Comparison of the adaptive potential of the Arthrobacter oxydans and Acinetobacter lwoffii isolates from permafrost sedimentary rock and the analogous collection strains

A comparative study was conducted on the adaptive mechanisms of the strains Arthrobacter oxydans K14 and Acinetobacter lwoffii EK30A isolated from permafrost subsoil sediments and of those of the analogous collection strains (Ac-1114 Type and BSW-27, respectively). In each pair of the strains compared, the strains differed in terms of (i) growth-related, physiological, and biochemical properties; (ii) resistance to stress factors; (iii) capacity for generation of dormant forms (DFs) under growth arrest conditions, and (iv) intrapopulation production of phase variants. The strains isolated from permafrost displayed a lower growth rate but were more resistant to repeated freezing-thawing treatment than the collection strains. Under the same growth conditions, the permafrost strains formed larger numbers of cystlike anabiotic DFs, extraordinarily small cells, and forms that became nonculturable during long-term storage. Resuscitation of the nonculturable forms resulted in a 2- to-7-fold increase in the percentage of FISH-detectable metabolically active cells. The permafrost strains were also distinguished by increased genome lability. This facilitated their dissociation into intrapopulation variants with phenotypically distinct colonial and morphological properties and different antibiotic resistance. The phenotypic variability was more prominent in Arthrobacter (for which it was not reported previously) than in Acinetobacter. In the populations produced by plating the dormant bacterial forms, the qualitative and quantitative characteristics of the phase variant spectra varied depending on the formation conditions and the composition of the solid media used for the plating. Thus, the permafrost isolates of A. oxydans and Ac. lwoffii were distinguished from their collection analogs by a more manifest adaptive potential including stress resistance, the intensity of DF generation under growth arrest conditions, and increased intrapopulation variability.     

Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst

Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47(phox), a major cytosolic component of this oxidase. Protein kinase C zeta (PKC zeta), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC zeta in p47(phox) phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47(phox) with recombinant PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K(m) value of 2 microM. Phosphopeptide mapping analysis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC zeta by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC zeta that correlated to that of p47(phox). A cell-permeant-specific peptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation of p47(phox) and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC(50) of 10 microM), but not that induced by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 microM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47(phox) is a substrate for PKC zeta and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.     

Characteristics of <Emphasis Type="Italic">Pseudomonas aurantiaca</Emphasis> DNA supramolecular complexes at various developmental stages

Differences in viscoelasticity (η) and molecular mass (M) values, as well as in the fatty acid profile of lipids in DNA supramolecular complexes (SC), isolated from Pseudomonas aurantiaca cultures at the exponential and stationary growth phases, were established for the first time. Typical characteristics of DNA SC from actively growing cells were the following: η = 315 ± 15 dl/g, M_DNA = 39 × 10⁶ Da, C_16:0 > C_18:0 > C_18:1 present as basic fatty acids (FA) in a pool of loosely DNA-bound lipids; the tightly DNA-bound lipid fraction consisted of only two acids C_18:0 > C_16:0. Significantly higher values of viscoelasticity η = 779 ± 8 dl/g and M_DNA = 198 × 10⁶ Da were observed for DNA SC of the stationary phase cells; one more FA, C_14:0, was detected in the loosely bound lipid fraction, while lipids tightly bound to DNA contained mainly C_16:0 > C_18:1 > C_18:0 > C_14:0 FA. The content of saturated FA in the DNA-bound lipids in the stationary phase cells was twice as high than in the exponential phase cells. The fraction of tightly bound lipids from the stationary phase cells contained nine times more unsaturated fatty acids than the fraction from proliferating cells. These differences in FA composition of DNA-bound lipids demonstrate the importance of lipids for the structural organization and functioning of genomic DNA during bacterial culture development.     

What Do Pneumocystis Organisms Tell Us about the Phylogeography of Their Hosts? The Case of the Woodmouse Apodemus sylvaticus in Continental Europe and Western Mediterranean Islands

Pneumocystis fungi represent a highly diversified biological group with numerous species, which display a strong host-specificity suggesting a long co-speciation process. In the present study, the presence and genetic diversity of Pneumocystis organisms was investigated in 203 lung samples from woodmice (Apodemus sylvaticus) collected on western continental Europe and Mediterranean islands. The presence of Pneumocystis DNA was assessed by nested PCR at both large and small mitochondrial subunit (mtLSU and mtSSU) rRNA loci. Direct sequencing of nested PCR products demonstrated a very high variability among woodmouse-derived Pneumocystis organisms with a total number of 30 distinct combined mtLSU and mtSSU sequence types. However, the genetic divergence among these sequence types was very low (up to 3.87%) and the presence of several Pneumocystis species within Apodemus sylvaticus was considered unlikely. The analysis of the genetic structure of woodmouse-derived Pneumocystis revealed two distinct groups. The first one comprised Pneumocystis from woodmice collected in continental Spain, France and Balearic islands. The second one included Pneumocystis from woodmice collected in continental Italy, Corsica and Sicily. These two genetic groups were in accordance with the two lineages currently described within the host species Apodemus sylvaticus. Pneumocystis organisms are emerging as powerful tools for phylogeographic studies in mammals.     

Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans–Induced Bone Resorption in a Rat Model for Periodontal Disease

Aggregatibacter actinomycetemcomitans a causative agent of periodontal disease in humans, forms biofilm on biotic and abiotic surfaces. A. actinomycetemcomitans biofilm is heterogeneous in nature and is composed of proteins, extracellular DNA and exopolysaccharide. To explore the role played by the exopolysaccharide in the colonization and disease progression, we employed genetic reduction approach using our rat model of A. actinomycetemcomitans-induced periodontitis. To this end, a genetically modified strain of A. actinomycetemcomitans lacking the pga operon was compared with the wild-type strain in the rat infection model. The parent and mutant strains were primarily evaluated for bone resorption and disease. Our study showed that colonization, bone resorption/disease and antibody response were all elevated in the wild-type fed rats. The bone resorption/disease caused by the pga mutant strain, lacking the exopolysaccharide, was significantly less (P < 0.05) than the bone resorption/disease caused by the wild-type strain. Further analysis of the expression levels of selected virulence genes through RT-PCR showed that the decrease in colonization, bone resorption and antibody titer in the absence of the exopolysaccharide might be due to attenuated levels of colonization genes, flp-1, apiA and aae in the mutant strain. This study demonstrates that the effect exerted by the exopolysaccharide in A. actinomycetemcomitans-induced bone resorption has hitherto not been recognized and underscores the role played by the exopolysaccharide in A. actinomycetemcomitans-induced disease.     

Osteoprotective effect of soybean and sesame oils in ovariectomized rats via estrogen-like mechanism

The purpose of the present study was to investigate the osteoprotective effects of soybean oil (SbO) and sesame oil (SO) in ovarictomized (OVX) rats. The results indicated that the OVX rats exhibited a significant decrease in Ca and P level in both serum and bone, the activities of the antioxidant enzymes SOD and CAT and the antioxidant biomarker GSH accompanied with a marked increase in the oxidative stress markers MDA and PC, the inflammatory indices (TNF-α, CRP levels, WBCs counts and ACP activity) in, both, bone and serum. Supplementating the diet of the OVX rats with SbO (15 % w/w) or SO (10 % w/w) for 2 months to resulted in modulation of the alterations in all tested parameters and succeeded to restore minerals, antioxidant enzymes, antioxidant biomarkers, oxidative stress markers, inflammatory indices, and WBCs counts. It could be concluded that the consumption of diets supplemented with SbO or SO might be useful for preventing bone loss caused by estrogen deficiency in ovariectomy status.     

Results of the population and genetic study in the Republic of Tatarstan

Using data on the distribution of frequent surnames, the Crow index and its components, and nine polymorphic DNA loci, it was shown that the Tatar population of the Republic of Tatarstan is divided into subethnic groups (Mishars, Teptyars, and Kazan Tatars). No subdivision within each of these groups was found.     

Chromosome pairing in triploid intergeneric hybrids ofFestuca pratensis withLolium multiflorum, revealed by GISH

Genomic in situ hybridisation (GISH) was used to reveal chromosome pairing in two partly fertile, triploid (2n = 3x = 21) hybrids obtained by crossing the diploid (2n = 2x = 14) Festuca pratensis Huds. (designated FpFp), used as a female parent, with the autotetraploid (2n = 4x = 28) Lolium multiflorum Lam. (designated LmLmLmLm), used as a male parent. The pattern of chromosome pairing calculated on the basis of the mean values of chromosome configurations identified in all 100 PMCs analysed, was: 0.71I Lm + 2.24I Fp + 2.18II Lm/Lm + 0.54II Lm/Fp + 4.18III Lm/Lm/Fp. A relatively high number of Lm/Lm bivalents and Fp univalents, and a low number of Lm/Fp bivalents and Lm univalents indicated that the pairing was preferential between L. multiflorum chromosomes. Other observations regarding chromosome pairing within the Lm/Lm/Fp trivalents also confirmed this preferential pairing in the analysed triploids, as the Fp chromosome was not randomly located in the chain- and frying-pan-shaped trivalents. The similarities and differences in chromosome pairing at metaphase I and the level of preferential pairing between Lolium chromosomes in the different triploid Lolium-Festuca hybrids are discussed.     

Synthesis and FTIR Conformational Studies of Peptides From the Basic Region of c-Jun: a Critical Analysis on the Basis of CD and NMR Data

Abstract

The peptide (35 residues) corresponding to the basic subdomain (bSD) of c-Jun (residues 252–281) and its fragments NP (N-terminal peptide, 1–19) and CP (C-terminal peptide, 1635) were synthesized in stepwise solid-phase using the tert-butyloxycarbonyl/benzyl strategy. In a previous paper, we have shown that during its binding to the DNA site CRE (cAMP- responsive element) the bSD structure was converted into α-helix from an initial random coil conformation [Krebs, D., Dahmani, B., El Antri, S., Monnot, M., Convert, O., Mauffret, O., Troalen, F. & Fermandjian, S. Eur. J. Biochem. 231, 370–380 (1995)]. Our results suggested both a high flexibility and a helical potential in bSD, these two properties seeming crucial for the accommodation of the basic subdomain of c-Jun to its specific DNA targets. In this work, we assessed the conformational variability of bSD through the study of the secondary structures of its NP and CP fragments in trifluoroethanol (TFE)/²H₂O mixtures, using Fourier transform infrared (FTIR) spectroscopy. The IR results were critically analyzed in light of our previously reported circular dichroism (CD) and NMR data [Krebs, D., Dahmani, B., Monnot, M., Mauffret, O., Troalen, F. & Fermandjian, S. Eur. J. Biochem. 235, 699–712 (1996)]. Upon addition of TFE, the relative areas of the seven components of the amide I band (1700–1620 cm^?1) reflected the conversion of a large amount of random coil conformation into α-helix for the two fragments and bSD. This effect was accompanied by more subtle variations of the less populated structures, in agreement with the results of CD and NMR experiments. The IR results stipulated the conservation of the parent bSD secondary structures in both fragments; however, NP and CP peptides did not display similar random-to-α-helix stabilization pattern upon additions of TFE to aqueous solutions. The profile from CD signal at 222 nm was found sigmoidal for NP and almost linear for CP, while that corresponding to the parent peptide bSD was just in between those of its fragments. Thus, the present study confirms the high flexibility and helix propensity of the c-Jun basic subdomain and suggests that the N- and C-terminal parts of the peptide do not follow the same random-to-helix conversion profile during their complexation with DNA.     

Aluminum mediates compositional alterations of polar lipid classes in maize seedlings

Changes in lipid composition were investigated on maize roots and shoots under aluminum stress. After 4d exposure to 100 microM Al, root growth was inhibited while shoot growth was not affected. In roots, the decrease of the DBI (double bond index) of total fatty acids may signal a decrease in membrane fluidity. The total lipids (TL) decreased by 49%, but phospholipids (PL), phosphatidylcholine (PC) and phosphatidylinositol (PI) increased to approximately 3-fold. The MGDG increased to 2-fold but no significant change was found in the DGDG. The steryl lipids (SL) increased by 69%. The SL/PL ratio decreased from 2.64 to 1.52 and the MGDG/DGDG ratio increased from 0.45 to 1.06 in roots of Al-stressed plants. Al leads to oxidative stress in roots of treated plants as indicated by the increase of malondialdehyde (MDA) concentrations. In shoots, changes in fatty acid composition were associated with an increase of the DBI in all lipid classes except that of the DGDG decreased. The PG was the lipid class which shows the large variation of fatty acid composition. No significant changes were found either for TL, PL, SL or MDA concentrations in shoots of Al-treated plants. While PE levels did not show significant change, PI and PG increased and PC decreased. However, the Al caused 87% decrease in the GL levels. The MGDG and DGDG decreased to 19- and 8-fold, respectively. The deleterious effects of Al on polar lipids could be caused by a direct intervention of Al on plasma membrane and/or alteration of cell metabolism.