Amplification of a single-locus variable-number direct repeats with restriction fragment length polymorphism (DR-PCR/RFLP) for genetic typing of Acinetobacter baumannii strains

In search of an effective DNA typing technique for Acinetobacter baumannii strains for hospital epidemiology use, the performance and convenience of a new target sequence was evaluated. Using known genomic sequences of Acinetobacter baumannii strains AR 319754 and ATCC 17978, we developed single-locus variable-number direct-repeat analysis using polymerase chain reaction-restriction fragment length polymorphism (DR-PCR/RFLP) method. A total of 90 Acinetobacter baumannii strains isolated from patients of the Clinical Hospital in Bydgoszcz, Poland, were examined. Initially, all strains were typed using macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE). Digestion of the chromosomal DNA with the ApaI endonuclease and separation of the fragments by PFGE revealed 21 unique types. Application of DR-PCR/RFLP resulted in recognition of 12 clusters. The results showed that the DR-PCR/RFLP method is less discriminatory than REA-PFGE, however, the novel genotyping method can be used as an alternative technique for generating DNA profiles in epidemiological studies of intra-species genetic relatedness of Acinetobacter baumannii strains.     

Mesostigmatic mites (Acari: Mesostigmata) in nests of the Eurasian griffon vulture (<Emphasis Type="Italic">Gyps fulvus</Emphasis>) in Croatia

We examined the species composition and community structure of mites of the order Mesostigmata (Acari) in nests of the Eurasian griffon vulture (Gyps fulvus Hablizl, 1783) in Croatia. Material collected from 18 nests included 565 mites belonging to seven species. The most abundant species were Leiodinychus orbicularis (C.L. Koch, 1839) (Trematuridae) and Androlaelaps casalis (Berlese, 1887) (Laelapidae). The results were compared with the community structure and frequency of dominant species of Mesostigmata in nests of 32 other bird species. Leiodinychus orbicularis occurred in the nests of 13 species of birds. It is a typical nidicolous species which occurs most frequently in the perennial nests of birds of prey. In contrast, A. casalis rarely occurs in the nests of birds of prey.     

The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS.     

Genotyping by REA-PFGE of Listeria monocytogenes isolated from clinical, environmental and food samples

Listeria monocytogenes strains isolated from clinical food and environmental samples were genotyped by Restriction Enzyme Analysis with Pulsed Field Gel Electrophoresis (REA-PFGE) using ApaI and AscI enzymes according to PulseNet Europe procedure. Analysis of DNA fragments profiles obtained by AscI digestion demonstrated presence of 62 REA-PFGE profiles grouped in 2 lineages (FI, FII). Diversity of strains source among both lineages was observed. Statistical analysis showed, that strains isolated from clinical samples more frequently are included to lineage FI, then lineage FII. Non-clinical strains were more frequently included to lineage FII. Combined analysis of REA-PFGE profiles for ApaI and AscI enzymes showed 8 unique pulsotypes characteristic for two or more L. monocytogenes isolates. Moreover researched L. monocytogenes strains were analyzed by multiplex-PCR according Doumith et al methodology. PCR-group 4B was most frequent among strains isolated from clinical samples. Correlation between PCR-group and pulsotype was observed only in few cases.     

Lack of stearoyl-CoA desaturase 1 upregulates basal thermogenesis but causes hypothermia in a cold environment

Stearoyl-CoA desaturase (SCD) is a microsomal enzyme involved in the biosynthesis of oleate and palmitoleate. Mice with a targeted disruption of the SCD1 isoform (SCD1-/-) exhibit reduced adiposity and increased energy expenditure. To address whether the energy expenditure is attributable to increased thermogenesis, we investigated the effect of SCD1 deficiency on basal and cold-induced thermogenesis. SCD1-/- mice have increased expression of uncoupling proteins in brown adipose tissue (BAT) relative to controls. The beta3-adrenergic receptor (beta3-AR) expression was increased and the phosphorylation of cAMP response element binding protein and the protein level of peroxisome proliferator-activated receptor-gamma coactivator-1alpha were increased in the SCD1-/- mice. Both lipolysis and fatty acid oxidation were increased in the SCD1-/- mice. When exposed to 4 degrees C, SCD1-/- mice showed hypothermia, hypoglycemia, and depleted liver glycogen. High levels of dietary oleate partially compensated for the hypothermia and rescued plasma glucose and liver glycogen. These results suggest that SCD1 deficiency stimulates basal thermogenesis through the upregulation of the beta3-AR-mediated pathway and a subsequent increase in lipolysis and fatty acid oxidation in BAT. The hypothermia and hypoglycemia in cold-exposed SCD1-/- mice and the compensatory recovery by oleate indicate an important role of SCD1 gene expression in thermoregulation.     

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.