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J Stanley

Publications and source records attributed to J Stanley.

At least 145 records · Page 8Linked to original sources
PubMed1992

Genetic relationships among strains of Salmonella enteritidis in a national epidemic in Switzerland.

A collection of Salmonella enteritidis strains isolated in Switzerland (1965-90) was characterized. The phage type and plasmid profile of isolates were compared with the copy number and insertion loci of the DNA insertion element IS200. Three clonal lines of S. enteritidis were identified by IS200 profile; the various phage types were subtypes reproducibly associated with one of these lines. All human and poultry isolates contained a 38 Mda plasmid which hybridized with a mouse virulence-associated gene probe. In S. enteritidis, the IS200 profile is a race-specific molecular marker of the chromosome, and may be particularly applicable for studying the epidemiology of less common serovars.

Bacterial Typing Techniques↗
PubMed1992

Characterization of a human renal cell carcinoma specific cytotoxic CD8+ T cell line.

Human renal cell carcinoma (RCC) is one of the tumors most sensitive to immunotherapy and in that regard it is similar to malignant melanoma. Clinical studies reporting responses to therapy suggested that a host immune response may be involved in the antitumor activity induced by immunotherapy in these tumors. Although detection of a specific T cell response to melanoma has been well documented, this has not been the case for RCC. The lytic response of interleukin-2 (IL-2) cultured tumor-infiltrating lymphocytes (TILs) from RCC has been nonspecific. However, in this report we describe a CD8+ TIL line derived from a primary RCC tumor that displays specificity for the autologous tumor. This line is lytic for autologous RCC but does not lyse autologous lymphoblasts, allogeneic RCC, or tumor cell lines of other histologic types. It also proliferates specifically to the autologous tumor in the absence of exogenous IL-2. However, the addition of low dose IL-2 to the cultures can significantly augment its proliferative response. When stimulated with autologous RCC but not allogeneic RCC the CD8+ line will produce interferon-gamma (IFN-gamma). It appears that recognition of RCC by this TIL line is through the TCR/CD3 complex because anti-CD3 antibody blocks the lytic activity, proliferation and IFN-gamma production of the line in response to the autologous tumor. Additional studies illustrate that cytotoxic T lymphocytes with apparent specificity for the autologous tumor are present in unseparated cultured TILs that can be detected by clonal analysis. Collectively these results suggest that there is a specific T cell response to human RCC.

CD8 Antigens↗
PubMed1992

Salmonella plasmids of the pre-antibiotic era.

In order to provide a profile of the plasmid gene pool in Salmonella prior to the clinical use of antibiotics, a molecular genetic analysis was made of plasmids in strains collected by E.D.G. Murray between 1917 and 1954. These pre-antibiotic era (PAE) salmonellae contain conjugative plasmids of the same incompatibility groups as contemporary enterobacterial plasmids. Upon analysis of total plasmid content, 42 plasmids, sized between 23 and 72 MDa, were found. We defined and investigated six groups of these PAE Salmonella plasmids in terms of three groups of genes; those involved in plasmid maintenance and incompatibility, DNA repair and virulence. Of the five groups, three were replicon-typed to groups IncI1, IncX and IncFII; one group exhibited no homology to contemporary Inc/Rep probes, and one group represented virulence plasmids containing a common plasmid-partitioning locus. The results indicated that most of the PAE groups were progenitors of contemporary R-plasmids, except for the virulence plasmids, which have generally not evolved as vectors of antibiotic resistance.

Bacteriophage Typing↗
PubMed1992

Campylobacter helveticus sp. nov., a new thermophilic species from domestic animals: characterization, and cloning of a species-specific DNA probe.

An atypical group of thermophilic catalase-negative Campylobacter strains, the 'CH' (Swiss) group, can be recovered from faeces of domestic cats and dogs after selection by filtration, or with the antibiotic cefoperazone. This group of strains shows no relative DNA homology with any species in rRNA superfamily VI (Vandamme et al., 1991, International Journal of Systematic Bacteriology 41, 88-103) except with four thermophilic Campylobacter species, notably C. upsaliensis. The group is homogeneous and possesses a DNA base composition, cellular morphology at the electron microscope level and phenotypic properties characteristic of Campylobacter. Nonetheless it is distinct from known species of Campylobacter in terms of conventional bacteriological tests, total cellular protein profile, rRNA gene profile, and genomic DNA homology. On the basis of an integrated study of phenotype and genotype, we conclude that these bacteria constitute a previously undescribed species for which we propose the name Campylobacter helveticus sp. nov. A species-specific recombinant DNA probe was cloned from the designated type strain (NCTC 12470) for use in identification and further analysis of the epidemiology, pathogenicity and transmission of C. helveticus.

Animals↗
PubMed1992

The insertion sequence IS200 fingerprints chromosomal genotypes and epidemiological relationships in Salmonella heidelberg.

In Salmonella heidelberg the copy number of the Salmonella-specific insertion element IS200 was found to vary from four to six. All strains tested contained at least one common insertion site which was serovar specific, and most strains contained three common sites. Concurrent analysis of plasmids indicated that all insertion sequence copies were chromosomally located, and also supported the equivalence of an IS200 fingerprint and clonality. Seven intra-serovar clonal lines were thereby identified. One of these was associated with human infections, including septicaemias. Another was associated with chicken as a host: all these strains also carried a unique plasmid of 23 MDa, which was typed as a member of the IncX group. The chromosomal fingerprint of a third clone showed it to be a descendant of the chicken line marked by a single IS200 transposition. One or two representatives of four other clonal lines were identified. These lines of S. heidelberg could be related by divergent evolution, and the most recent relatives conformed to a continuous branching process model of IS200 transposition. This insertion sequence provided a highly discriminatory molecular marker of the S. heidelberg chromosome, and two of the seven clonal lines so identified were associated with distinct clinical/epidemiological contexts.

Animals↗
PubMed1992

16S rrn gene copy number in Helicobacter pylori and its application to molecular typing.

The copy number of the genes encoding 16S ribosomal RNA was analysed for the genomes of geographically diverse strains of Helicobacter pylori, and restriction site variation within and around the genes was characterized. A DNA probe of 550 bp was amplified by the polymerase chain reaction from genomic DNA of the type strain NCTC 11637. This probe constituted a sequence internal to the 3' end of the 16S rrn gene. Homology profiles were compared for genomic Southern blots made with four restriction enzymes cutting within and outside the probe sequence. A copy number of two was established for all 12 strains analysed. This approach yielded significantly simpler data than does conventional 'ribotyping ' of H. pylori. It was equally discriminatory, however, and provided strain-specific 16S rrn gene 'signatures'. These represent both fundamental physical-genetic information and a novel approach to typing this gastric pathogen.

Bacterial Typing Techniques↗
PubMed1992

Cloning of a probe for a previously undescribed enterobacterial tetracycline resistance gene.

An 8.35 kb BamHI fragment was cloned from the plasmid R714a. It encoded resistances to chloramphenicol, streptomycin, spectinomycin and tetracycline. Tetracycline resistance was determined by a locus without homology to the known enterobacterial gene classes, TetA-TetE. Further subcloning of the fragment located an unusual tetracycline resistance gene on a 4.7 kb BamHI-BglII fragment. A physical-genetic map of this fragment indicated that the gene was bisected by a PstI site. Deletion analysis and insertion mutagenesis were used to define a suitable probe. An intragenic PstI-AvaI fragment of 1.2 kb was identified, and used as a non-radioactive probe, being specific for this previously undescribed enterobacterial Tet gene.

Cloning, Molecular↗
PubMed1992

Serum gastrin concentrations in colorectal cancer patients.

Fasting serum gastrin concentrations were shown to be elevated in colorectal cancer patients compared with controls (P = 0.0037), which was mainly accounted for by a subgroup of patients who had significantly elevated levels. In cancer patients there was no difference in gastrin concentrations in blood taken from a tumour-draining mesenteric vein and from a peripheral vein at the time of colonic resection. Serum gastrin concentrations were significantly lower after apparently curative resection for colorectal cancer (P = 0.028), suggesting that the elevated serum gastrin seen in these patients may be due, at least in part, to secretion of gastrin by the tumour.

Adult↗
PubMed1991

Molecular analysis of an essential gene upstream of rpoN in Rhizobium NGR234.

Rhizobium sp. NGR234 is a broad-host range strain. The rpoN gene of this organism encodes a sigma factor which is a primary co-regulator of endosymbiosis. We characterized the locus upstream of rpoN, and identified a contiguous open reading frame, here termed ORF1. DNA sequence analysis of this ORF showed that it encoded a polypeptide highly conserved with a corresponding ORF of Rhizobium meliloti. The gene product contained two ATP/GTP binding pockets. Codon usage in the ORF and the nitrogenase operon nifKDH of NGR234 was similar. Although we used a non-transposable cassette flanked by appropriate sized DNA fragments, we were unable to isolate site-directed mutants in the ORF, whose ATP/GTP binding protein product is thus probably of essential biological function. ORF1 and rpoN exhibited conserved linkage among diverse rhizobia, and in Azotobacter vinelandii. Intragenomic and interspecific homology studies confirmed directly that ORF1 (NGR234) belonged to a large family of ATP-binding protein genes.

Adenosine Triphosphate↗
PubMed1991

Evolutionary lines among Salmonella enteritidis phage types are identified by insertion sequence IS200 distribution.

A survey was made of the presence, copy number and location of the Salmonella-specific DNA insertion element IS200, within the genomes of the 27 phage type strains of Salmonella enteritidis. All the phage type strains contained copies of IS200 revealed by genomic Southern blot hybridizations with a 300-bp DNA probe internal to the element. Restriction site variation around IS200 insertion sites was examined. Three fundamental patterns of hybridization corresponding to chromosomal IS200 loci were found. In terms of population genetics, these 'IS200 profiles' correspond to clonal lineages of recent evolutionary origin, and underline the phage-typing scheme for epidemiological subdivision of S. enteritidis. The molecular analysis is consistent with genetic selection pressures which are apparent in the observed epidemiological distribution of S. enteritidis, since each clonal lineage contained one of the phage types of major clinical importance in the U.K.

Biological Evolution↗
PubMed1991

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).

Chromatography↗
PubMed1991

Site-directed mutagenesis and DNA sequence of pckA of Rhizobium NGR234, encoding phosphoenolpyruvate carboxykinase: gluconeogenesis and host-dependent symbiotic phenotype.

We have cloned and sequenced the pckA gene of Rhizobium sp. NGR234, a broad host-range strain. The gene encodes phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of gluconeogenesis. The locus was isolated and subcloned from a genomic library of NGR234 employing hybridization with an R. meliloti pck gene probe and complementation of a Tn5 mutant in this species. The DNA sequence of pckA (NGR234) was determined and encoded a PEPCK protein of 535 amino acids with a molecular weight of 58.4 kDa. The deduced polypeptide sequence was compared to those of three known ATP-dependent PEPCKs. Slightly higher homology was observed with yeast and trypanosome polypeptides than with that of Escherichia coli. We have identified several regions that are conserved in all four PEPCK proteins. A mutant constructed in the pck gene by site-directed mutagenesis with interposon omega failed to grow on succinate, malate and arabinose but grew on glucose and glycerol as sole carbon sources. These data show that NGR234 requires PEPCK-driven gluconeogenesis to grow on TCA cycle intermediates. A host-dependent effect of the pckA mutation was observed on nodule development and nitrogen fixation. Nodules formed by the site-directed mutant on Leucaena leucocephala and Macroptilium atropurpureum were FixRed, but on Vigna unguiculata were Fix-. The expression of the gene was positively regulated in free-living cells of NGR234 by either succinate or host-plant exudates, and was subject to catabolite repression by glucose.

Amino Acid Sequence↗
PubMed1991

African cassava mosaic virus DI DNA interferes with the replication of both genomic components.

Natural infections of the geminivirus African cassava mosaic virus (ACMV) are known to be associated with low levels of defective interfering (DI) DNAs. Recently it has been demonstrated that extrachromosomal copies of the DI DNA, mobilized and amplified from an integrated DI DNA dimer, can ameliorate ACMV symptoms in transformed Nicotiana benthamiana, providing a possible means for the control of cassava mosaic disease. To further understand the molecular basis of the interference phenomenon, we have compared the ability of ACMV and tomato golden mosaic virus (TGMV) genomic components to replicate in leaf discs derived from DI DNA-transformed and control plants. Results indicate that the ACMV DI DNA interferes with the replication of both genomic components of ACMV to a similar extent. TGMV DNA A replicates to normal levels in transformed leaf discs and plants because it is unable to mobilize and amplify ACMV DI DNA. Differences in the relative levels of ACMV genomic components in transformed leaf discs and plants are discussed in terms of DNA replication and the availability of the genomic components for spread throughout the plant.

Blotting, Southern↗
PubMed1991

Phase I trial of continuous infusion interleukin-2 and doxorubicin in patients with refractory malignancies.

A phase I trial was performed to assess the immunomodulatory activities, maximum tolerated doses, and the toxicity of recombinant interleukin-2 (rIL-2) administered in combination with doxorubicin to patients with refractory malignancies. Therapy was administered to successive cohorts of four to six patients who were treated at three different dose levels (1A, 1B, 2A). Levels 1-2 refer to doxorubicin (40 or 60 mg/m2) given as an intravenous (i.v.) bolus on day 1, and levels A-B refer to rIL-2 (1.0 or 3.0 x 10(6) U/m2) given as a continuous i.v. infusion on days 2-5, 9-12, and 16-19. Cycles were repeated every 28 days. Seventeen patients were entered in the trial. Dose limiting toxicity consisted of neutropenia, and the maximum tolerated dose (MTD) of the combination was doxorubicin 40 mg/m2 and rIL-2 3.0 x 10(6) U/m2. No objective responses were observed. Lymphocytosis related to rIL-2 occurred and flow cytometry demonstrated significant increases in the following subsets: CD3+CD25+HLADr+ and CD11b-CD16c+CD8-. Natural killer cell activity and lymphokine-activated killer (LAK) cell precursors were increased in patients treated at dose levels 1A and 1B (40 mg/m2 doxorubicin), but no consistent changes in LAK activity were noted. No clinical responses were seen and the overall toxicity of this combination was moderate to severe. Administration of doxorubicin prior to rIL-2 does not enhance the immunologic effects of rIL-2.

Adult↗
PubMed1991

Mutational analysis of complementary-sense genes of African cassava mosaic virus DNA A.

We have investigated the ability of African cassava mosaic virus DNA A mutants, containing disrupted complementary-sense genes, to infect Nicotiana benthamiana and to replicate in Nicotiana tabacum protoplasts. Three overlapping open reading frames (ORFs) with the capacity to encode proteins with an Mr greater than 10K (AC1, AC2 and AC3) are highly conserved between geminiviruses that infect dicotyledonous plants and one (AC4) is less well conserved. Of these, only AC1 is a prerequisite for DNA replication; disruption of this ORF rendered the DNA noninfectious in plants and prevented DNA replication in protoplasts. Disruption of ORF AC2 prevented plant infection but mutants were capable of autonomous replication and replicated DNA B in trans in protoplasts to produce DNA forms that comigrated with wild-type virus DNAs. The AC2 mutant phenotype suggests that the product of this ORF is involved in virus spread within the plant. Mutants in which ORF AC3 had been disrupted retained the ability to replicate and to infect plants systemically although symptom development was delayed and attenuated, and mutant DNA accumulated to much lower levels (10 to 20%) in comparison with wild-type infection. Typical geminate virus particles were observed in extracts of plants infected with ORF AC3 mutants indicating that this gene is not essential for coat protein synthesis or virus assembly but possibly acts by modulating virus levels in infected tissues. Disruption of ORF AC4 had no effect on infectivity or symptom development suggesting that this ORF is maintained only because it overlaps the highly conserved ORF AC1.

DNA Mutational Analysis↗
PubMed1991

Dioxins and dibenzofurans in adipose tissue of US Vietnam veterans and controls.

The primary reason for concern about the adverse effects of exposure to Agent Orange is attributable to its toxic contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or dioxin. We studied adipose tissues from 36 Vietnam veterans, a similar group of 79 non-Vietnam veterans, and 80 civilians; the tissue specimens were selected from the 8,000 archived tissues collected from the non-institutionalized general population by the US Environmental Protection Agency. The geometric mean (+/- standard deviation) dioxin levels in adipose tissue for Vietnam veterans, non-Vietnam veterans, and civilian controls were 11.7 (+/- 1.7), 10.9 (+/- 1.7), and 12.4 (+/- 1.9) parts per trillion on a lipid weight basis, respectively. The mean levels for these groups were not significantly different from each other with or without adjustment for age of individuals, body mass index, and specimen collection year. In addition, none of the surrogate measures of Agent Orange exposure such as military branch, service within specific geographic region, military occupation, and troop location in relation to recorded Agent Orange spray was associated with the dioxin levels in adipose tissue of Vietnam veterans. Our results suggest that heavy exposure to Agent Orange or dioxin for most US troops was unlikely.

Adipose Tissue↗