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Biomedical subjects

G Frankel

Publications and source records attributed to G Frankel.

At least 19 recordsLinked to original sources
PubMed1992

Mono- and bi-phasic Salmonella typhi: genetic homogeneity and distinguishing characteristics.

Several lines of evidence indicate a relatively low genetic heterogeneity in the natural Salmonella typhi population. However, some S. typhi isolates found in Indonesia express, instead of the usual fliC-d flagellin gene, a different flagellar gene fliC-j. In addition, Indonesian strains may have a second flagellar antigen fliC-z66. We have previously suggested, on the basis of the flagellar antigen constitution, that S. typhi evolved in an isolated human population in Indonesia. In order to test this hypothesis, we have gathered S. typhi isolates from around the world and tested the genetic heterogeneity among them. In general, polymorphism was greater in isolates from the Far East, as was indicated by Southern hybridizations with rDNA and fliC DNA probes. Gene fliC-j was not found in S. typhi isolates, other than those from Indonesia. However, the one-clone origin of S. typhi was indicated by a common DNA fingerprint pattern and by the occurrence, in the 5' end region of the fliC gene, of 10 scattered nucleotides that differ from the corresponding 10 nucleotides in other fliC alleles studied. These nucleotides were present in all isolates tested but did not change the amino acid sequence of the flagellin polypeptide.

Africa↗
PubMed1991

Diffuse-adhering Escherichia coli (DAEC) as a putative cause of diarrhea in Mayan children in Mexico.

Diarrhea is a major cause of infantile morbidity and mortality in developing countries. A community-based, case control study was conducted in a southern Mexican Mayan village for 3 weeks during the peak diarrhea period to prospectively identify the infectious agents associated with childhood diarrheal disease. Several enteropathogens were isolated from stools of 34 of 58 cases, although none was significantly associated with diarrhea. For the 24 cases from which no enteropathogens were isolated, diffuse-adhering Escherichia coli (DAEC) strains were significantly associated with diarrheal disease (P less than .02; odds ratio = 6; 95% confidence limit, 1.08-99.0). DAEC were highly heterogeneous with respect to plasmid content and serotype. Three DNA probes designed to differentiate E. coli exhibiting localized, diffuse, or aggregative adherence were compared with results from a standard HeLa cell binding assay to assess the utility of these probes in the field. This study provides evidence for the potential pathogenic capacity of DAEC and underscores the variety of diarrheal agents operating within a community.

Bacterial Adhesion↗
PubMed1991

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Physicians↗
PubMed1990

Detection of Shigella in feces using DNA amplification.

A rapid diagnostic method employing a polymerase chain reaction procedure (PCR) was used to identify Shigella and enteroinvasive Escherichia coli. This procedure amplified a region of the invasive-associated locus (ial) from a crude DNA extract of feces. A synthetic 21-base oligonucleotide corresponding to the ial gene sequence was shown to specifically hybridize only with enteroinvasive E. coli (EIEC) strains and Shigella species. Upon PCR amplification, a 320-base pair fragment was generated in DNA extracted from feces reconstituted with EIEC or Shigella flexneri but not in DNA from 70 normal stools lacking these organisms and could be readily detected by the ial probe. For identifying Shigella and EIEC, the PCR assay was 10(5)- and 10(2)-fold more sensitive than standard biochemical tests and the macrocolony hybridization assay, respectively. These findings demonstrate a novel methodology for rapid, sensitive, and culture-independent diagnosis of diarrhea caused by these pathogens and underscores the utility of PCR in the diagnostic laboratory.

Base Sequence↗
PubMed1989

Intragenic recombination in a flagellin gene: characterization of the H1-j gene of Salmonella typhi.

Salmonella typhi, the etiologic agent of typhoid fever, typically has only a phase-1 flagellar antigen, d, but some isolates, found only in Indonesia, have antigen j instead, and may have a second flagellar antigen, z66. It appears that intragenic recombination involving a directly repeated 11 bp sequence in the H1-d flagellin gene changed the flagellar antigen to j, by deleting 261 bp in its central, antigenically determinant, part. Sequencing of the hypervariable regions of genes H1-d and H1-j, and hybridization of such genes, after amplification by the polymerase chain reaction, with oligonucleotide probes specific for the deleted segment or for the sequence produced by the recombination confirmed that all the j alleles have the postulated deletion. By applying the polymerase chain reaction to study S. typhi isolates from Jakarta, not previously tested in respect to flagellar antigen, we showed that gene H1-j was nearly as common as H1-d in these isolates.

Antigens, Bacterial↗
PubMed1989

Unique sequences in region VI of the flagellin gene of Salmonella typhi.

The H1 (now renamed fliC; lino et al., 1988) alleles specifying antigenically different Salmonella flagellins are identical at their ends but differ greatly towards the middle, where there are two hypervariable segments (regions IV and VI). The flagellar antigen, d, of Salmonella typhi, is found also as phase-1 antigen in many other Salmonella species. We cloned the H1-d gene of a strain of S. typhi and determined the nucleotide sequence of its two hypervariable regions. Comparison with gene H1-d of Salmonella muenchen showed substantial differences in region VI: four scattered amino acid differences and ten adjacent amino acids in the inferred S. typhi sequence, all of which differ from the corresponding nine amino acids in the S. muenchen sequence. The results of polymerase chain reaction amplification indicated the presence of the S. typhi version in all of 18 additional S. typhi strains and the presence of the S. muenchen version in all four non-S. typhi species with flagellar antigen d. The difference in amino acid sequence in segment VI may be responsible for the minor serological differences between antigens d of S. typhi and antigen d of S. muenchen.

Amino Acid Sequence↗
PubMed1989

Multi-gene amplification: simultaneous detection of three virulence genes in diarrhoeal stool.

Enterotoxigenic Escherichia coli (ETEC) and Shigella account for a substantial proportion of acute diarrhoeal illnesses among Third-World children. Rapid detection of these infectious agents in faeces followed by the prompt implementation of public health measures could help reduce their spread during the early phase of epidemics. Towards this end, three pairs of synthetic oligonucleotide primers were prepared and shown to hybridize specifically to the genes encoding the heat-stable (ST) and the heat-labile (LT) enterotoxins of ETEC and to invasion-associated loci (ial) of the large Shigella virulence plasmid. When the three primer pairs were used together in the polymerase chain reaction (PCR), the three corresponding genetic loci could be simultaneously amplified using DNA extracted directly from stool; the amplified products were readily detected by ST-, LT- and ial-specific, alkaline phosphatase-labelled oligonucleotide probes (AP probes). The performance of this system was evaluated in a Mayan community in southeastern Mexico, where diarrhoeal illnesses are a common cause of childhood morbidity and mortality. Using only simple and inexpensive laboratory equipment, multigene amplification with these primers and probes led to the identification of ETEC and/or Shigella in the stools of 20 out of 71 children with diarrhoea; the procedure could be completed in seven hours and was more sensitive than conventional diagnostic tests or DNA probes used without amplification.

Base Sequence↗
PubMed1987

Fractionation of Theiler's virus-infected BHK21 cell homogenates: isolation of virus-induced membranes.

A purified fraction containing unique membranes entrapping virions was isolated from homogenates of cells infected with the DA strain of Theiler's virus, after high-speed centrifugation through a sucrose gradient. This fraction, sedimented at 45-50% sucrose, was only found in cells infected with the DA strain but not in cells infected with the GDVII strain of Theiler's virus or in mock-infected cells. Immunogold staining of the membranes entrapping virions, using antivirus IgG antibodies, revealed that the membranes entrapping virions did not incorporate viral capsid antigens.

Animals↗
PubMed1987

Synthesis of long viral complementary DNA from 7.5 Kb poly A+ RNA templates.

The poly A+ RNA of the WW and GDVII virus isolates, belonging to the Theiler's murine encephalomyelitis virus group, were used as templates for cDNA synthesis. Since several secondary structures were present along these viral RNAs the reverse transcriptase was prematurely displaced from the RNA templates and only short cDNA molecules could be synthesized. Therefore a reliable and reproducible procedure for the synthesis of long cDNA transcripts, that can be directly used for cloning into respective plasmid or phage vectors, was developed. The precise conditions and kinetics of the several enzymatic reactions were studied. The use of methylmercury hydroxide for first strand synthesis, a correct choice of Klenow polymerase for second strand synthesis and the use of vertical gel electrophoresis in combination with zone centrifugation for removal of the excess linkers were found to be of paramount importance for the synthesis of long, up to intact, 7.5 Kb cDNA transcripts.

Animals↗
PubMed1987

Detection of disease-specific restriction fragment length polymorphisms in pemphigus vulgaris linked to the DQw1 and DQw3 alleles of the HLA-D region.

Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment length polymorphism analysis was undertaken with DQ beta, DQ alpha, and DR beta cDNA probes. Hybridization with the DQ beta probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQ alpha and DR beta probes failed to identify disease-specific restriction fragment length polymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment length polymorphism that is highly associated with susceptibility to autoimmune disease.

Alleles↗
PubMed1987

Human leukocyte antigens (HLA) class I and class II on sperm cells studied at the serological, cellular, and genomic levels.

The expression of human leukocyte antigens (HLA) on highly purified human ejaculated sperm cells was studied using the sensitive enzyme-linked immunosorbent assay (ELISA) technique and a wide panel of monoclonal antibodies to class I and class II HLA. In addition, the stimulatory capacity of these cells was tested in mixed cultures of lymphocytes and spermatozoa, and the levels of RNA homologous to the HLA class I and class II genes were determined. The results obtained using the ELISA indicate that the class I and class II HLA serologically defined antigens are weakly expressed on the cell surface of the mature spermatozoa. Highly purified sperm cells consistently stimulated heterologous lymphocytes but not when HLA-DR compatibility was observed between stimulator and responder. The proliferative response of lymphocytes induced by sperm cells was lower than the response obtained in a lymphocyte-lymphocyte combination, though the kinetics of the response were similar in both cases. In addition, it was found that spermatozoa contained RNA species homologous to HLA class II DR beta and DQ beta genes sequences but not to HLA class I sequences. The levels of these RNA species were significantly reduced after interferon stimulation. Lymphocytes that served as positive control were found to contain RNA complementary to both HLA class I and class II genes.

Antibodies, Monoclonal↗
PubMed1987

Monoclonal anti-I-A antibody reverses chronic paralysis and demyelination in Theiler's virus-infected mice: critical importance of timing of treatment.

Susceptibility to demyelination caused by the WW isolate of Theiler's murine encephalomyelitis viruses is linked to class II genes of the major histocompatibility complex. SJL/J (H-2s) mice, expressing only I-As class II gene products of the major histocompatibility complex, are highly susceptible to Theiler's murine encephalomyelitis virus infection with the WW virus isolate, with chronic paralysis and severe inflammation and demyelination in the central nervous system. The effect of in vivo administration of anti-I-As monoclonal antibodies on Theiler's murine encephalomyelitis virus infection was observed. SJL/J mice were treated in various protocols pre- or postinfection. Anti-I-As monoclonal antibody reversed chronic paralysis and reduced inflammation and demyelination when given after the establishment of persistent infection. The effect was long lasting, but clinical signs, inflammation, and demyelination recurred 2 months after treatment ceased. Anti-I-As antibodies had no effect on viral titers within the central nervous system. The timing of the administration of monoclonal antibodies was critical. Administration of anti-I-As before the establishment of the persistent infection resulted in fatal encephalitis.

Animals↗