Biomedical subjects

R S Stephens

Publications and source records attributed to R S Stephens.

At least 73 records · Page 4Linked to original sources

PubMed1991

Prevalence of gene sequences coding for hypervariable regions of Opa (protein II) in Neisseria gonorrhoeae.

Opas (protein IIs) are a family of surface-exposed proteins of Neisseria gonorrhoeae. Each strain of N. gonorrhoeae has multiple (10-11) genes encoding for Opas. Identifiable elements in opa genes include the coding repeat within the signal sequence, conserve 5' and 3' regions, and hypervariable regions (HV1 and HV2) located within the structural gene. N. gonorrhoeae strains appear to have many biological properties in common that are either HV-region-mediated or associated with the presence of specific HV regions, suggesting that HV regions could be found in many clinical isolates. Oligonucleotides from three source strains representing three conserved regions of opa, 12 HV1 regions, and 14 HV2 regions were used by dot blot analysis to probe 120 clinical isolates of N. gonorrhoeae. The probe for the coding repeat hybridized to all 120 strains, the 3' conserved-region probe reacted with 98% of the strains, and the 5' conserved-region probe with 90% of the strains. Nine HV1 probes hybridized to 3.3-39.2% of the strains, and 13 of the HV2 probes hybridized to 1.7-25% of the isolates. Analysis of the number of probes that hybridized to each of the isolates showed that 19% did not hybridize with any of the HV1 probes and 25% did not hybridize with any of the HV2 probes. Approximately three-quarters of the isolates hybridized with one, two or three of the HV1 probes or one, two or three of the HV2 probes; 89% of the isolates hybridized to least one HV1 or one HV2 probe. The data indicate that some genes encoding HV regions of N. gonorrhoeae Opa proteins are widely distributed in nature.

Bacterial Outer Membrane Proteins↗

PubMed1991

Cloning and sequence of the gene for heat shock protein 60 from Chlamydia trachomatis and immunological reactivity of the protein.

We isolated and sequenced the gene for the chlamydial heat shock protein 60 (HSP-60) from a Chlamydia trachomatis genomic library by molecular genetic methods. The DNA sequence derived revealed an operon-like gene structure with two open reading frames encoding an 11,122- and a 57,956-Da protein. The translated amino acid sequence of the larger open reading frame showed a high degree of homology with known sequences for HSP-60 from several bacterial species as well as with plant and human sequences. By using the determined nucleotide sequence, fragments of the gene were cloned into the plasmid vector pGEX for expression as fusion proteins consisting of glutathione S-transferase and peptide portions of the chlamydial HSP-60. HSP-60 antigenic identity was confirmed by an immunoblot with anti-HSP-60 rabbit serum. Sera from patients that exhibited both high antichlamydial titers and reactivity to chlamydial HSP-60 showed reactivity on immunoblots to two fusion proteins that represented portions of the carboxyl-terminal half of the molecule, whereas fusion proteins defining the amino-terminal half were nonreactive. No reactivity with the fusion proteins was seen with sera from patients that had been previously screened as nonreactive to native chlamydial HSP-60 but which had high antichlamydial titers. Sera from noninfected control subjects also exhibited no reactivity. Definition of recognized HSP-60 epitopes may provide a predictive screen for those patients with C. trachomatis infections who may develop damaging sequelae, as well as providing tools for the study of immunopathogenic mechanisms of Chlamydia-induced disease.

Amino Acid Sequence↗

PubMed1991

Direct sequence evaluation of the major outer membrane protein gene variant regions of Chlamydia trachomatis subtypes D', I', and L2'.

The nucleotide sequences of variable segments (VS) 1, 2, and 4 for the major outer membrane protein gene (omp1) of Chlamydia trachomatis were determined for serologically defined subtypes D', I', and L2'. Asymmetric DNA amplification was used to produce single-stranded DNA for direct sequencing. Amino acid substitutions were detected in VS1, VS2, and VS4 for I', in VS2 for L2', and in VS4 for D'. DNA sequencing of omp1 variant regions may be an important method for evaluating the molecular epidemiology of Chlamydia spp.

Amino Acid Sequence↗

PubMed1990

Chlamydia trachomatis RNA polymerase major sigma subunit. Sequence and structural comparison of conserved and unique regions with Escherichia coli sigma 70 and Bacillus subtilis sigma 43.

We identified and sequenced the gene for the Chlamydia trachomatis RNA polymerase major sigma subunit. The gene encodes a 66,141-dalton protein (sigma 66), intermediate in size between the major sigma subunits of Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). The C. trachomatis sigma 66 subunit had extensive amino acid homology with the sigma 70 and sigma 43. The sigma subunit regions purportedly involved in core enzyme binding and DNA promoter recognition were also highly conserved, despite the lack of a DNA promoter consensus sequence between E. coli and C. trachomatis promoters and the inability of E. coli holoenzyme to specifically transcribe chlamydial genes. Compared with E. coli sigma 70, there were some major differences in the chlamydial sigma 66 sequence, including a gap of 63 amino acids and an additional 16 amino acids at the carboxyl terminus, which may play some role in modifying the sigma-DNA interaction, such that a promoter sequence unique to C. trachomatis is recognized. Monoclonal antibodies specific for E. coli sigma 70 were used to probe for homologous structures between sigma 70 and sigma 66; only one of seven antibodies bound specifically to sigma 66, suggesting minimal conservation of antigenic sites. The chlamydial sigma 66 was present in elementary bodies and was expressed throughout the developmental cycle, which implied that this gene encodes the major vegetative sigma subunit. Because the ability to study the genetics of C. trachomatis is currently limited, this work provides a tool for more detailed study of chlamydial promoter structure and of coordinate gene expression during the developmental cycle.

Bacillus subtilis↗

PubMed1990

Differential human serologic response to two 60,000 molecular weight Chlamydia trachomatis antigens.

Chlamydia trachomatis causes sexually transmitted diseases and is associated with serious long-term sequelae such as tubal infertility and ectopic pregnancy. There have been suggestions that chlamydial antigens of approximately 57,000-60,000 Mr may be involved in the immunopathology. Two important chlamydial antigens of 57,000-60,000 Mr are a Triton X-100-soluble antigen, which induces hypersensitivity in ocular models, and a sarcosyl-insoluble cysteine-rich structural protein, omp2. In this study, a 57,000 Mr Triton X-100-soluble protein was characterized as the chlamydial homolog of groEL, a heat shock protein. Using protein fractions, antibody responses of pelvic inflammatory disease (PID) and ectopic pregnancy patients to chlamydial groEL and omp2 were differentiated. Nearly all patients in both groups were reactive to omp2. Of those with titers greater than or equal to 1:512, 31% of PID sera and 81% of ectopic pregnancy sera were positive for chlamydial groEL (P = .004). This selectivity suggests that women with PID who develop chronic sequelae are those with antibody to groEL.

Antibodies, Bacterial↗

PubMed1990

A soluble 60 kiloDalton antigen of Chlamydia spp. is a homologue of Escherichia coli GroEL.

Two major 60 kD protein species can be separated by differential detergent extraction in Chlamydia spp. A Sarkosyl-soluble 60 kD protein is (i) structurally and antigenically distinct from the previously characterized 60 kD Omp2 outer membrane protein; and (ii) antigenically related to a bacterial common antigen of similar molecular weight which includes a 65 kD mycobacterial antigen and the GroEL heat-shock protein of Escherichia coli. Among GroEL homologues, the chlamydial protein (chl-GroEL) uniquely displays affinity towards immobilized thiol groups. The significance of this property is discussed with respect to the synthesis and assembly of the chlamydial disulphide-rich cell wall late in the growth cycle. Chl-GroEL is identical to the Triton X-100-soluble, ocular delayed-type hypersensitivity agent (Morrison et al., 1989), an essential component in the development of blinding trachoma. An autoimmune mechanism for chronic chlamydial diseases based on chl-GroEL homology to host proteins is hypothesized.

Antigens, Bacterial↗

PubMed1990

Developmental-stage-specific plasmid supercoiling in Chlamydia trachomatis.

Chlamydia trachomatis elementary body (EB) and reticulate body (RB) developmental stages have polymorphic plasmid DNA. Several plasmid forms separated by gel electrophoresis were identified as topoisomers by treatment with topoisomerase I. Among these topoisomers was one form unique to EBs and one form unique to RBs. The unique EB plasmid topoisomer was characterized as highly supercoiled, on the basis of band migrations by gel electrophoresis and its appearance by electron microscopy. The unusual physical state of this topoisomer was probably mediated, in part, by DNA-specific structural proteins. The unique RB plasmid topoisomer was a supercoiled form of lower superhelical density than the other identified topoisomers. Developmental-stage-specific differences in super-helical density of plasmid DNA suggest cause-and-effect relationships between DNA topology and metabolic activity in RBs and metabolic quiescence in EBs.

Blotting, Southern↗

PubMed1990

Cysteine-rich outer membrane proteins of Chlamydia trachomatis display compensatory sequence changes between biovariants.

Two cysteine-rich proteins of Chlamydia trachomatis are essential structural components of the unique outer membrane of the infectious elementary body. These 58,000 (outer membrane protein 2; OMP2) and 15,000 (OMP3) proteins also differ structurally and chemically between biovariants that differ in invasive capability. We have identified the gene for OMP3 and sequenced both trachoma and lymphogranuloma venereum (LGV) omp3 genes. We have previously sequenced omp2 from the LGV biovar and now describe the omp2 sequence for a trachoma biovariant. Amino acid sequence differences between biovariants were few but, significantly, these changes have altered the charge of both OMP2 and OMP3 such that the net charge of each protein differs between biovariants. These compensatory charge alterations have implications for the outer membrane organization of these proteins. In addition, examination of the OMP3 sequence suggests that OMP3 may be a lipoprotein.

Amino Acid Sequence↗

PubMed1989

A rapid chemiluminescent DNA hybridization assay for the detection of Chlamydia trachomatis.

With an estimated 3-4 million new cases per year, human infections from Chlamydia trachomatis are probably the most prevalent sexually transmitted disease (STD) in the United States. Diagnosis of Chlamydia is usually conducted by tissue culture methods. Direct immunofluorescence and ELISA tests have become available, but there remains a need for a test with better specificity and sensitivity. In response to this need, we have developed a rapid DNA hybridization assay using synthetic oligonucleotide probes to detect the presence of the Chlamydia trachomatis specific 7.4 kb plasmid. The assay involves solution phase hybridization of unlabelled probes, rapid capture of the probe-target duplex onto a microtitre dish surface, a new signal amplification technique that employs chemically cross-linked oligonucleotides, and an alkaline phosphatase labelled probe. Signal is obtained by reacting the labelled probe-target complex with an enzyme triggerable dioxetane substrate. Detection of the chemiluminescent output is performed either with a luminometer or by exposure to instant film. All 15 serovars of Chlamydia trachomatis react positively, while organisms known to co-inhabit the human urogenital tract react negatively.

Alkaline Phosphatase↗

PubMed1989

Identification by sequence analysis of two-site posttranslational processing of the cysteine-rich outer membrane protein 2 of Chlamydia trachomatis serovar L2.

The 60,000-molecular-weight cysteine-rich outer membrane protein (OMP2) from Chlamydia trachomatis participates in the disulfide-mediated outer-membrane organization unique to this organism. In addition, this protein is a primary focus of the host immune response. We cloned and sequenced the gene for OMP2 from C. trachomatis serovar L2. A lambda gt11 recombinant that expressed an antigenic portion of this protein was selected by antibody screening and provided a probe for the selection in lambda 1059 of a clone containing the entire gene. DNA sequencing of this clone identified one open reading frame of 1,641 base pairs, starting with a methionine codon and coding for a polypeptide with a molecular weight of 58,792. Amino-terminal protein sequencing and analysis of the translated DNA sequence demonstrated that processing at alternate signal peptide cleavage sites accounts for the molecular-weight polymorphism of this protein. The mature proteins had a net positive charge and contained 24 cysteine residues.

Amino Acid Sequence↗

PubMed1988

High-resolution mapping of serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis.

The principal surface protein antigen of Chlamydia trachomatis is the major outer membrane protein (MOMP). The MOMP is antigenically complex. Among the 15 serovars of C. trachomatis, mAbs define serovar-, subspecies-, and species-specific determinants on MOMP. The molecular basis of the antigenic diversity of these proteins is reflected in amino acid variable sequence domains. We have mapped the dominant topographic antigenic determinants of MOMP that are defined by mAbs. Using recombinant DNA approaches we have identified the linear distribution of two antigenic domains. One domain contains a serovar-specific determinant and the other contains subspecies- and species-specific determinants. These antigenic domains correspond to two amino acid sequence variable domains. Synthetic peptides were immunogenic and these resolved the serovar-specific determinant within a 14-amino acid peptide. The subspecies- and species-specific determinants were overlapping within a 16-amino acid peptide.

Amino Acid Sequence↗

PubMed1988

Developmental-form-specific DNA-binding proteins in Chlamydia spp.

We identified DNA-binding proteins specific to the elementary body (EB) developmental form of Chlamydia spp. Chlamydial proteins from whole lysates were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with nick-translated chlamydial DNA. By this method, C. trachomatis serovar L2 EBs had three unique protein bands of 58,000, 25,700, and 17,000 molecular weight not seen in the reticulate bodies. The 17,000-molecular-weight protein and the 25,700-molecular-weight protein were identified in an acid-soluble protein fraction and were resistant to high-salt elution, similar to other procaryotic nucleoproteins. The 17,000-molecular-weight protein was also detected in preparations with isolated chromosomes from EBs. Preliminary characterization indicated that the protein-DNA interaction was principally charge related.

Bacterial Proteins↗

PubMed1988

Developmental regulation of tandem promoters for the major outer membrane protein gene of Chlamydia trachomatis.

Chlamydia trachomatis has a biphasic developmental cycle which is characterized by qualitative and quantitative changes in protein expression. The molecular mechanisms that mediate these changes are unknown. Evidence for transcriptional regulation of the chlamydial major outer membrane protein gene (omp1) was found by Northern hybridization of RNA isolated sequentially during the chlamydial developmental cycle. Early in the growth cycle a single transcript was detected, which was followed hours later in the cycle by an additional transcript. Mapping of the initiating nucleotide for each transcript suggested that this gene is regulated by differential transcription from tandem promoters.

Bacterial Outer Membrane Proteins↗

PubMed1987

Responses to depressed interpersonal behavior: mixed reactions in a helping role.

We placed 144 female subjects in a helping role and randomly assigned them to interact with a confederate in a 3 X 3 X 2 X 2 (Psychopathology X Blaming X Advice Seeking X Sex of Confederate) factorial design. In order to study behaviors that mediate interpersonal responses to depression, male and female confederates enacted depressed, anxious, or normal roles and blamed themselves, others, or no one for their problems. The confederates requested advice in half of the conditions. Results indicated that depressed confederates were rejected more on questionnaire measures; however, depressed confederates received more conversational advice and support from subjects than did the equally disturbed anxious confederates. The self-blaming and advice-seeking manipulations did not interact with depression to produce more negative reactions in subjects. There was no evidence of a negative mood induction in subjects, nor did the sex of the confederate have important interpersonal consequences. Results are discussed in terms of theoretical and methodological issues in studies of interpersonal factors in depression.

Adult↗

PubMed1987

Diversity of Chlamydia trachomatis major outer membrane protein genes.

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (omp1) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of omp1 gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.

Amino Acid Sequence↗

PubMed1987

Verbal report methods in clinical research on alcoholism: response bias and its minimization.

Verbal report procedures, such as interviews, tests and questionnaires, have become the dominant method to obtain clinical data on alcohol abuse and its modification through treatment. The extent to which this method provides reliable and valid information for research purposes, and how its accuracy and usefulness can be enhanced, is examined. A review of methodological studies in the alcohol literature shows that although the information obtained from alcoholics and heavy drinkers tends to be reliable and valid, there can be considerable variability in accuracy, depending on the sensitivity of the information sought, the specificity of the validation criteria, the personal characteristics of the respondents and the demand characteristics of the task. It is suggested that the question of whether verbal report procedures are valid or invalid is less important than the issue of how they can be improved to the point that confidence can be placed in their findings. To facilitate this process, methodological techniques likely to enhance validity are reviewed.

Alcoholism↗

PubMed1986

Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2.

The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced. A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C. trachomatis serovar L2. Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes. The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases. Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing. The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons. The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence. The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues.

Amino Acid Sequence↗

PubMed1985

Immunotyping of Chlamydia trachomatis with monoclonal antibodies.

Rapid and precise immunotyping of Chlamydia trachomatis was achieved by testing whole organisms (elementary bodies) in the microimmunofluorescence test with monoclonal antibodies. Monoclonal antibodies were produced with standard techniques by using an immunization schedule that encouraged the development of immunotype-specific antibodies. Fifteen monotypic or multitypic (subspecies) monoclonal antibodies were chosen for use in a two-step typing system that required strains of C. trachomatis to be tested against six to eight monoclonal antibodies for classification. Immunotyping with monoclonal antibodies was studied by testing 313 strains, typed with the previous method that utilized immunized mouse antisera, that represented each of the 15 established serovars. The two-step monoclonal antibody method resulted in a classification similar to the current one. Only one strain typed differently with the two methods. With the monoclonal antibody method, available lymphogranuloma venereum (LGV) serovars L1 and L3 could not be differentiated from trachoma serovars E and G, respectively, unless the strains had been identified as LGV. Monoclonal antibody typing was simpler to perform and more precise; it allowed easy differentiation between closely related serovars. Three new types were discovered among the strains previously classified as serovars D, I, and L2. These are tentatively being considered subtypes and are labeled D', I', and L2'.

Animals↗