evidence. Knowledge and use of these concepts was tested in a tutorial program directed toward students who were beginning graduate studies in the field of social work. Some of the difficulties encountered by these students, especially in the clinical aspects of their training, were directly related to an inadequate understanding of these logical forms of reasoning. It is suggested that a portion of the clinical training in mental health fields be directed towards a deeper understanding and utilization of these basic concepts.

Curriculum↗