Activation of human meiosis-specific recombinase Dmc1 by Ca2+

Rad51 and its meiotic homolog Dmc1 are key proteins of homologous recombination in eukaryotes. These proteins form nucleoprotein complexes on single-stranded DNA that promote a search for homology and that perform DNA strand exchange, the two essential steps of genetic recombination. Previously, we demonstrated that Ca2+ greatly stimulates the DNA strand exchange activity of human (h) Rad51 protein (Bugreev, D. V., and Mazin, A. V. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9988-9993). Here, we show that the DNA strand exchange activity of hDmc1 protein is also stimulated by Ca2+. However, the mechanism of stimulation of hDmc1 protein appears to be different from that of hRad51 protein. In the case of hRad51 protein, Ca2+ acts primarily by inhibiting its ATPase activity, thereby preventing self-conversion into an inactive ADP-bound complex. In contrast, we demonstrate that hDmc1 protein does not self-convert into a stable ADP-bound complex. The results indicate that activation of hDmc1 is mediated through conformational changes induced by free Ca2+ ion binding to a protein site that is distinct from the Mg2+.ATP-binding center. These conformational changes are manifested by formation of more stable filamentous hDmc1.single-stranded DNA complexes. Our results demonstrate a universal role of Ca2+ in stimulation of mammalian DNA strand exchange proteins and reveal diversity in the mechanisms of this stimulation.     

Substrate specificity of human ceramide kinase

Previous studies in our laboratory have established ceramide kinase (CERK) as a critical mediator of eicosanoid synthesis. To date, CERK has not been well characterized in vitro. In this study, we investigated the substrate specificity of CERK using baculovirus-expressed human CERK (6 x His) and a newly designed assay based on mixed micelles of Triton X-100. The results indicate that the ability of CERK to recognize ceramide as a substrate is stereospecific. A minimum of a 12 carbon acyl chain was required for normal CERK activity, and the 4-5 trans double bond was important for substrate recognition. A significant discrimination by CERK was not observed between ceramides with long saturated and long unsaturated fatty acyl chains. Methylation of the primary hydroxyl group resulted in a loss of activity, confirming that CERK produces ceramide-1-phosphate versus ceramide-3-phosphate. In addition, methylation of the secondary hydroxyl group drastically decreased the phosphorylation by CERK. These results also indicated that the free hydrogen of the secondary amide group is critical for substrate recognition. Lastly, the sphingoid chain was also required for substrate recognition by CERK. Together, these results indicate a very high specificity for substrate recognition by CERK, explaining the use of ceramide and not sphingosine or diacylglycerol as substrates.     

Prolonged moderate elevation of corticosterone does not affect hippocampal anatomy or cell proliferation rates in mountain chickadees (Poecile gambeli)

Chronic stress and corresponding chronic elevations of glucocorticoid hormones have been widely assumed to have deleterious effects on brain anatomy and functions such as learning and memory. In particular, it has been suggested that chronic elevations of glucocorticoid hormones result in death of hippocampal neurons and in reduced rates of hippocampal neurogenesis. It is not clear, however, if any increase in glucocorticoid levels has negative effects on hippocampal anatomy as many animals regularly maintain moderately elevated levels of glucocrticoids over long periods of time under natural energetically demanding conditions. We used unbiased stereological methods to investigate whether mountain chickadees (Poecile gambeli) implanted for 49 days with continuous time-release corticosterone pellets, designed to approximately double the baseline corticosterone levels, differed from placebo-implanted chickadees in their hippocampal anatomy and cell proliferation rates. We found no significant differences between corticosterone and placebo-implanted birds in either telencephalon volume, volume of the hippocampal formation, or the total number of hippocampal neurons. Cell proliferation rates, measured as the total number of BrdU-labeled cells in the ventricular zone adjacent either to the hippocampus or to the mesopallium, were also not significantly different between corticosterone and placebo-implanted chickadees. Our results suggest that prolonged moderate elevation of corticosterone might not provide the suggested deleterious effects on hippocampal anatomy and neurogenesis in food-caching birds and, as we have shown previously, it actually enhances spatial memory.     

Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.     

Drug susceptibility of Pseudomonas aeruginosa strains isolated from patients of a hospital and specialistic outpatients clinics of the SP ZOZ in Nidzica

The aim of this study was to evaluate a frequency of isolation and antimicrobial susceptibility testing (AST) of Pseudomonas aeruginosa strains cultured from clinical specimens collected from patients hospitalized in wards and specialistic outpatients clinics of a hospital in Nidzica (01. 09. 2000 -31. 12. 2003). During over three years 392 Pseudomonas aeruginosa strains were cultured from 16346 clinical samples provided to bacteriological laboratory. P. aeruginosa strains were isolated from 2.5% of examined specimens. Susceptibility of Pseudomonas aeruginosa strains to antimicrobial agents was tested. The highest in vitro activity against clinical P. aeruginosa strains demonstrated imipenem. One strain was resistant to imipenem. This strain was isolated from a patient of a surgical department. Metalo-beta-lactamase was not detected (MBL-negative strain).Twenty nine strains were ESBL producer (7.4% of all strains). The contribution of Pseudomonas aeruginosa strains to the etiology of nosoconial and ambulatory infections increases. In vitro activity of antibacterial agents against P. aeruginosa strains should be monitored during therapy of infections. Resistance to antibiotics/chemothe-rapeutics may be acquired during treatment with antibacterial agent to which P. aeruginosa strain was susceptible according to the antibiogram.     

Application of four variants of the diagnostic disc test (CD01, CD02, CD03, CD04) for detection of ESBL-positive strains of gram-negative rods

The aim of this study was to evaluate the usefulness of four variants of the diagnostic disc test (DD) to detect extended-spectrum beta-lactamases (ESBLs) in nosocomial strains of gram-negative rods. Also, the diagnostic disc test (DD) was compared with the double-disc synergy test (DDST) for the effectivity of ESBLs identification. A total number of 111 ESBL-positive (DDST-positive) strains of gram-negative rods isolated from hospitalized patients in 2004 was examined. Ninety nine strains belonged to enteric rods (89.2%) and twelve strains--to nonfermentative rods (10.8%). Two reference strains: E. coli ATCC 25922 (ESBL-negative one) and K. pneumoniae ATCC 700603 (ESBL-positive one) were included in the study. Four variants of the diagnostic disc test (DD, Oxoid Ltd, UK) were applied for ESBLs detection: CPD/CD01, CAZ/CD02, CTX/CD03 and CPO/CD04. All examined strains (111) were DDST-positive. Positive results in the DD test (Oxoid Ltd) were as follows: CPD/CD01--59 strains (53.2%), CAZ/CD02--80 strains (72.1%), CTX/CD03--92 strains (82.9%) and CPO/CD04--110 strains (99.1%). Discs containing cefpirome (CPO) and cefpirome with clavulanic acid (CD04) were the best set for detection of ESBLs in our collection of clinical gram-negative rods. Results of this variant of the DD test were the most consistent with the results of the DDST. Application of several disc diffusion methods to detect ESBL producers increases the probability of proper identification of these strains.     

Bacteriophage contamination: is there a simple method to reduce its deleterious effects in laboratory cultures and biotechnological factories?

Infection of bacterial cultures by bacteriophages as well as prophage induction in the host cells are serious problems in both research and biotechnological laboratories. Generally, prevention strategies (like good laboratory/factory hygiene, sterilisation, decontamination and disinfection) are necessary to avoid bacteriophage contamination. However, it is well known that no matter how good the laboratory/factory practice and hygiene are, bacteriophage infections occur from time to time. The use of immunised or resistant bacterial strains against specific phages may be helpful, but properties of the genetically modified strains resistant to phages are often worse (from the point of view of a researcher or a biotechnological company) than those of the parental, phage-sensitive strains. In this article we review recent results that may provide a simple way to minimise deleterious effects of bacteriophage infection and prophage induction. It appears that low bacterial growth rates result in a significant inhibition of lytic development of various bacteriophages. Moreover, spontaneous prophage induction is less frequent in slowly growing bacteria.     

Microbiologic analysis of results from blood cultures

The aim of our investigations was the microbiological analysis together with the evaluation of sensitivity of bacteria frequently isolated from blood cultures. Blood samples were taken from patients with symptoms suggesting bacteremia in Rydygier's Hospital in Cracow. A total of 11,170 blood samples taken from 1997 to 2000 were tested. Automatic VITAL system (bioMerieux) was applied to culture and detect microorganisms. Bacteria were identified by ATB system (bioMerieux). Susceptibility was detected by ATB and disc diffusion method. Percentage of positive results relating to detection of microorganisms of clinical significance was 16.9% (1891 cultures). Staphylococcus spp. (Staph. epidermidis in range 22.8% to 21.9%), Enterococcus spp. and Streptococcus spp. were most frequently isolated species among aerobic Gram-positive bacteria. In 2000, compared to 1997 the number of isolates of MRSA increased considerably (from 1.8% to 6.8%). In blood infections the increase of frequency of E. coli bacteria was also noted: 6.1% and 11.4% in 1997 and 2000, respectively. Among non-fermentant bacilli the percentage of occurrence of P. aeruginosa in the period of 4 years was comparable in the range 7.3% in 1997 to 7.2% in 2000. The increase in the frequency of blood infections of A. baumanii was also noticed (respectively from 4.8% to 9.9%). Susceptibility of P. aeruginosa strains to selected beta-lactame antibiotics and aminoglycosydes increased in 2000 in comparison to 1999. A. baumanii strains were 100% sensitive only to imipenem.