Biomedical subjects

S F Hayes

Publications and source records attributed to S F Hayes.

At least 37 records · Page 2Linked to original sources

PubMed1997

Natural proteoglycan receptor analogs determine the dynamics of Opa adhesin-mediated gonococcal infection of Chang epithelial cells.

Many bacterial pathogens possess a complex machinery for the induction and/or secretion of factors that promote their uptake by mammalian cells. We searched for the molecular basis of the 60- to 90-min lag time in the interaction of Neisseria gonorrhoeae carrying the heparin-binding Opa adhesin with Chang epithelial cells. Infection assays in the presence of chloramphenicol demonstrated that the Opa-mediated gonococcal infection of Chang cells required bacterial protein synthesis when the microorganisms were derived from GC agar but not when grown in liquid media. Further analysis indicated that contact with agar ingredients rather than the growth state of the microorganisms determined the infection dynamics. DEAE chromatography of GC agar extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and testing of collected fractions in infection assays identified negatively charged high-molecular-weight polysaccharides in the agar as inhibitors of the cellular infection. Electron microscopy showed that agar-grown gonococci were surrounded by a coat of alcian blue-positive material, probably representing accreted polysaccharides. Similar antiphagocytic material was isolated from bovine serum, indicating that in biological fluids gonococci producing the heparin-binding Opa adhesin may become covered with externally derived polysaccharides as well. Binding assays with gonococci and epithelial proteoglycan receptors revealed that polysaccharides derived from agar or serum compete with the proteoglycans for binding of the heparin-binding Opa adhesin and thus act as receptor analogs. Growth of gonococci in a polysaccharide-free environment resulted in optimal proteoglycan receptor binding and rapid bacterial entry into Chang cells. The recognition that gonococci with certain phenotypes can recruit surface polysaccharides that determine in vitro infection dynamics adds a different dimension to the well-recognized biological significance of genetic variation for this pathogen.

Animals↗

PubMed1997

Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.

The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins corresponding to surface loops 3 and 4 of CPV contain linear epitopes that are located on the external surface of the ADV capsid. Furthermore, these linear epitopes contain neutralizing determinants. Computer comparisons with the CPV crystal structure suggest that these sequences may be adjacent to the threefold axis of symmetry of the viral particle.

Aleutian Mink Disease Virus↗

PubMed1996

Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture.

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.

Animals↗

PubMed1995

Low-efficiency (macro-)pinocytic internalization of non-pathogenic Escherichia coli into HEp-2 cells.

HEp-2 cells internalize non-pathogenic Escherichia coli bacteria by a low-efficiency internalization mechanism which is upregulated in Pho-derepressed strains (as shown by Sinai and Bavoil in 1993), and is independent of microfilament integrity but requires functional microtubules. Here, we further characterize the microtubule requirement of this pathway using various effectors of microtubule integrity and function. Furthermore, we show that internalization is enhanced upon treatment with monodansylcadaverine, a specific inhibitor of receptor mediated endocytosis, and is insensitive to brefeldin A, which promotes the microtubule-dependent reorganization of the endosome. An assay system is also described to directly evaluate the contribution of pinocytosis to this pathway based on the ability of the bacteria to cointernalize and consequently colocalize with the fluid-phase marker, Texas-red-conjugated dextran (TRD). Using this assay, Hoescht-stained bacteria were observed in TRD-containing vesicles in numbers that are consistent with their observed internalization rate. Overall, these data are strongly supportive of the existence of a low-efficiency macropinocytic mechanism of entry for these non-pathogenic bacteria. Moreover, the observed requirements for host tyrosine kinase and protein kinase C activities suggest that it is inducible.

Antifungal Agents↗

PubMed1994

Characterization of KpsT, the ATP-binding component of the ABC-transporter involved with the export of capsular polysialic acid in Escherichia coli K1.

The 17-kilobase kps gene cluster of Escherichia coli K1 contains all the information necessary for the expression of capsular polysaccharide. Region 3 of the cluster encodes two genes, kpsM and kpsT, whose products belong to the ATP-Binding Cassette (ABC)-transporter protein family. The KpsMT system is involved with the export of capsular polysaccharide in E. coli. Earlier work indicated that interaction between KpsT and ATP is important for transport. In this study, we report that KpsT, a peripheral inner membrane protein, can be photolabeled by the ATP analog, 8-N3[gamma-32P]ATP. The derivatization of KpsT by this reagent is inhibited by cold ATP or ATP gamma S. Furthermore, the protein seems to require a membrane environment for efficient photolabeling, but does not require any other kps gene products. Results obtained from saturation mutagenesis of the ATP-binding consensus sequence of KpsT and the phenotypes of strains with defined mutations in the chromosomal gene, are consistent with the view that ATP-binding by KpsT is required for transport of polymer across the inner membrane. The structure of KpsT was compared to a model developed for other ABC-transport proteins, and important functional regions were determined. The results obtained from chemical mutagenesis of kpsT are consistent with the model and revealed characteristics particular to capsule transporters.

ATP-Binding Cassette Transporters↗

PubMed1994

Challenging heterosexism in college health service delivery.

The empowerment and affirmation of lesbian, bisexual, and gay students is long overdue. This article explores how human immunodeficiency virus and acquired immune deficiency syndrome (HIV/AIDS), substance abuse, violence and hate-related crimes, suicide, and heterosexism all adversely affect the physical and emotional health of nonheterosexual college students. College health services must expand their current scope and practice and assume a leadership role in combating all forms of oppression by actively incorporating and addressing the unique health issues and needs of the lesbian, bisexual, and gay population. This article provides a brief overview of the relevant healthcare issues for lesbians, bisexuals, and gays; examples of heterosexism in college health services; and recommendations for institutional and personal and professional change.

Bisexuality↗

PubMed1994

Nucleotide sequence and mutational analysis of the gene encoding KpsD, a periplasmic protein involved in transport of polysialic acid in Escherichia coli K1.

The 17-kb kps gene cluster encodes proteins necessary for the synthesis, assembly, and translocation of the polysialic acid capsule of Escherichia coli K1. We previously reported that one of these genes, kpsD, encodes a 60-kDa periplasmic protein that is involved in the translocation of the polymer to the cell surface. The nucleotide sequence of the 2.4-kb BamHI-PstI fragment accommodating the kpsD gene was determined. Sequence analysis showed an open reading frame for a 558-amino-acid protein with a typical N-terminal prokaryotic signal sequence corresponding to the first 20 amino acids. KpsD was overexpressed, partially purified, and used to prepare polyclonal antiserum. A chromosomal insertion mutation was generated in the kpsD gene and results in loss of surface expression of the polysialic acid capsule. Immunodiffusion analysis and electron microscopy indicated that polysaccharide accumulates in the periplasmic space of mutant cells. A wild-type copy of kpsD supplied in trans complemented the chromosomal mutation, restoring extracellular expression of the K1 capsule. However, a kpsD deletion derivative (kpsD delta C11), which results in production of a truncated KpsD protein lacking its 11 C-terminal amino acids, was nonfunctional. Western blot (immunoblot) data from cell fractions expressing KpsD delta C11 suggest that the truncated protein was inefficiently exported into the periplasm and localized primarily to the cytoplasmic membrane.

Amino Acid Sequence↗

PubMed1993

Directional actin polymerization associated with spotted fever group Rickettsia infection of Vero cells.

Members of the spotted fever group (SFG) of rickettsiae spread rapidly from cell to cell by an unknown mechanism(s). Staining of Rickettsia rickettsii-infected Vero cells with rhodamine phalloidin demonstrated unique actin filaments associated with one pole of intracellular rickettsiae. F-actin tails greater than 70 microns in length were seen extending from rickettsiae. Treatment of infected cells with chloramphenicol eliminated rickettsia-associated F-actin tails, suggesting that de novo protein synthesis of one or more rickettsial proteins is required for tail formation. Rickettsiae were coated with F-actin as early as 15 min postinfection, and tail formation was detected by 30 min. A survey of virulent and avirulent species within the SFG rickettsiae demonstrated that all formed actin tails. Typhus group rickettsiae, which do not spread directly from cell to cell, lacked F-actin tails entirely or exhibited only very short tails. Transmission electron microscopy demonstrated fibrillar material in close association with R. rickettsii but not Rickettsia prowazekii. Biochemical evidence that actin polymerization plays a role in movement was provided by showing that transit of R. rickettsii from infected cells into the cell culture medium was inhibited by treatment of host cells with cytochalasin D. These data suggest that the cell-to-cell transmission of SFG rickettsiae may be aided by induction of actin polymerization in a fashion similar to that described for Shigella flexneri and Listeria monocytogenes.

Actin Cytoskeleton↗

PubMed1992

Nucleoid condensation in Escherichia coli that express a chlamydial histone homolog.

Chlamydial cell types are adapted for either extracellular survival or intracellular growth. In the transcriptionally inert elementary bodies, the chromosome is densely compacted; in metabolically active reticulate bodies, the chromatin is loosely organized. Condensation of the chlamydial nucleoid occurs concomitant with expression of proteins homologous to eukaryotic histone H1. When the Chlamydia trachomatis 18-kilodalton histone homolog Hc1 is expressed in Escherichia coli, a condensed nucleoid structure similar to that of chlamydiae is observed with both light and electron microscopy. These results support a role for Hc1 in condensation of the chlamydial nucleoid.

Blotting, Western↗

PubMed1992

Characteristics of the internalization and intracellular survival of Campylobacter jejuni in human epithelial cell cultures.

The characteristics associated with the internalization and intracellular behavior of Campylobacter jejuni during short-term and long-term cultivation with INT 407 cells were examined. The internalization of C. jejuni by INT 407 cells was inhibited by cytochalasin dansylcadaverine, chemicals that disrupt microfilament formation and inhibit receptor cycling, respectively. Ammonium chloride and methylamine, two chemicals that inhibit endosomal acidification, did not affect C. jejuni internalization. Once internalized, C. jejuni were found exclusively with membrane-bound vacuoles. With regard to intracellular survival, a decline in the number of viable intracellular bacteria, as determined by protection from gentamicin, occurred during the initial phase of infection and when a low level of the antibiotic was maintained in the culture medium. However, the number of intracellular C. jejuni increased markedly after the removal of the antibiotic. In the absence of antibiotic, the infection led to the deterioration of the cell monolayers, indicating that C. jejuni is able to survive within epithelial cells and elicit a cytotoxic effect. The ability of C. jejuni to enter and exert deleterious effects on cells may reflect a pathogenic mechanism associated with enteritis caused by this organism.

Cadaverine↗

PubMed1992

Translocation of Campylobacter jejuni across human polarized epithelial cell monolayer cultures.

The ability of Campylobacter jejuni isolates to translocate across an epithelial cell barrier was investigated by using polarized Caco-2 cell monolayers grown on microporous membrane filters. The 4 C. jejuni isolates tested all traversed the Caco-2 cell monolayers and displayed similar translocation kinetics. The number of bacteria crossing the polarized cell monolayers continued to increase with time until 4 h after inoculation, at which time a maximum rate of translocation was observed. Transmission electron microscopy revealed that C. jejuni translocated across polarized Caco-2 cell monolayers by passing both through and between cells. Chloramphenicol, an inhibitor of bacterial protein synthesis, reduced the translocation of C. jejuni. Bacterial attachment, internalization, and translocation were inhibited at low temperature. These data indicate that adherence, penetration, and translocation of C. jejuni require active bacterial and target cell processes and further suggest a role for cellular translocation in the pathogenesis of C. jejuni-mediated enteritis.

Campylobacter jejuni↗

PubMed1992

Group B streptococcal opacity variants.

Colony opacity variants were detected for type III group B streptococci (GBS). Transparent colonies predominate in the parent GBS, with occasional colonies having opaque portions. Two stable opaque variants (1.1 and 1.5) were compared with three transparent clones (1.2, 1.3, and 1.4). All grew well on blood agar and on GC medium, but variant 1.1 failed to grow on Todd-Hewitt medium. Scanning and transmission electron microscopy demonstrated that colony opacity correlated with bacterial aggregation status, with opaque variants forming longer and more organized chains. Opaque-transparent switches were observed in both directions for most variants, with transparent to opaque noted most frequently, but 1.5 did not switch at all. Switching of the opacity phenotype was observed both in vitro and in neonatal mice. Relationships between colony opacity and several cell surface phenomena were explored. (i) Opaque variant 1.1 had two surface proteins (46 and 75 kDa) that were either unique or greatly overexpressed. (ii) Variant 1.1 was deficient in type III polysaccharide, while 1.5 lacked group B antigen. Diminished capsular polysaccharide of variant 1.1 was reflected in reduced negative electrophoretic mobility and in increased buoyant density. (iii) Transparent variant colonies growing closest to a penicillin disk were opaque, but colonial variants did not differ in their sensitivity to penicillin. These data indicate that GBS can exist in both opaque and transparent forms, with opaque appearance occurring by multiple routes. Opaque variants grow poorly on Todd-Hewitt medium generally used for isolation of GBS, so any possible relationships between opacity variation and pathogenesis of GBS infection are unknown.

Bacterial Capsules↗

PubMed1992

Identification of the tick-borne relapsing fever spirochete Borrelia hermsii by using a species-specific monoclonal antibody.

Borrelia hermsii causes a relapsing fever in humans and is one of several species of tick-borne spirochetes known to occur in the western United States. Spirochetes observed in the peripheral blood of patients acutely ill have been presumptively identified in the past by the geographic location of exposure and the probable species of tick vector. We describe a monoclonal antibody (H9826) that bound to the flagellar protein of B. hermsii but not to those of any of the other species tested, which included B. parkeri, B. turicatae, B. coriaceae, B. anserina, B. burgdorferi, and Leptospira interrogans serovar ballum. This antibody bound efficiently to B. hermsii in an indirect immunofluorescence assay and was used to rapidly detect and identify this spirochete in the peripheral blood of experimentally infected mice and in the central ganglia of Ornithodoros hermsi ticks. H9826 can rapidly confirm the identification of B. hermsii to increase our understanding concerning the geographic distribution, vector specificity, and epidemiological significance of this zoonotic human pathogen.

Antibodies, Bacterial↗

PubMed1992

Expression of Aleutian mink disease parvovirus capsid proteins by a recombinant vaccinia virus: self-assembly of capsid proteins into particles.

A portion of a cDNA clone containing coding sequences for both structural proteins (VP1 and VP2) of Aleutian mink disease parvovirus (ADV) was inserted into recombinant vaccinia viruses, VV:ADSP. Immunohistochemical staining of VV:ADSP-infected cells revealed that the ADV antigen was readily detected and localized in the nuclei of infected cells. Analysis of VV:ADSP-infected cell lystates indicated that both VP1 and VP2 were produced and comigrated with authentic VP1 and VP2 from ADV-infected Crandell feline kidney cells. These results suggested, therefore, that both VP1 and VP2 were synthesized from a single cloned transcript. CsCl density gradient centrifugation of partially purified VV:ADSP-infected cell lysates indicated that the majority of the antigen was located in a fraction with a density near 1.33 g/ml, indicative of empty ADV particles. Subsequent electron microscopic examination revealed the presence of 27-nm icosahedral virion-like structures at the same density, suggesting that the proteins self-assembled into empty virions. Furthermore, sera from eight of eight mice inoculated with VV:ADSP contained ADV-specific antibodies and two of these eight serum samples had neutralizing activity, indicating that the particles produced in VV:ADSP-infected cells were immunogenic. Finally, when lysates from VV:ADSP-infected cells were compared with standard ADV antigens in counterimmunoelectrophoresis assays, a similar pattern of specific reactivity was observed for sera from normal and infected mink.

Aleutian Mink Disease↗

PubMed1991

Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy.

A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.

Acetylglucosamine↗

PubMed1991

Substitution of leucine for isoleucine in a sequence highly conserved among retroviral envelope surface glycoproteins attenuates the lytic effect of the Friend murine leukemia virus.

Friend murine leukemia virus is a replication-competent retrovirus that contains no oncogene and that exerts lytic and leukemogenic properties. Thus, newborn mice inoculated with Friend murine leukemia virus develop severe early hemolytic anemia before appearance of erythroleukemia. To identify the retroviral determinants regulating these effects, we used chimeric infectious constructions and site-directed point mutations between a virulent Friend murine leukemia virus strain and a naturally occurring variant attenuated in lytic and leukemogenic effects. We found that severe hemolytic anemia was always associated with higher numbers of blood reticulocytes with budding retroviral particles. Furthermore, a remarkably conservative leucine to isoleucine change in the extracellular SU component of the retroviral envelope was sufficient to attenuate this lytic effect. Also, this leucine at position 348 of the envelope precursor protein was located within the only stretch of five amino acids that is conserved in the extracellular SU component of all murine, feline, and primate type C and type D retroviral envelopes. This observation suggested an important structural function for this yet undescribed conserved sequence of the envelope. Lastly, we observed that lytic and leukemogenic effects were attenuated by a deletion of a second repeat in the transcriptional enhancer region of the viral long terminal repeats of the variant strain.

Amino Acid Sequence↗

PubMed1991

Production of monoclonal antibodies reactive with a denatured form of the Friend murine leukemia virus gp70 envelope protein: use in a focal infectivity assay, immunohistochemical studies, electron microscopy and western blotting.

Four monoclonal antibodies were selected for their ability to recognize the envelope protein of Friend murine leukemia virus (F-MuLV) in methanol-fixed tissue culture cells. Each of these monoclonal antibodies was found to react only with F-MuLV. By using recombinant retroviruses, it was determined that each of the monoclonal antibodies recognized the C-terminal one-third of the F-MuLV gp70 envelope protein. The monoclonal antibodies were effective in radioimmunoprecipitation of F-MuLV proteins, and one of the antibodies, 720, was also effective in Western blotting. The ability of antibody 720 to react with F-MuLV in methanol-fixed cells facilitated the use of a sensitive immunoperoxidase method with a focal virus infectivity assay. In immunohistochemical studies using light microscopy, antibody 720 could specifically label F-MuLV-infected cells in acetone-fixed tissue sections from F-MuLV-infected animals. Finally, in immuno-gold labelling studies using electron microscopy, antibody 720 could be used to distinguish F-MuLV from amphotropic MuLV.

3T3 Cells↗

PubMed1991

Murine retrovirus-induced spongiform encephalopathy: productive infection of microglia and cerebellar neurons in accelerated CNS disease.

We have examined the pathological lesions and sites of infection in mice inoculated with a highly neurovirulent recombinant wild mouse ecotropic retrovirus (FrCasE). The spongiform lesions appeared initially as swollen postsynaptic neuronal processes, progressing to swelling in neuronal cell bodies, all in the absence of detectable gliosis. Infection of neurons in regions of vacuolation was not detected. However, high level infection of cerebellar granule neurons was observed in the absence of cytopathology, wherein viral protein was found associated with both axons and dendrites. Infection of ramified and amoeboid microglial cells was associated with cytopathology in the brain stem, and endothelial cell-pericyte infection was found throughout the CNS. No evidence of defective retroviral expression was observed. These results are consistent with an indirect mechanism of retrovirus-induced neuropathology.

Animals↗