SSCP analysis of the tyrosine kinase domain of the insulin receptor gene: Polymorphisms detected in South African black and white subjects

The frequency of DNA polymorphisms in the tyrosine kinase domain (exons 17–21) of the insulin receptor gene was assessed in 30 black and 30 white South Africans, using single-stranded conformation polymorphism and direct sequencing analysis. A comparison of the frequencies of the normal versus the combined polymorphic alleles, found only in exon 17, showed a significant difference between black and white groups (P = 0.037). Received: 25 May 1995 / Revised: 1 September 1995     

HSP25 in isolated perfused rat hearts: Localization and response to hyperthermia

Recent investigations concentrate on the correlation between the myocardial expression of the inducible 70-kDa heat shock protein (HSP70i) by different stress conditions and its possible protective effects. Only few studies have focused on the involvement of small heat shock proteins in this process. We analyzed the location of the small heat shock protein HSP25 in isolated cardiomyocytes as well as its location and induction in isolated perfused hearts of rats. By immunofluorescence microscopy HSP25 was found to colocalize with actin in the I-band of myofibrils in cardiomyocytes of isolated perfused hearts as well as in isolated neonatal and adult cardiomyocytes. Hyperthermic perfusion of isolated hearts for 45 min resulted in modulation of different parameters of heart function and in induction of HSP25 and HSP70i. Temperatures higher than 43°C (44–46°C) were lethal with respect to the contractile function of the hearts. Compared to control hearts perfused at 37°C, significant increases during hyperthermic perfusion at 42°C and 43°C were obtained for heart rate, contraction velocity and relaxation velocity. In response to hyperthermia at 43°C and after subsequent normothermic perfusion for 135 min at 37°C, left ventricular pressure, contraction velocity and relaxation velocity remained significantly elevated. However, heart rate returned to control values immediately after the period of heat treatment. HSP25 is constitutively expressed even in normothermic perfused hearts as shown by Western blotting. Hyperthermia increased the content of HSP25 only in the left ventricular tissue. In contrast, HSP70i was strongly induced in all analyzed parts of the myocardium (left ventricle, right ventricle, septum). Our findings suggest a differential regulation of HSP25 and HSP70i expression in response to hyperthermia in isolated perfused hearts. The constitutively expressed HSP25 seems to be located adjacent to the myofibrils which implies a specific role of this protein even under unstressed conditions for the contractile function of the myocardium.     

Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria.

A hierarchical set of five 16S rRNA-targeted oligonucleotide DNA probes for phylogenetically defined groups of autotrophic ammonia- and nitrite-oxidizing bacteria was developed for environmental and determinative studies. Hybridization conditions were established for each probe by using temperature dissociation profiles of target and closely related nontarget organisms to document specificity. Environmental application was demonstrated by quantitative slot blot hybridization and whole-cell hybridization of nitrifying activated sludge and biofilm samples. Results obtained with both techniques suggested the occurrence of novel populations of ammonia oxidizers. In situ hybridization experiments revealed that Nitrobacter and Nitrosomonas species occurred in clusters and frequently were in contact with each other within sludge flocs.     

Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies.

Universal oligonucleotide hybridization probes targeting the small-subunit rRNA are commonly used to quantify total microbial representation in environmental samples. Universal probes also serve to normalize results obtained with probes targeting specific phylogenetic groups of microorganisms. In this study, six universal probes were evaluated for stability of probe-target duplexes by using rRNA from nine organisms representing the three domains of Bacteria, Archaea, and Eucarya. Domain-specific variations in dissociation temperatures were observed for all probes. This could lead to a significant bias when these probes are used to quantify microbial populations in environmental samples. We suggest lowering the posthybridization wash stringency for two of the universal probes (S-*-Univ-1390-a-A-18 and S-*-Univ-1392-a-A-15) examined. These two probes were evaluated with traditional and modified hybridization conditions to characterize defined mixtures of rRNAs extracted from pure cultures and rRNA samples obtained from anaerobic digester samples. Probe S-*-Univ-1390-a-A-18 provided excellent estimations of domain-level community composition of these samples and is recommended for future use in microbial ecology studies.     

Competition and Coexistence of Sulfate-Reducing and Methanogenic Populations in Anaerobic Biofilms

The microbial population structure and function of natural anaerobic communities maintained in laboratory fixed-bed biofilm reactors were tracked before and after a major perturbation, which involved the addition of sulfate to the influent of a reactor that had previously been fed only glucose (methanogenic), while sulfate was withheld from a reactor that had been fed both glucose and sulfate (sulfidogenic). The population structure, determined by using phylogenetically based oligonucleotide probes for methanogens and sulfate-reducing bacteria, was linked to the functional performance of the biofilm reactors. Before the perturbation, the methanogenic reactor contained up to 25% methanogens as well as 15% sulfate-reducing bacteria, even though sulfate was not present in the influent of this reactor. Methanobacteriales and Desulfovibrio spp. were the most abundant methanogens and sulfate-reducing bacteria, respectively. The presence of sulfate-reducing bacteria (primarily Desulfovibrio spp. and Desulfobacterium spp.) in the absence of sulfate may be explained by their ability to function as proton-reducing acetogens and/or fermenters. Sulfate reduction began immediately following the addition of sulfate consistent with the presence of significant levels of sulfate-reducing bacteria in the methanogenic reactor, and levels of sulfate-reducing bacteria increased to a new steady-state level of 30 to 40%; coincidentally, effluent acetate concentrations decreased. Notably, some sulfate-reducing bacteria (Desulfococcus/Desulfosarcina/Desulfobotulus group) were more competitive without sulfate. Methane production decreased immediately following the addition of sulfate; this was later followed by a decrease in the relative concentration of methanogens, which reached a new steady-state level of approximately 8%. The changeover to sulfate-free medium in the sulfidogenic reactor did not cause a rapid shift to methanogenesis. Methane production and a substantial increase in the levels of methanogens were observed only after approximately 50 days following the perturbation.     

The phage lambda orf gene encodes a trans-acting factor that suppresses Escherichia coli recO, recR, and recF mutations for recombination of lambda but not of E. coli.

Bacteriophage lambda can recombine in recBC sbcB sbcC mutant cells by using its own gene orf, the Escherichia coli recO, recR, and recF genes, or both. Expression of an orf-containing plasmid complements the recombination defects of orf mutant phage. However, this clone does not complement a recO mutation for conjugational recombination or recO, recR, or recF mutations for repair of UV-induced DNA damage. A plasmid clone of orf produces a protein with an apparent molecular mass of 15 kDa.     

Comparison of phylogenetic relationships based on phospholipid fatty acid profiles and ribosomal RNA sequence similarities among dissimilatory sulfate-reducing bacteria

Abstract Twenty-five isolates of dissimilatory sulfate-reducing bacteria were clustered based on similarity analysis of their phospholipid ester-linked fatty acids (PLFA). Of these, 22 showed that phylogenetic relationships based on the sequence similarity of their 16S rRNA directly paralleled the PLFA relationships. Desulfobacter latus and Desulfobacter curvatus grouped with the other Desulfobacter spp. by 16S rRNA comparison but not with the PLFA analysis as they contained significantly more monoenoic PLFA than the others. Similarly, Desulfovibrio africanus clustered with the Desulfovibrio spp. by 16S rRNA but not with them when analyzed by PLFA patterns because of higher monoenoic PLFA content. Otherwise, clustering obtained with either analysis was essentially congruent. The relationships defined by PLFA patterns appeared robust to shifts in nutrients and terminal electron acceptors. Additional analyses utilizing the lipopolysaccharide-lipid A hydroxy fatty acid patterns appeared not to shift the relationships based on PLFA significantly except when completely absent, as in Gram-positive bacteria. Phylogenetic relationships between isolates defined by 16S rRNA sequence divergence represent a selection clearly different from the multi-enzyme activities responsible for the PLFA patterns. Determination of bacterial relationships based on different selective pressures for various cellular components provides more clues to evolutionary history leading to a more rational nomenclature.