Strong asymmetric mutation bias in endosymbiont genomes coincide with loss of genes for replication restart pathways

A large majority of bacterial genomes show strand asymmetry, such that G and T preferentially accumulate on the leading strand. The mechanisms are unknown, but cytosine deaminations are thought to play an important role. Here, we have examined DNA strand asymmetry in three strains of the aphid endosymbiont Buchnera aphidicola. These are phylogenetically related, have similar genomic GC contents, and conserved gene order structures, yet B. aphidicola (Bp) shows a fourfold higher replication-induced strand bias than B. aphidicola (Sg) and (Ap). We rule out an increase in the overall substitution frequency as the major cause of the stronger strand bias in B. aphidicola (Bp). Instead, the results suggest that the higher GC skew in this species is caused by a different spectrum of mutations, including a relatively higher frequency of C to T mutations on the leading strand and/or of G to A mutations on the lagging strand. A comparative analysis of 20 gamma-proteobacterial genomes revealed that endosymbiont genomes lacking recA and other genes involved in replication restart processes, such as priA, which codes for primosomal helicase PriA, displayed the strongest strand bias. We hypothesize that cytosine deaminations accumulate during single-strand exposure at arrested replication forks and that inefficient restart mechanisms may lead to high DNA strand asymmetry in bacterial genomes.     

Serum malondialdehyde correlates with therapeutic efficiency of high activity antiretroviral therapies (HAART) in HIV-1 infected children

Serum malondialdehyde (MDA) levels are increased in human immunodeficiency virus (HIV)-infected children, as it happens also in infected adult individuals. Introduction of high activity antiretroviral therapy (HAART) has promoted an intense decline in morbidity and mortality of these patients. Here we present data on the effect of HAART on serum MDA of HIV+ children and compare them with levels prior to HAART. MDA levels reflect, as other markers do, the HAART-induced clinical improvement and probably also the pro-oxidant/antioxidant side effects of the different drugs used. The results herein allow the proposal of including serum MDA levels as an additional parameter for the clinical management of HIV+ children.     

Genotoxic effects of ethylene oxide,propylene oxide and epichlorohydrin in humans: update review (1990-2001)

Ethylene oxide (EtO), propylene oxide (PO) and epichlorohydrin (ECH) are important industrial chemicals widely used as intermediates for various synthetic products. EtO and PO are also environmental pollutants. In this review we summarize data published during the period 1990-2001 concerning both the genotoxic and carcinogenic effects of these epoxides in humans. The use of DNA and hemoglobin adducts as biomarkers of exposure and the role of polymorphism, as well as confounding factors, are discussed. We have also included recent in vitro data comprising genotoxic effects induced by EtO, PO and ECH in mammalian cells. The uncertainties regarding cancer risk estimation still persist, in spite of the large database collected.     

Genetic serum protein markers (HP,TF, GC and PI) in eight Slovakian population samples

1194 individuals from eight different regions of Slovakia have been typed for haptoglobin (HP) types and for transferrin (TF), group specific component (GC) and alpha-1-antitrypsin (PI) subtypes. Whereas the HP allele frequencies do not show a remarkable regional variability within Slovakia, this could be demonstrated concerning the TF, GC and PI allele frequencies. The reason for these distribution heterogeneities seems to be due to the incomplete panmixia of the Slovakian population by which local variations in the distribution of genetic markers could be maintained.     

Probing the active site of glyoxalase I from human erythrocytes by use of the strong reversible inhibitor S-p-bromobenzylglutathione and metal substitutions

Glyoxalase I from human erythrocytes was studied by use of the strong reversible competitive inhibitor S-p-bromobenzylglutathione. Replacements of cobalt, manganese and magnesium for the essential zinc in the enzyme were made by a new procedure involving 10% methanol as a stabilizer of the enzyme. The K_m value for the adduct of methylglyoxal and glutathione was essentially unchanged by the metal substitutions, whereas the inhibition constant for S-p-bromobenzylglutathione increased from 0.08μm for the Zn-containing enzyme to 1.3, 1.7 and 2.4μm for Co-, Mn- and Mg-glyoxalase I respectively. Binding of the inhibitor to the enzyme caused quenching of the tryptophan fluorescence of the protein, from which the binding parameters could be determined by the use of non-linear regression analysis. The highest dissociation constant was obtained for apoenzyme (6.9μm). The identity of the corresponding kinetic and binding parameters of the native enzyme and the Zn²⁺-re-activated apoenzyme and the clear differences from the parameters of the other metal-substituted enzyme forms give strong support to the previous identification of zinc as the natural metal cofactor of glyoxalase I. Binding to apoenzyme was also shown by the use of S-p-bromobenzylglutathione as a ligand in affinity chromatography and as a protector in chemical modification experiments. The tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl bromide caused up to 85% inactivation of the enzyme. After blocking of the thiol groups (about 8 per enzyme molecule) 6.1 2-hydroxy-5-nitrobenzyl groups were incorporated. Inclusion of S-p-bromobenzylglutathione with the modifying reagent preserved the catalytic activity of the enzyme completely and decreased the number of modified residues to 4.4 per enzyme molecule. The findings indicate the presence of one tryptophan residue in the active centre of each of the two subunits of the enzyme. Thiol groups appear not to be essential for catalytic activity. The presence of at least two categories of tryptophan residues in the protein was also shown by quenching of the fluorescence by KI.     

Temperature-Dependent Variation in API 50 CH Fermentation Profiles of Lactobacillus Species

API 50 CH fermentation profiles of 45 Lactobacillus, one Atopobium, and three Weissella strains incubated at 30°C and 37°C were evaluated. Atopobium uli and ten species of Lactobacillus showed stable patterns despite the change in temperature. The rest of the type strains showed discrepancy between the two incubation temperatures: 18 strains lost, 12 additionally fermented another sugar, and 7 others fermented a different one in lieu. The variation was maximum in L. delbrueckii subsp. delbrueckii. L. malefermentans failed to ferment any of the substrates at 37°C. Majority of the food and plant-associated strains (mainly heterofermenters) retained distinctive traits at 30°C, while most of the animal-associated strains (mostly homofermenters) did so at 37°C. No general trend was observed; 30°C appeared to promote heterofermentation, while 37°C favored homofermenters. Use of API 50 CH profiles for taxonomic purpose in most lactobacilli appears reproducible if a specific temperature for a species is strictly followed. Received: 10 December 1999 / Accepted: 10 January 2000     

Determinative Value of a Portion of the nifH Sequence for the Genera Nostoc and Anabaena (Cyanobacteria)

The taxonomic positions of Nostoc and Anabaena strains are currently disputed. We selected three Nostoc and Anabaena strains, using the classic criteria of morphology and life cycle. DNA sequences of a part of the nifH gene were determined from these strains and aligned with homologous sequences from 10 other Nostoc/Anabaena strains in the public databases. Phylogenetic reconstructions were carried out to test the consistency of the taxonomic placement of these strains. The phylogenetic trees do not separate these strains into distinct groups. Our results are in agreement with other molecular-based phylogenies that also fail to differentiate the Nostoc-Anabaena groups. The data suggest that the currently recognized genera Nostoc and Anabaena may in fact belong within a single, broadly defined genus. Received: 14 February 2000 / Accepted: 21 March 2000     

Characterization of a ribonuclease from Anacystis nidulans infected with cyanophage as-1

In Anacystis nidulans the ribonuclease (RNase) activity is very low but is greatly increased upon phage-infection. A RNase was isolated and purified over 300-fold from A. nidulans cells infected by cyanophage AS-1. The enzyme did not attack single- or double-stranded DNA, was inactive on p-nitrophenyl phosphate or bis-p-nitrophenyl phosphate as substrates, and had neither 3′- nor 5′-nucleotidase activity. The approximate MW of the enzyme was 12000. Maximal enzyme activity was at pH 7.5. No absolute requirement for metal ions was observed, but Fe³⁺ stimulated and Co²⁺ and Ni²⁺ inhibited enzyme activity. The enzyme is an endonuclease which, upon exhaustive hydrolysis, produces mainly oligonucleotides (average chain-length: 3) with 3′-P termini. Analysis of the base composition of these oligonucleotides and determination of their 3′-terminal nucleosides, together with the investigation of the rate of hydrolysis of synthetic polyribonucleotides, have shown that the enzyme has a relative specificity for uridylic acid.     

Multiple displacement amplification of DNA from human colon and rectum biopsies: bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis

Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis; the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens.