Use of subtractive hybridization to identify a diagnostic probe for a cystic fibrosis epidemic strain of Pseudomonas aeruginosa

A multiresistant strain of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients attending clinics in Liverpool, United Kingdom. Suppression subtractive hybridization was used to identify sequences present in the Liverpool CF epidemic strain but absent from strain PAO1. Using dot blot and PCR amplification assays, the prevalence of such sequences among a panel of CF isolates was determined. Several sequences were found only in the Liverpool epidemic strain. Some sequences were present in the Liverpool epidemic strain and in a minority of other isolates, including sequences with homology to genes implicated in O6 serotype and siderophore production. The Liverpool epidemic strain and 81% of nonepidemic isolates contained a sequence identified as part of the PAGI-1 genomic island. Other strains implicated in epidemic spread, which were from Manchester, United Kingdom, and Melbourne, Australia, were also screened. None of the sequences identified was present in the Manchester strain. However, one of two Melbourne strains contained some of the sequences found in the Liverpool epidemic strain. All isolates implicated in epidemic spread and 76% of sporadic isolates contained the exoS gene. A sequence present in all isolates of the Liverpool epidemic strain was used to develop a diagnostic PCR test for identification of the strain from colonies or directly from sputum samples.     

Observations on rat collagenase-separated islets and long-term composite islet allografts

Collagenase digest preparations of isolated rat islets and long-surviving allogeneic rat islets implanted beneath the kidney capsule of a composite kidney and islet allograft have been studied immunocytochemically and by electron microscopy. The four main endocrine cell types were identified in the collagenase preparations with minimal evidence of cell damage. In contrast, there were only granulated B cells in the composite grafts and amongst the granules of these cells there was pleomorphism. The granule appearances may support previous suggestions that paracrine control of insulin secretion is lacking in these composite grafts.     

Development of monoclonal mouse antibodies specific for allergenic components in ryegrass (Lolium perenne) pollen

The development of monoclonal mouse antibodies against ryegrass (Lolium perenne) pollen allergens is described. Hybridoma colonies secreting antibodies specific for allergenic components were detected using an enzyme-linked immunosorbent assay. Positive colonies were cloned and expanded. The pollen components with which the monoclonal antibodies interact were identified and characterised following sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose. In this paper six monoclonal mouse antibodies are described. Three antibodies interact with a single molecule of between 30,000 and 35,000 daltons. One antibody interacts with a component of 16,000 daltons whereas the remaining two antibodies react with more than one component, one reacting with two components at 28,000 and 30,000, and the other with five components having molecular weights between 18,000 and 71,000 daltons.     

Hybridization studies of cultured human pituitary prl and gh producing adenoma cells: Effects of thyrotropin-releasing hormone,somatostatin, and phorbol ester

The effects of the hypothalamic hormones, thyrotropin-releasing hormone (TRH), and somatostatin (SRIH), and of phorbol 12-myristate 13-acetate (PMA) on PRL and GH secretion and messenger RNA (mRNA) levels were analyzed in 10 GH and/or PRL producing adenomas after culturing the tumor cells in the presence of these secretagogues for 7 days. The expression of chromogranin A and B mRNAs was also examined. All four of the clinically diagnosed GH adenomas expressed or secreted both GH and PRL while four of six clinically diagnosed prolactinomas produced or secreted both PRL and GH. Prolactinomas had less than 10% of tumor cells expressing chromogranin A mRNA while more than 40% of the adenoma cells expressed chromogranin B mRNA. TRH stimulated PRL secretion and increased PRL mRNA levels while SRIH decreased GH secretion and mRNA expression in some cases. Unexpectedly, PMA stimulated PRL mRNA levels four- to sevenfold above control levels in two adenomas and generally stimulated chromogranin A and B mRNA expression but not GH mRNA, as determined by Northern hybridization and in situ hybridization analyses. These results indicate that cultured prolactinoma cells express significantly more chromogranin B mRNA than chromogranin A mRNA, and that PMA increases PRL mRNA expression in some prolactinomas, although the effect of PMA on various adenomas reflects the heterogeneity of these tumors with respect to protein kinase C stimulation.     

THE RELATION OF THYROID AUTO-IMMUNITY TO ROUND-CELLED INFILTRATION OF THE THYROID GLAND

The relationship between thyroid auto-immunity and the presence of lymphocytes in the thyroid gland has been analysed in patients with Graves' disease, toxic adenoma, and non-toxic nodular goitre. In all these conditions circulating antibody to thyroglobulin was significantly associated with lymphocytic infiltration. No similar correlation was established in the case of the complement-fixing antibody.     

Experimental reproduction of the runting-stunting syndrome of broiler chickens

A model for the reproduction of the runting-stunting syndrome (RSS) of broiler chickens is described. In this model, groups of at least 90 day-old broiler chickens were inoculated (per os) with various tissue homo-genates or virus preparations. During the first week post-inoculation, birds were examined for the development of histopathological changes in their intestines. At day 14 post-inoculation, the remaining birds were weighed and tested for elevations in plasma amylase activity and examined for the development of pancreatic atrophy. Bacteria-free intestinal and pancreatic homogenates from chickens of different ages, taken from flocks which developed RSS, regularly induced a lower mean live-weight in treated birds. Of these, only intestinal homogenates prepared from 5-day-old birds induced intestinal lesions, lowered mean live-weight and increased the incidence of both elevated plasma amylase activity and pancreatic atrophy. These changes were more marked in birds exposed to short periods of sub-optimal temperatures during the first week post-inoculation. An ultracentri-fuged pellet prepared from this intestinal homogehate, was also found to induce an increased incidence of pancreatic atrophy in treated birds. These studies suggest that the causative agent(s) of RSS is an as yet unidentified virus, and that the effects of this infection are greater in birds subjected to stress, such as sub-optimal temperature exposure, within the first week of hatch.     

Life expectancy in British Marfan syndrome populations

A total of 206 patients with Marfan syndrome were ascertained throughout genetic clinics in Wales and Scotland during the period 1970–1990. There were 45 deaths representing 22% of the cohort. Mean age at death was 45.3 ± 16.5 years. 50% median cumulative survival in the total cohort (n = 206) was 53 years for males and 72 years for females. Multivariate analysis confirmed severity as the best independent indicator of survival. These findings and survival curves will assist in the counselling of British families and individuals with Marfan syndrome.     

Molecular determinants of glycine receptor αβ subunit sensitivities to Zn2+-mediated inhibition

Glycine receptors exhibit a biphasic sensitivity profile in response to Zn²⁺-mediated modulation, with low Zn²⁺ concentrations potentiating (< 10 μ m ), and higher Zn²⁺ concentrations inhibiting submaximal responses to glycine. Here, a substantial 30-fold increase in sensitivity to Zn²⁺-mediated inhibition was apparent for the homomeric glycine receptor (GlyR) α1 subunit compared to either GlyR α2 or α3 subtypes. Swapping the divergent histidine (H107) residue in GlyR α1, which together with the conserved H109 forms part of an intersubunit Zn²⁺-binding site, for the equivalent asparagine residue present in GlyR α2 and α3, reversed this phenotype. Co-expression of heteromeric GlyR α1 or α2 with the ancillary β subunit yielded receptors that maintained their distinctive sensitivities to Zn²⁺ inhibition. However, GlyR α2β heteromers were consistently 2-fold more sensitive to inhibition compared to the GlyR α2 homomer. Comparative studies to elucidate the specific residue in the β subunit responsible for this differential sensitivity revealed instead threonine 133 in the α1 subunit as a new vital component for Zn²⁺-mediated inhibition. Further studies on heteromeric receptors demonstrated that a mutated β subunit could indeed affect Zn²⁺-mediated inhibition but only from one side of the intersubunit Zn²⁺-binding site, equivalent to the GlyR α1 H107 face. This strongly suggests that the α subunit is responsible for Zn²⁺-mediated inhibition and that this is effectively transduced, asymmetrically, from the side of the Zn²⁺-binding site where H109 and T133 are located.     

Association of polymorphisms of the estrogen receptor gene with anxiety-related traits in children and adolescents: a longitudinal study

Anxiety problems and associated temperamental traits are multifactorial, determined by the interaction of genetic and environmental factors. Genetic effects may involve both neurotransmitters and hormones. A good candidate gene for association with anxiety-related traits is the estrogen receptor (ESRalpha). Estrogen exerts an effect on mood and behavior in humans through gene regulation on binding to estrogen receptor protein. Association between ESRalpha polymorphism and anxiety-related traits was investigated in a cohort of 680 Australian adolescents studied from 4-8 months to 15-16 years of age. Genotype frequencies were estimated for polymorphic PvuII and XbaI restriction sites in intron 1 and a microsatellite [(TA)(n)] locus 5' of ESRalpha. There was strong linkage disequilibrium between the three loci and a significant sex difference was observed in allele (for (TA)(n), PvuII) and genotype (for XbaI) frequencies. There were no significant allelic or genotypic differences in anxiety-related traits for the three loci tested. However, some significant associations were found for PvuII/(TA)(n) and XbaI/(TA)(n) two-locus genotypes and anxiety, accounting for between 1.6% and 2.8% of the total variance for anxiety in this population. The discordance in Hardy-Weinberg proportions at the XbaI locus between the sexes is an important finding, perhaps indicating a sex-specific role for ESRalpha in fetal survival.