Biomedical subjects

J Stanley

Publications and source records attributed to J Stanley.

At least 127 records · Page 7Linked to original sources

PubMed1993

Molecular comparison of the IncX plasmids allows division into IncX1 and IncX2 subgroups.

We have investigated molecular relationships and evolution of plasmids classified genetically to incompatibility (Inc) group X, in particular by comparison of plasmids from the pre-antibiotic era (PAE) with contemporary R-plasmids. On the basis of restriction analysis, R6K, the best-described and 'prototype' plasmid of the IncX group, exhibited little similarity with the other plasmids in this Inc group. Other contemporary IncX R-plasmids exhibited a substantial degree of interrelationship, and were also related to PAE IncX plasmids. When the origin of plasmid replication of R6K was used as a replicon probe, R6K was the only plasmid tested which exhibited homology. Other contemporary and PAE IncX plasmids exhibited homology with the origin of plasmid R485. These data suggest that the IncX group should be subdivided. R485 may be regarded as representative of the major subgroup present before and after the advent of antibiotic selection pressure. Plasmids of this subgroup, IncX1, possess an internal region which yields five characteristic EcoRV fragments. R6K may be regarded as representative of subgroup IncX2, of which it is presently the sole well-described member. The antibiotic resistances encoded by contemporary IncX R-plasmids are due to insertion of identifiable transposons in progenitor plasmids identical to the R485 subgroup of PAE IncX plasmids.

Biological Evolution↗

PubMed1993

Genotypes and phylogenetic relationships of Salmonella typhimurium are defined by molecular fingerprinting of IS200 and 16S rrn loci.

Molecular fingerprints of chromosomal genotypes in Salmonella typhimurium were generated by analysis of variation at the 16S rrn gene loci and the sites of the insertion sequence IS200. Genetic and reference strains of S. typhimurium were compared with clinical phage type strains from cases of human salmonellosis. Three 16S rrn profiles, one of which was predominant, were found. The copy number of the Salmonella-specific insertion sequence IS200 varied from 6 to 12, and all insertions were chromosomal. Three of the insertion sites shared by all strains were serovar-specific for S. typhimurium. Thirteen distinct profiles of IS200 were detected, providing a high level of intraserovar strain discrimination. Profiles were generally more conserved among genetic and reference strains; representatives of clinical phage type strains, which are recent human isolates, showed much greater diversity of IS200 profiles. Irrespective of their origin, strains could be assigned to IS200 profile groups, phylogenetically related lines identified by combinations of conserved insertion sites. Hybridization profiles of this mobile element are markers of intermediate and short-term evolution in S. typhimurium. They provide a fingerprinting scheme for the purposes of genetics, and delineate a molecular typing scheme for the purposes of epidemiology.

Base Sequence↗

PubMed1993

Genetic diversity of Helicobacter pylori indexed with respect to clinical symptomatology, using a 16S rRNA and a species-specific DNA probe.

DNA probes are described which identify group and fingerprint strains of the human gastric pathogen Helicobacter pylori, on the basis of well-defined band homologies. A 544 bp internal fragment of the 16S ribosomal RNA gene was generated by polymerase chain reaction (PCR) with primers derived from the Escherichia coli rRNA gene sequence. In genomic Southern blots this probe detected restriction site variation around these loci, generating simple but strain-specific molecular fingerprints. A small conserved chromosomal fragment of 1.2 kbp, Hps, species-specific for H. pylori, was obtained by cloning random HindIII fragments into pUC19. It was useful for dot-blot identification, and also separated isolates into one major and two minor groups. When results for these two probes were combined, a baseline characterization of genotype was obtained. A band-matching database of molecular fingerprints for the type strain and 63 clinical isolates of H. pylori from asymptomatic, ulcer and gastritis contexts is presented. No significant association between the genotypes at this level of definition and the associated clinical symptomatology of the isolates was detected.

DNA Primers↗

PubMed1993

Novel Campylobacter-like organism resembling Helicobacter fennelliae isolated from a boy with gastroenteritis and from dogs.

We isolated a Campylobacter-like organism resembling Helicobacter fennelliae from a 5 1/2-year-old boy with gastroenteritis. Similar strains had been found previously in fecal specimens from healthy and diarrheic dogs. These isolates could be differentiated from H. fennelliae by a lack of catalase and arylsulfatase activities. This group of organisms seems to be homogeneous by a nonradioactive dot blot DNA hybridization assay.

Animals↗

PubMed1993

Posterior truncal vagotomy and stapling of the anterior stomach wall in 30 patients with duodenal ulcer: acid inhibition, gastric emptying, and endoscopic dye spraying. Prospects for endoscopic vagotomy.

The use of a stapling instrument (TA90) to effect a transection of the anterior gastric wall with posterior truncal vagotomy but preserving innervation to the anterior wall of the antrum for duodenal ulcer is described. The operation resulted in the endoscopic healing of all the ulcers; however, recurrent ulceration was seen in 3 of 25 patients. Basal acid output was reduced from 7.84 (SEM 1.73) to 3.49 (SEM 1.15) mmol/h (t = 2.09, df = 15.7, p = 0.05), while peak acid output was reduced from 38.67 (SEM 3.11) to 19.26 (SEM 2.3) mmol/h (t = 5.01, df = 23.9, p = 0.0000). Solid and liquid gastric emptying studies were performed, and while some delay in solid emptying was seen, only one patient complained of transient gastric stasis. Endoscopic Congo red staining was performed postoperatively and a tongue of innervated mucosa along the lesser curve was seen in each case. The procedure was easily and rapidly performed, and early clinical results are good, with 25 of 30 patients having a good (Visick I) result.

Adult↗

PubMed1992

RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus.

The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression.

DNA, Viral↗

PubMed1992

Chromosomal genotypes (evolutionary lines) of Salmonella berta.

The copy number and location of the insertion sequence IS200, a mobile DNA element, was established across a collection of Salmonella berta. All strains contained one common site, assumed to be present in the evolutionary ancestor of this serovar. With one exception, all strains, including recent outbreak isolates from the UK and sporadic isolates of world-wide distribution, were representatives of a single genotypic clone which carried three common IS200 insertion sites. This clone has acquired diverse combinations of plasmids, reflecting its actual or recent distribution and host. A single isolate, belonging to a second, minor genotypic clone was characterised by two IS200 insertion sites.

Animals↗

PubMed1992

Molecular phylogenetic typing of pandemic isolates of Salmonella enteritidis.

Salmonella enteritidis is now the most common Salmonella serovar in many countries. We have used cloned DNA probes to analyze genome interrelationships between strains chosen to represent the current S. enteritidis pandemic, and included designated type strains of the seven subspecies of Salmonella in order to compare the levels of discrimination of probes. DNA sequence divergence and rearrangements were analyzed in and around the rfa, fim and umuDC loci, and around insertion sites of the Salmonella-specific DNA insertion element, IS200. The S. enteritidis isolates showed a high degree of genome homogeneity. Chromosomal genetic loci exhibited characteristic DNA sequence divergence between subspecies of Salmonella, but no intraserovar divergence or difference with the subspecies I type strain was observed for S. enteritidis. The locus umuDC was not found in S. enteritidis. S. enteritidis contains a conserved and a variable site of insertion of insertion sequence IS200 and the analysis of DNA rearrangements around the second of these sites showed that three distinct evolutionary lines or races exist within pandemic isolates associated with human gasteroenteritis. IS200 profiles of a range of U.K. isolates of the epidemic phage type PT4 showed that all belonged to a single clonal line.

Animals↗

PubMed1992

Determinants of tomato golden mosaic virus symptom development located on DNA B.

Infectious clones have been constructed from two strains of the bipartite geminivirus tomato golden mosaic virus. The common strain and the yellow vein strain show marked phenotypic differences in Nicotiana benthamiana which are reproduced following infection with the cloned viral genomes. Pseudorecombinants between the two strains, produced by exchange of genome components (DNAs A and B), established that the difference in symptoms in several species of the Solanaceae is determined by DNA B. Recombinants produced in vitro between the DNA B components showed that determinants of symptom development map to the common region and gene BL1. DNA B is known to carry functions necessary for spread of viral DNA through the host plant. Our results emphasize the link between symptom type and virus spread.

Cloning, Molecular↗

PubMed1992

Mutational analysis of the monopartite geminivirus beet curly top virus.

Mutants of the monopartite geminivirus beet curly top virus have been screened for infectivity and symptom development in Nicotiana benthamiana and Beta vulgaris, for replication competence in N. benthamiana leaf discs, and for transmission by the leafhopper Circulifer tenellus. Disruption of open reading frame (ORF) V2 by the introduction of a termination codon resulted in symptomless infection of N. benthamiana associated with low levels of virus and reduced single-stranded (ss) DNA and prevented systemic infection of B. vulgaris. Reduced levels of ssDNA were produced by the mutant in N. benthamiana leaf discs, suggesting that V2 affects the synthesis or accumulation of this viral DNA form. Mutants in which ORF C2 had been truncated by the introduction of termination codons or by frame-shifting remained highly infectious and induced severe symptoms in both N. benthamiana and B. vulgaris. Similarly, a mutant containing a termination codon within ORF C3 was highly infectious and induced severe symptoms in N. benthamiana although infectivity in B. vulgaris was greatly reduced, symptoms were extremely mild, and virus levels were low. A synergistic effect of a double mutation in ORFs C2 and C3, manifested by the inability of mutants to systemically infect N. benthamiana and the production of reduced amounts of ssDNA in N. benthamiana leaf discs, suggests that both ORFs are functional in this host. A mutant containing a termination codon within the 5' terminus of ORF C4 produced severe symptoms in both N. benthamiana and B. vulgaris resembling those induced by wild-type virus. Comparison with the phenotypes of previously characterized ORF C4 mutants suggests that a conserved core sequence of this ORF is an important symptom determinant. ORF C2, C3, and C4 mutants produced virus particles and were transmitted by C. tenellus, eliminating agroinoculation as a contributory factor to the mutant phenotypes. Our results are compared with those derived from mutagenesis studies on related bipartite geminiviruses.

Amino Acid Sequence↗

PubMed1992

Inhibition of African cassava mosaic virus systemic infection by a movement protein from the related geminivirus tomato golden mosaic virus.

Plant viruses encode proteins that mediate their movement through the host plant leading to the establishment of a systemic infection. We have analyzed the effect of tomato golden mosaic virus (TGMV) genes BL1 and BR1, which are thought to be involved in the process of virus movement, on the infectivity of African cassava mosaic virus (ACMV) in Nicotiana benthamiana. Recombinant genomes were constructed by replacing the ACMV coat protein coding sequence with those of either BL1 or BR1. Replication of recombinants containing BL1 and BR1 coding sequences in the sense orientation with respect to the coat protein promoter was detected in the inoculated leaves only when the constructs were co-inoculated, suggesting that both genes are being expressed and act in a cooperative manner. Co-inoculated recombinants induced localized symptoms on inoculated leaves but did not spread systematically, either because of a defect in BL1 and/or BR1 expression or due to the inability of the TGMV gene products to functionally complement their ACMV counterparts. Systemic spread of ACMV was inhibited when the recombinant containing the BL1 coding sequence in the sense, but not in the antisense, orientation was co-inoculated with ACMV DNA B. Disruption of the BL1 coding sequence by a frameshift mutation restored the ability of the recombinant to spread systemically, suggesting that the gene product is responsible for the inhibitory effect. The inhibitory phenotype was mimicked by a chimera containing amino-terminal sequences of TGMV BL1 and carboxy-terminal sequences of its ACMV homologue, BC1. The chimera has characteristics of a dominant negative mutant. We suggest that dominant negative mutants of virus movement genes may provide a novel source for virus resistance genes.

Base Sequence↗

PubMed1992

Characterization of beet curly top virus subgenomic DNA localizes sequences required for replication.

Subgenomic viral DNA is accumulated in Nicotiana benthamiana and Beta vulgaris plants agroinoculated with the geminivirus beet curly top virus. The subgenomic DNA is more abundant in N. benthamiana and is distributed between two broad size groups in this host. Six unique examples, ranging in size from 887 to 1311 nucleotides, have been cloned from viral double-stranded DNA purified from N. benthamiana and analyzed by sequence determination. Deletions are distributed throughout most of the genome and only nucleotides 2946-410 are represented in all subgenomic DNAs. Comparison with a previously characterized subgenomic DNA suggests that cis-acting signals necessary for viral DNA replication are located in a predominantly intergenic region between nucleotides 2946-308.

Base Sequence↗

PubMed1992

A symptom variant of beet curly top geminivirus produced by mutation of open reading frame C4.

Two beet curly top virus (BCTV) mutants have been constructed in vitro that contain G-to-T transversions either at nucleotide 2682 or at nucleotide 2802 within the overlapping open reading frames (ORFs) C1 and C4. The mutations introduce termination codons in ORF C4 without affecting the amino acids encoded by ORF C1. When agroinoculated into Nicotiana benthamiana the mutants caused stunting and yellowing of the plant and downward leaf curl but not the vein swelling and upward leaf curl symptoms that are characteristic of wild-type BCTV infection in this host. Levels of viral single- and double-stranded DNA forms were similar in mutant and wild-type infections. Symptoms induced by one such mutant in Nicotiana clevelandii and Datura stramonium were less severe than those in wild-type infections and were again qualitatively distinct. The mutants caused symptomless infections in Beta vulgaris, contrasting with stunting, severe leaf curl, and vein swelling symptoms associated with wild-type infection of this host. The levels of mutant DNA in newly expanding asymptomatic leaves frequently reached those of wild-type virus in leaves showing severe symptoms. The results suggest that ORF C4 encodes a protein that is a major determinant of pathogenesis that might affect the hyperplastic response of the host to BCTV infection.

Cloning, Molecular↗

PubMed1992

Subchronic toxicity study of Caramel Colour II in F344 rats.

Caramel Colour II is a distinct type of colourant with a pronounced reddish hue. It is made with sulphite reactants but without ammonia. The red colour and a high alcohol solubility provide functional characteristics that are important in foods or beverages containing natural flavour extractives. Caramel Colour II is widely used in ice creams and liqueurs; however, it represents less than 1% of total caramel colour manufacture. The toxicity of Caramel Colour II was evaluated in a 13-wk study in Fischer-344 (F344) rats. The test material was mixed with demineralized water and the solutions were given to the animals ad lib. in the drinking fluid. The concentrations of caramel colour in the drinking fluid were adjusted periodically to achieve the desired caramel colour intake/kg body weight/day. Groups of 20 rats/sex were given Caramel Colour II at levels of 0, 4, 8, 12 or 16 g/kg for at least 13 wk. There were no deaths in any of the groups fed Caramel Colour II. All rats fed caramel colour had soft faeces. All treated groups also had lower fluid consumption that was attributed to poor palatability of the high concentrations of caramel colour that were fed. A number of changes observed (reduced food consumption in all treatment groups except males given 4 g/kg; significantly lower body weights for males given 12 g/kg or more and for females given 8 g/kg or more; lower urine volume and higher specific gravity) were attributed to the reduced water intake and not considered to be toxicologically significant. There were no consistent treatment-related alterations in haematology or blood chemistry variables, and random changes noted were not associated with macroscopic or microscopic pathological alterations. There were no toxicologically important pathological findings. Based on this study, Caramel Colour II was not toxic in F344 rats treated for 13 wk. The highest dose level tested in this study (16 g/kg) was considered to be the no-observed-adverse-effect level.

Animals↗

PubMed1992

Toxicity studies of Caramel Colour III and 2-acetyl-4(5)-tetrahydroxybutylimidazole in F344 rats.

Caramel Colour III is used as a colour additive in beers and a variety of foods. Beer is the most important single source of Caramel Colour III in the diet although consumption of dark beers has been decreasing in recent years. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established an acceptable daily intake of 200 mg/kg/day for Caramel Colour III. The safety of Caramel Colour III has been questioned during recent years following feeding studies in the rat that were associated with reduced white cell and lymphocyte counts. These effects have been attributed to the presence of 2-acetyl-4(5)-tetrahydroxybutylimidazole (THI) in this class of caramel colour. Short-term oral toxicity studies were conducted on low-THI and high-THI samples of Caramel Colour III (13 wk) and on a sample of THI (28 days). In both studies, the test materials were mixed with demineralized water and the solutions were given to the animals ad lib. in the drinking fluid. In the 13-wk subchronic toxicity study of Caramel Colour III, groups of 20 rats/sex were given concentrations of caramel colour equivalent to intakes of 0, 10, 15 or 20 g low-THI caramel colour/kg body weight/day or 20 g/kg of a high-THI caramel colour. In the 4-wk toxicity study with THI, groups of 20 rats/sex were given 0, 8 or 64 ppm THI (equivalent to approx. 0, 0.9 or 7.2 mg/kg/day) and 10 rats/sex were given 1, 2, 4, 16 or 32 ppm THI (equivalent to approx. 0.1, 0.2, 0.5, 1.9 or 3.7 mg/kg/day) for 4 wk followed by a 2-wk recovery phase for 10 rats/sex in the 0, 8 and 64 ppm groups. Rats given Caramel Colour III had soft faeces; there were no other treatment-related clinical observations and no treatment-related deaths occurred. All treated groups given Caramel Colour III had lower food and fluid consumption than controls. Males given 15 or 20 g low-THI caramel colour/kg or 20 g high-THI caramel colour/kg and females given 20 g/kg of either type had lower body weights than controls. In the 4-wk toxicity study with THI, there were no treatment-related ante-mortem observations, and no effects on body weights or food consumption. Fluid consumption by males and females treated with 64 ppm THI was lower than that of controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration, Oral↗

PubMed1992

Toxicity and carcinogenicity studies of Caramel Colour IV in F344 rats and B6C3F1 mice.

Caramel Colour IV, a type of caramel colour used in the manufacture of cola soft drinks, was evaluated for subchronic and chronic toxicity in rats, and carcinogenicity in Fischer-344 (F344) rats and B6C3F1 mice. In each of the studies, Caramel Colour IV was mixed with demineralized water and the solutions given to the animals ad lib. in the drinking fluid. The concentrations of Caramel Colour IV in the drinking fluid were adjusted periodically to achieve the desired caramel colour intake per kg body weight. In the range-finding studies, groups of 30 rats/sex were given Caramel Colour IV at levels of 0, 15, 20, 25 or 30 g/kg for 13 wk, and groups of 10 male rats were given levels of 0, 2.5, 5, 10 or 15 g/kg for 6 wk followed, for some dose groups, by a 2-wk withdrawal period, and then re-initiation of dosing for another 2 wk. In the rat chronic toxicity study, levels of Caramel Colour IV of 0, 2.5, 5, 7.5 or 10 g/kg were given to groups of 25 rats/sex for 12 months. The test groups in the rat and mouse carcinogenicity studies were composed of 50 animals/sex and each species was given the caramel colour at levels of 0, 0, 2.5, 5 or 10 g/kg for 24 months. In each of the studies, treated animals tended to have dose-related lower water consumption than controls. This was attributed to poor palatability of the drinking fluid, and was generally associated with decreased food consumption and body weights. Rats given caramel colour often had soft or liquid malodorous faeces although there were no treatment-related ante-mortem observations in mice. Blood biochemical changes in the rat (i.e. reduced blood urea nitrogen, alkaline phosphatase and total serum protein) appeared to be related to dietary influences and were not considered toxicologically significant. There were no treatment-related alterations in haematological variables or treatment-related differences in survival or in the incidence of benign or malignant tumours among treated and control groups and no toxicologically important pathological findings. On the basis of these studies, Caramel Colour IV was not toxic or carcinogenic in F344 rats or B6C3F1 mice. The highest dose level tested in the long-term studies (10 g/kg) was considered to be the no-observed-adverse-effect level (NOAEL).

Administration, Oral↗

PubMed1992

Molecular typing of Helicobacter pylori by chromosomal and plasmid DNA organization.

Diverse strains of Helicobacter pylori were examined in order to initiate a molecular epidemiological typing scheme for this agent of human gastritis. Twelve differently-sized plasmids from 1.8 to 63 kbp were identified in those strains harbouring extrachromosomal DNA. Recombinant DNA probes were cloned randomly from the chromosome of the (plasmid-free) type strain (NCTC 11637), and used to probe genomic Southern blots for restriction site variation in and around homologous loci. Genus-specific probe DNAs were obtained which grouped strains on the bases of DNA base substitution or rearrangements. On the basis of the four probes examined, all strains exhibited intraspecific chromosomal divergence, indicating that H. pylori is highly diverse genetically, but nonetheless susceptible to chromosome and plasmid molecular typing.

Bacterial Typing Techniques↗

PubMed1992

Enterobacterial tetracycline resistance in relation to plasmid incompatibility.

Sixty-eight well-characterized antibiotic resistance (R) plasmids belonging to 19 Incompatibility groups were screened with probes representing heterologous classes (A-E) of the enterobacterial tetracycline resistance (TcR) determinant. The Class B determinant was shown to be predominant in the IncF and IncH complexes and the IncC group. The Class A determinant was shown to be predominant in the IncP and IncM groups. There was no correlation between distribution of the class of TcR gene and the genus or the species of the host bacterial strain. The plasmids R714a (IncFI) and pHH1465 (IncC) contained TcR determinants which had no homology with any of these five probes.

Enterobacteriaceae↗