Marine amoebae from waters of northwest Spain, with comments on a potentially pathogenic euryhaline species

Of 17 species of free-living amoebae identified in various samples of salt water, only 1, Acanthamoeba polyphaga, is known to be a potential pathogen. While no deaths occurred when laboratory animals were inoculated with A. polyphaga to test for pathogenicity, the protozoa were present in the brain, liver and lungs of some but not all of the animals.     

Linkage disequilibrium studies in multiple endocrine neoplasia type 1 (MEN1)

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroids, pancreas and anterior pituitary. The MEN1 gene has been localised to a 2-Mb region of chromosome 11q13 by meiotic mapping studies in MEN1 families. Such studies may have a limited resolution of approximately 1 cM (i.e. 1 Mb) and we have therefore investigated 96 MEN1 families (40 British, 17 French, 12 Finnish, 7 Swedish, 7 Dutch, 7 North American, 2 Australian, 1 New Zealand, 1 German, 1 Spanish and 1 Danish) for linkage disequilibrium, in order to facilitate a finer mapping resolution. We have utilised five microsatellite DNA sequence polymorphisms from the candidate region and have accurately determined their allele sizes, which ranged from 161 bp to 272 bp. The heterozygosity and number of alleles (given in brackets), respectively, at the loci were: D11S1883 (76%, 11), D11S457 (55%, 5), PYGM (94%, 18), D11S1783 (10%, 4) and D11S449 (87%, 16). Allelic association was assessed by Chi-square 2 ×n contingency tables, by Fisher exact 2 ×n contingency tables and by a likelihood-based approach. The results of haplotype analysis revealed 91 different affected haplotypes in the 96 families, an identical affected haplotype being observed in no more than two families. These results indicate the absence of an ancestral affected haplotype. Significant linkage disequilibrium (P < 0.005) could be established amongst the microsatellite loci but not between the loci and MEN1 in either the total population or in any of the geographical sub-populations. The absence of linkage disequilibrium between MEN1 and the polymorphic loci is probably the result of the occurrence of multiple different disease-causing mutations in MEN1. Received: 1 April 1997 / Accepted: 25 June 1997     

Mitochondrial function in oncogene-transfected rat fibroblasts isolated from multicellular spheroids

Twomitochondrion-specific fluorochromes,10-N-nonyl acridineorange (NAO) and rhodamine 123 (Rh123), were used todetermine the mechanism responsible for alterations in energymetabolism of transformed rat embryo fibroblast cells isolated fromdifferent locations within multicellular spheroids. Accumulation ofRh123 depends on intact mitochondrial membrane potential, whereas NAO is taken up by mitochondria independently of their function and thusrepresents mitochondrial distribution only. A reproducible selectivedissociation procedure was used to isolate cells from differentlocations within the spheroids. After isolation, cells weresimultaneously stained with one mitochondrial stain and the DNA dyeHoechst 33342, and several parameters, including cell volume, weremonitored via multilaser-multiparameter flow cytometry. Our dataclearly show a decrease in the uptake of Rh123 in cells from theperiphery to the inner regions of the tumor spheroids, reflecting apersistent alteration in mitochondrial function. However, NAO stainingexperiments showed no reduction in the total mitochondrial mass perunit cell volume. Because cells were exposed to stain under uniformconditions after isolation from the spheroid, these data indicate thatdownregulation of mitochondrial function is associated with cellquiescence rather than a transient effect of reduced nutrientavailability. This result, which is in accordance with data from twoother cell lines (EMT6 and 9L), might reflect a general phenomenon inmulticellular spheroids, supporting the hypothesis that quiescent cellsin the innermost viable spheroid layer stably reduce theirmitochondrial function, presumably to compensate for lower nutrientsupply and/or decreased energy demand.

     

The utilization of organosulphonates by soil and freshwater bacteria

Utilization of the biogenic aliphatic organosulphonates taurine, isethionate, sulphoacetaldehyde and sulphoacetate was investigated in 100 soil and freshwater bacteria isolated on modified complete mineral salts medium. More than 90% could use all the compounds as sole sulphur sources, and some 10% used taurine and isethionate as sole carbon and energy, or sole carbon, energy and sulphur sources. None could mineralize sulphoacetaldehyde or sulphoacetate; however, two isolates capable of growth on sulphoacetate as sole carbon, energy and sulphur source were obtained by enrichment culture. The results suggest that in the majority of environmental bacteria the pathways of organosulphonate biodegradation may be independently controlled by the supply of carbon and sulphur to the cell, and that a number of routes may exist for cleavage of the organosulphonate C–S bond.     

An ELISA for detection of apoptosis.

We describe a simple and convenient enzyme-linked immunosorbent assay (ELISA) for the detection of apoptosis in tissue culture. An early event in apoptosis is DNA fragmentation followed by release of nucleosomes into the cytoplasm. Our sandwich assay uses a pair of monoclonal antibodies specific for two nucleosomal epitopes to capture and detect cytoplasmic nucleosomes onto the ELISA plate. Our assay is about 500 times more sensitive than the detection of apoptotic DNA ladder by agarose electrophoresis and is especially suited for the testing of large numbers of samples.     

Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum

Expression of virulence genes in Ralstonia solanacearum , a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs. Production of several virulence determinants is co-ordinately reduced by inactivation of phcB , but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wild-type strains of R. solanacearum . The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography. Gas chromatography and mass spectroscopy identified 3-hydroxypalmitic acid methyl ester (3-OH PAME) as the major component in the single peak of VEF activity. Authentic 3-OH PAME and the purified VEF were active at ≤1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant. Authentic 3-OH PAME also increased the production of three virulence factors by a phcB mutant over 20-fold to wild-type levels, restored normal cell density-associated expression of eps and increased expression of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small-molecule S -adenosylmethionine-dependent methyltransferase, which might catalyse synthesis of 3-OH PAME from a naturally occurring fatty acid. Biologically active concentrations of extracellular 3-OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild-type R. solanacearum . Other than acyl-homoserine lactones, 3-OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long-distance intercellular communication.     

Poverty or income inequality as predictor of mortality: longitudinal cohort study.

OBJECTIVE: To determine the effect of inequality in income between communities independent of household income on individual all cause mortality in the United States. DESIGN: Longitudinal cohort study. SUBJECTS: A nationally representative sample of 14,407 people aged 25-74 years in the United States from the first national health and nutrition examination survey. SETTING: Subjects were followed from initial interview in 1971-5 until 1987. Complete follow up information was available for 92.2% of the sample. MAIN OUTCOME MEASURES: Relation between both household income and income inequality in community of residence and individual all cause mortality at follow up was examined with Cox proportional hazards survival analysis. RESULTS: Community income inequality showed a significant association with subsequent community mortality, and with individual mortality after adjustment for age, sex, and mean income in the community of residence. After adjustment for individual household income, however, the association with mortality was lost. CONCLUSIONS: In this nationally representative American sample, family income, but not community income inequality, independently predicts mortality. Previously reported ecological associations between income inequality and mortality may reflect confounding between individual family income and mortality.