Biomedical subjects

L R Hendrix

Publications and source records attributed to L R Hendrix.

7 recordsLinked to original sources

PubMed2000

Contact-dependent hemolytic activity distinct from deforming activity of Bartonella bacilliformis.

Although Bartonella bacilliformis causes a severe anemia in humans, this study presents the first report of hemolytic activity by B. bacilliformis. The activity was not apparent in culture supernatants but was reliably detected when B. bacilliformis cells were centrifuged onto erythrocytes prior to incubation. Abrogation of hemolytic activity by proteinase K treatment suggested the hemolysin was a Bartonella protein. Even though hemolysis required relatively long incubation times, de novo protein synthesis was not required to produce the protein. A preparation containing factors released by B. bacilliformis, including deformin, a B. bacilliformis protein able to induce pits and invaginations in erythrocyte membranes, had some ability to lyse erythrocytes. However, pre-deformed erythrocytes did not lyse faster or to a greater extent than control erythrocytes after the addition of B. bacilliformis cells. Inhibition of deformation caused by B. bacilliformis cells with the erythrocyte ATPase inhibitor, vanadate, did not affect hemolytic activity. This study suggests hemolytic activity and deforming activity are attributable to different B. bacilliformis proteins.

Animals↗

PubMed1999

Differential expression of translational elements by life cycle variants of Coxiella burnetii.

Coxiella burnetii replicates as distinct morphological forms, which may allow potential life cycle variants to survive the harsh environment of the phagolysosome. Monoclonal antibodies (MAbs) were compared by Western blotting for reactivity with large cell variant (LCV) and small cell variant (SCV) antigens to characterize proteins differentially expressed by C. burnetii. MAb NM7.3 reacted with a approximately 32-kDa LCV-upregulated antigen, and MAb NM183 reacted with a approximately 45-kDa LCV-specific antigen. MAb NM7.3 was used to screen a lambdaZapII C. burnetii DNA expression library, and an immunoreactive clone was identified with sequence similarity to the Escherichia coli tsf gene, which encodes elongation factor Ts (EF-Ts). Since a similar screen with MAb NM183 did not identify immunoreactive clones, an alternate strategy was devised to clone the reactive antigen based on observations of cross-reactivity with the 45-kDa elongation factor Tu (EF-Tu) protein from Chlamydia trachomatis. The highly conserved nature of EF-Tu among eubacteria allowed PCR amplification of a tuf gene fragment (encoding approximately 95% of the predicted EF-Tu open reading frame) from C. burnetii using degenerate primers. The product of the cloned tuf gene fragment reacted with MAb NM183 in Western blot analysis, confirming the identity of the 45-kDa LCV-specific antigen. Identification of two proteins differentially expressed by C. burnetii, EF-Tu and EF-Ts, both essential components of the translational machinery of the cell, supports the hypothesis that LCVs are metabolically more active than SCVs.

Amino Acid Sequence↗

PubMed1993

Cloning and sequencing of Coxiella burnetii outer membrane protein gene com1.

The gene for an approximately 27-kDa outer membrane-associated, immunoreactive protein was cloned from the rickettsial pathogen Coxiella burnetii. The gene, designated com1 for Coxiella outer membrane protein 1, was expressed in Escherichia coli, presumably by its own promoter. The complete nucleotide sequence of the gene was determined. The deduced amino acid sequence of 252 residues includes a putative leader sequence. The leader sequence is recognized in and removed by E. coli on the basis of the difference in the molecular mass of the protein produced in an in vitro transcription-translation system (27.6 kDa) and that of the protein immunoprecipitated from an iodinated E. coli clone (25.7 kDa). The Com1 protein expressed in E. coli was proteinase K sensitive in nondisrupted cells and soluble in 1% Sarkosyl, suggesting a loose association with the outer membrane. While the complete predicted sequence of the Com1 protein does not show any overall similarity to those of previously described proteins, a region which includes the only two cysteines in Com1 is homologous to the catalytic site of protein disulfide oxidoreductases.

Amino Acid Sequence↗

PubMed1991

Defective intracellular transport as a common mechanism limiting expression of inappropriately paired class II major histocompatibility complex alpha/beta chains.

Distinct combinations of class II major histocompatibility complex (MHC) alpha and beta chains show widely varying efficiencies of cell surface expression in transfected cells. Previous studies have analyzed the regions of the class II chains that are critically involved in this phenomenon of variable expression and have shown a predominant effect of the NH2-terminal domains comprising the peptide-binding site. The present experiments attempt to identify the post-translational defects responsible for this variation in surface class II molecule expression for both interisotypic alpha/beta combinations failing to give rise to any detectable cell membrane molecules (e.g., E alpha A beta k) and intraisotypic pairs with inefficient surface expression (e.g., A alpha d A beta k). The results of metabolic labeling and immunoprecipitation experiments using L cell transfectants demonstrate that in both of these cases, the alpha and beta chains form substantial amounts of stable intracellular dimers. However, the isotype- and allele-mismatched combinations do not show the typical post-translational increases in molecular weight that accompany maturation of the N-linked glycans of class II MHC molecules. Studies with endoglycosidase H reveal that no or little progression to endoglycosidase H resistance occurs for these mismatched dimers. These data are consistent with active or passive retention of relatively stable and long-lived mismatched dimers in a pre-medial-Golgi compartment, possibly in the endoplasmic reticulum itself. This retention accounts for the absent or poor surface expression of these alpha/beta combinations, and suggests that conformational effects of the mismatching in the NH2-terminal domain results in a failure of class II molecules to undergo efficient intracellular transport.

Alleles↗

PubMed1991

MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding.

Class II major histocompatibility complex molecules undergo a change in structure upon stable binding of peptide antigen. Analysis of the site and extent of this change among class II molecules of splenic antigen-presenting cells reveals the preference of class II for peptide acquisition outside the endoplasmic reticulum and indicates that the class II presentation system is not saturated with self peptides. There are numerous empty class II molecules on the cell surface and peptide antigen is evidently important in regulating surface class II expression.

Animals↗

PubMed1991

Differentiation of Coxiella burnetii isolates by analysis of restriction-endonuclease-digested DNA separated by SDS-PAGE.

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.

Animals↗

PubMed1990

Identification and cloning of a 27-kDa Coxiella burnetii immunoreactive protein.

Patients with acute Q fever develop Coxiella burnetii-specific antibody, and chronic disease patients often develop extremely high levels of C. burnetii-specific antibody. Antibody-reactive LPS has been well characterized, but only a few immunoreactive proteins have been identified. An immunoreactive ca. 62-kDa protein has been cloned, sequenced, and shown to be related to an Escherichia coli heat-shock protein. A ca. 27-kDa immunoreactive outer membrane protein(s) has also been identified. We have begun characterizing C. burnetii immunoreactive proteins by gene-cloning methods. A gene bank of total C. burnetii Nine Mile phase 1 (acute strain) DNA was created using the lambda vector EMBL3. This bank was screened for plaques which reacted with E. coli-pre-absorbed rabbit antisera specific for purified, Nine Mile C. burnetii whole cells. Twenty-three immunoreactive plaques were identified from a screening of 3000 plaques. Twenty-two plaques produced a ca. 60-65-kDa immunoreactive protein. One plaque produced a ca. 27-kDa immunoreactive protein, as well as a ca. 60-65-kDa immunoreactive protein. Phage extracts of this plaque were used to enrich the antisera to produce a ca. 27-kDa-specific antisera. This enriched sera reacted with a ca. 27-kDa protein in all C. burnetii isolates tested, comprising isolates from both chronic and acute strains. The DNA contained in the immunoreactive clone was C. burnetii-specific, as shown by DNA hybridization. We are currently subcloning the ca. 27-kDa protein-coding region for sequencing to determine if this gene encodes the previously identified ca. 27-kDa immunodominant protein. This protein may ultimately have both diagnostic and vaccinogenic potential.

Antigens, Bacterial↗