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Biomedical subjects

M H Vodkin

Publications and source records attributed to M H Vodkin.

30 records · Page 2Linked to original sources
PubMed1992

Detection of St. Louis encephalitis virus in mosquitoes by use of the polymerase chain reaction.

We recently developed an assay using the polymerase chain reaction (PCR) for the specific detection of St. Louis encephalitis (SLE) virus RNA. This assay was tested in a blind study on 7 samples of pooled mosquitoes (50 mosquitoes/pool) which were also characterized for SLE virus by plaque assay in Vero cell culture. One sample was positive for the SLE virus as determined by both the PCR assay and a combination of the plaque assay and the indirect fluorescent antibody assay. The remaining 6 samples were negative for the presence of SLE virus as determined by both methods. These data indicate that this PCR assay can be used to monitor for the presence of SLE virus in pools of homogenized mosquitoes. This approach could provide early data on which to base disease control decisions.

Animals↗
PubMed1991

Amplification of repetitive DNA for the specific detection of Naegleria fowleri.

By using hybridization at low C0t values, a genomic library on Naegleria fowleri was screened for clones containing repetitive DNA. Partial sequence information from a repetitive clone, Nf9, showed sequence homologies with the mitochondrial ATPase 6 subunit from yeasts and other organisms. Synthetic DNA primers were selected and tested in amplification reactions. Nonstringent hybridization conditions were defined which allowed amplification of N. fowleri DNA and reduced amplification of DNA from nonpathogenic Naegleria species. Stringent conditions were selected which allowed detection only of N. fowleri. Identity of the amplified DNA was confirmed by using internal restriction sites and an internal primer. In a blind study, tissue from mice experimentally infected with N. fowleri was specifically detected by using stringent hybridization conditions.

Amebiasis↗
PubMed1988

The Mycobacterium tuberculosis 65-kilodalton antigen is a heat shock protein which corresponds to common antigen and to the Escherichia coli GroEL protein.

Monoclonal hybridoma antibodies directed against a 65-kilodalton (kDa) mycobacterial protein could detect similarly sized antigens in many other bacterial species. In Pseudomonas aeruginosa, the cross-reacting protein corresponded to a 62-kDa antigen that has been called Common Antigen. The mycobacterial 65-kDa antigen and Common Antigen are similar in that both (i) are highly immunoreactive molecules, (ii) contain species-specific and genus-specific epitopes in addition to the broadly cross-reactive epitopes, (iii) can be isolated as homomultimers of greater than 240 kDa, and (iv) have similar amino acid compositions. In Escherichia coli, the cross-reactive protein corresponded to the GroEL protein. Both the GroEL protein and the mycobacterial 65-kDa protein are expressed as heat shock proteins.

Antigens, Bacterial↗
PubMed1988

A heat shock operon in Coxiella burnetti produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli.

A gene library from the DNA of Coxiella burnetii has been constructed in the cosmid vector pHC79. A particular clone, pJB196, reacted strongly with Coxiella-specific antibodies elicited in a number of different species of animals. This clone produced two abundant C. burnetii-specific polypeptides, a 14-kilodalton nonimmunoreactive protein and a 62-kilodalton immunoreactive protein. Sequencing identified two open reading frames, encoding polypeptides of 10.5 and 58.3 kilodaltons. The only transcriptional control element observed on the 5' side of the initiation codon resembled a heat shock promoter. This heat shock promoter was functionally regulated in Escherichia coli, since both proteins were produced by growth conditions at 37 degrees C and neither protein was detected at 23 degrees C. There were four sequences from the literature that were highly homologous (greater than 50%) to the 62-kilodalton protein from C. burnetii. Three were from Mycobacterium species and represent the immunodominant antigen of this genus. The other was from E. coli, detected as a gene that complements or suppresses a temperature-sensitive RNase activity. Since the recombinant protein was immunogenic, it may serve as an efficacious vaccine against C. burnetii and other pathogenic microorganisms that express the conserved antigen.

Amino Acid Sequence↗
PubMed1987

Use of conversion adaptors to clone antigen genes in lambda gt11.

A strategy has been devised and tested to employ EcoRI conversion adaptors for cloning 5' cohesive-ended restriction fragments into the unique EcoRI site of the lambda gt11 expression vector. Five lambda gt11 chromosomal libraries were constructed with Rickettsia tsutsugamushi genomic DNA digested with restriction enzymes generating five different 5' cohesive ends. Recombinant phage yields as high as 10(7) plaque forming units were achieved without amplification of the five libraries. Sequences encoding epitopes of all eight R. tsutsugamushi polypeptide antigens, previously identified by Western blot analysis, were obtained in the five lambda gt11 expression libraries. Recombinant antigen expression was dependent on lambda gt11 lac promoter induction in 39% of the recombinants assayed. This method significantly improves the efficiency of genomic lambda gt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides.

Antigens, Bacterial↗
PubMed1987

Metabolism and genetics of chlamydias and rickettsias.

Chlamydial and rickettsial diseases pose a hazard to man and to domesticated and wild animals. The virulence mechanisms which aid the establishment of these obligate intracellular parasites in the eukaryotic host are still not within our grasp. Recent knowledge of the biochemical stratagem, the metabolic capabilities and the genetic diversity of these microbes illustrate fundamental differences in ecology and evolutionary divergence. The preferred site of intracellular residence determines the strategy for uptake, for nutrient assimilation and also for evasion of the host's immunological defenses. The Chlamydia, Rickettsia, and Coxiella are the most extensively studied of the genera. Whereas the Ehrlichia and Cowdria are poorly understood, they are also the most intriguing of the Rickettsiae. A number of antigenically and genetically distinct species are identified for the genera Chlamydia, Rickettsia, and Ehrlichia, whereas the Coxiella and Cowdria may not represent such a wide diversity. Recent information on the genetic heterogeneity of the chromosomal and plasmid DNAs of the strains of Coxiella suggest the diversity is greater than was originally envisioned. New information regarding the antigenic structure of Cowdria and their cellular tropisms suggests that they are closely related to the Ehrlichia. In this review we compare the metabolic capabilities and the genetic diversity of these different intracellular bacteria.

Animals↗
PubMed1986

Genetic heterogeneity among isolates of Coxiella burnetii.

Chromosomal and plasmid DNA have been extracted from six isolates of Coxiella burnetii, the aetiological agent of Q fever. Restriction fragment length polymorphisms detected after HaeIII digestions of chromosomal DNA revealed four different patterns that distinguished the American from the European isolates, and the Nine Mile phase I prototype strain from a spontaneously derived, isogenic phase II nonrevertant variant. At least one of the HaeIII fragments visible in the pattern from Nine Mile phase I and not in that from Nine Mile phase II could not be detected by DNA-DNA hybridization, and thus may have been deleted during the phase transition. Comparison of Nine Mile phase II, which does not survive animal passage, with Grita M44 phase II, which does, indicated that the HaeIII fragment was present in the Grita strain. These results suggest that this HaeIII fragment may be concerned with functions necessary to survive the cellular immune response in vivo. Isolates from two human endocarditis cases showed the greatest divergence from all the other isolates, having at least five fragments of unique mobility in the HaeIII digestion pattern of their chromosomal DNA. Also, a plasmid obtained from these two isolates was 2 to 3 kb larger than the plasmid present in the other five isolates, and its restriction pattern could be distinguished from that of the other plasmids by several endonucleases. Detection of chromosomal and plasmid restriction fragment length polymorphisms among strains of phase I or phase II C. burnetii from various geographical locations and environmental sources will facilitate Q fever diagnosis and strain identification.

Autoradiography↗
PubMed1986

Overlapping deletion in two spontaneous phase variants of Coxiella burnetii.

Chromosomal DNA from the Nine Mile phase I strain of Coxiella burnetii (CB9MIC7) was cloned into the cosmid vector pHC79. The resulting gene library was probed with a radiolabelled HaeIII fragment present in the parental strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). The insert, which includes the missing HaeIII fragment, was 38.5 kb in length. When DNA from this cosmid clone was hybridized to genomic DNA of the parental CB9MIC7 and its derivative CB9MIIC4, a number of fragments were missing or altered in the latter strain. Restriction mapping localized the fragments to a contiguous portion of the chromosomal DNA fragment. The data were consistent with an 18 kb deletion in the chromosome of CB9MIIC4. Another intrastrain spontaneous derivative, CB9MI514, also lacked the sentinel HaeIII fragment and carried a deletion of approximately 29 kb within the same cloned insert. Both deletions appeared to share a common terminus, within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed intact. The strains examined were representative of various stages of phase variation. The relationship between the observed deletions and the mechanism of phase transition in Nine Mile strains is discussed.

Chromosome Deletion↗
PubMed1983

Cloning of the protective antigen gene of Bacillus anthracis.

The tripartite protein toxin of Bacillus anthracis consists of protective antigen (PA), edema factor (EF), and lethal factor (LF). As a first step in developing a more efficacious anthrax vaccine, recombinant plasmids containing the PA gene have been isolated. A library was constructed in the E. coli vector pBR322 from Bam HI-generated fragments of the anthrax plasmid, pBA1. Two clones producing PA were identified by screening lysates with ELISA (enzyme-linked immunosorbent assay). Western blots revealed a full-size PA protein in the recombinant E. coli, and a cell elongation assay demonstrated biological activity. Both positive clones had a 6 kb insert of DNA, which mapped in the Bam HI site of the vector. The two inserts are the same except that they lie in opposite orientations with respect to the vector. Thus PA is encoded by the plasmid pBA1.

Animals↗
PubMed1973

A nucleic acid associated with a killer strain of yeast.

A crude membrane fraction was isolated from both killer M(k) and isogenic nonkiller M(o) strains of yeast labeled with [(3)H]adenine. Nucleic acids were extracted from this fraction and centrifuged to equilibrium in an ethidium bromide-Cs(2)SO(4) solution. A peak of radioactivity coincident with a single fluorescent band was present in the membrane fraction isolated from cells of the killer strain but was absent from that isolated from nonkiller cells. This material has been characterized as double-stranded RNA by treatment with various nucleases and by chromatography on a cellulose CF-11 column. Sedimentation in a sucrose gradient indicates an S value of 8-10.

Carbon Isotopes↗
PubMedDate not supplied

Identification of Acanthamoeba at the generic and specific levels using the polymerase chain reaction.

We have adapted the polymerase chain reaction to identify strains of Acanthamoeba. Using computer-assisted analysis, primers were designed from an anonymous repetitive sequence and from published sequences of 18S and 5S ribosomal RNA genes of A. castellanii. Amplification of a short ribosomal DNA target (272 base pairs) at restrictive annealing conditions (greater than 50 degrees C) resulted in a single band that was unique for the genus and distinguished Acanthamoeba from Naegleria. This assay functioned with fresh and formalin-fixed cells as starting material. Amplification of longer targets (400-700 base pairs) at less restrictive annealing conditions (less than 47 degrees C) led to more than one band. This multiple banding pattern could reproducibly classify Acanthamoeba at the strain level and was, in certain cases, diagnostic for known pathogenic strains. However, these assays need to be further refined to make them relevant for clinical purposes.

Acanthamoeba↗