Hepatic and renal cadmium accumulation is associated with mass-specific daily metabolic rate in the bank vole (Clethrionomys glareolus)

Previous study has shown that photoperiod and age affect tissue accumulation of cadmium (Cd) in a small rodent, the bank vole. Since the body mass is also influenced by these factors, the present study was designed to determine whether mass-specific daily metabolic rate might be responsible for differential accumulation of Cd in the liver and kidneys of the short- and long-photoperiod bank voles as well as of the young and old animals. One- and five-month old male bank voles were held under short (8 h light/16 h dark) or long (16 h light/8 h dark) photoperiods and exposed to dietary Cd (100 microg/g) for 6 weeks. The bank voles raised under the short photoperiod and those injected subcutaneously with melatonin (7 micromol/kg/day) under the long photoperiod showed significantly higher concentrations of Cd in the liver (43-60%) and kidneys (40-47%) than the age-matched long-photoperiod animals. The old bank voles accumulated significantly less Cd in both organs than the young animals. These differences in Cd accumulation appeared not to be associated with the relative Cd intake. However, the hepatic and renal Cd levels followed a pattern similar to that of the mass-specific daily metabolic rate (or energy expenditure) and energy assimilation efficiency. These data indicate that mass-specific daily metabolic rate and energy assimilation efficiency (an indicative of digestive and absorptive processes) may be responsible for differential tissue Cd accumulation in the bank vole.     

Contribution of Pseudomonas Aeruginosa strains to infections in patients of specialistic outpatients clinics of the SP ZOZ in Nidzica

The aim of this study was to examine a frequency of isolation and analysis of drug susceptibility o P. aeruginosa strains cultured from clinical specimens obtained from patients treated in specialistic outpatient clinics of the Samodzielny Publiczny Zespó? Opieki Zdrowotnej (SP ZOZ) in Nidzica durin 40 months (01. 09. 2000 - 31. 12. 2003). Ninety six P. aeruginosa strains were cultured out of 829 clinical samples collected from ambulatory patients and processed in the Bacteriological Laboratory of SP ZOZ in Nidzica during over three years. P. aeruginosa strains were isolated from 11.6% of examined specimens. The greatest number of strains (49.0%) were cultured from urine samples obtained from children. Identification of strains was performed using biochemical tests (Becton Dickinson, Emapol, bio-Merieux). Susceptibility of strains to antimicrobial agents was determined with disc diffusion method according to NCCLS recommendations. Special tests were applied to detect extended-spectrum beta-lactamases (ESBL). The most active in vitro against isolated P. aeruginosa strains was a carbapenem - imipenem. All strains were susceptible to this antibiotic. Ciprofloxacin (94.8% of susceptible strains), ceftazidime (89.6%), gentamicin (86.5%), piperacillin (84.4%) and aztreonam (76.0%) were active against the majority of P. aeruginosa strains isolated from ambulatory patients. Six strains (6.25% of all strains) producing extended--spectrum beta--lactamases (ESBL) were detected. It is alarming, that the majority of P. aeruginosa strains from outpatients were cultured out of pediatric samples (61.5%). Because of an increase in resistance and appearance of new mechanisms of resistance to antibiotics/chemotherapeutics in P. aeruginosa strains, it is necessary to monitor a drug susceptibility of these strains causing infections in ambulatory patients.     

In vitro activity of cefepime against ESBL-positive clinical strains of gram-negative rods

The aim of this study was to determine an in vitro activity of cefepime against ESBL-positive clinical strains of Gram-negative rods isolated from hospitalized patients. Experiments were performed with 100 ESBL-positive strains of Gram-negative rods isolated from clinical samples in 2004. Strains were identified with the use of automatic ATB Expression system and biochemical ID 32 GN tests (bioMdrieux sa). Extended-spectrum beta-lactamases (ESBLs) were detected by means of disc diffusion methods: the double-disc synergy test (DDST) and the diagnostic disc test (DD, Oxoid Ltd, UK). Susceptibility in vitro of ESBL producers to 4th generation--cefepime was determined with gradient diffusion method Etest (AB Biodisk, Solna, Sweden). MIC value of cefepime was assessed for each strain. Among 100 ESBL-producing strains, 94--belonged to enteric rods and 6--to nonfermentative rods. The greatest number of strains belonged to the species Serratia marcescens (27% of all strains) and next--to the species Enterobacter cloacae (21%). Fourteen strains were susceptible (S) in vitro to cefepime, 12--intermediately susceptible (I) and 74--resistant (R). Application of cefepime in a therapy of infections caused by ESBL-positive strains of Gram-negative rods highly susceptible in vitro to this antibiotic, should be considered.     

Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples

MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50-200 ng of PCR product and 1-3 microg of Thermus thermophilus his6-MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET, hMSH2, hMLH1, BRCA1, BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations.     

HIV-1 viral escape in infancy followed by emergence of a variant-specific CTL response

Mutational escape from the CTL response represents a major driving force for viral diversification in HIV-1-infected adults, but escape during infancy has not been described previously. We studied the immune response of perinatally infected children to an epitope (B57-TW10) that is targeted early during acute HIV-1 infection in adults expressing HLA-B57 and rapidly mutates under this selection pressure. Viral sequencing revealed the universal presence of escape mutations within TW10 among B57- and B5801-positive children. Mutations in TW10 and other B57-restricted epitopes arose early following perinatal infection of B57-positive children born to B57-negative mothers. Surprisingly, the majority of B57/5801-positive children exhibited a robust response to the TW10 escape variant while recognizing the wild-type epitope weakly or not at all. These data demonstrate that children, even during the first years of life, are able to mount functional immune responses of sufficient potency to drive immune escape. Moreover, our data suggest that the consequences of immune escape may differ during infancy because most children mount a strong variant-specific immune response following escape, which is rarely seen in adults. Taken together, these findings indicate that the developing immune system of children may exhibit greater plasticity in responding to a continually evolving chronic viral infection.     

Modulation of transforming growth factor-beta (TGF-beta) signaling by endogenous sphingolipid mediators

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that plays a critical role in tissue repair and fibrosis. Sphingolipid signaling has been shown to regulate a variety of cellular processes and has been implicated in collagen gene regulation. The present study was undertaken to determine whether endogenous sphingolipids are involved in the TGF-beta signaling pathway. TGF-beta treatment induced endogenous ceramide levels in a time-dependent manner within 5-15 min of cell stimulation. Using human fibroblasts transfected with a alpha2(I) collagen promoter/reporter gene construct (COL1A2), C(6)-ceramide (10 microm) exerted a stimulatory effect on basal and TGF-beta-induced activity of this promoter. Next, to define the effects of endogenous sphingolipids on TGF-beta signaling we employed ectopic expression of enzymes involved in sphingolipid metabolism. Sphingosine 1-phosphate phosphatase (YSR2) stimulated basal COL1A2 promoter activity and cooperated with TGF-beta in activation of this promoter. Furthermore, overexpression of YSR2 resulted in the pronounced increase of COL1A1 and COL1A2 mRNA levels. Conversely, overexpression of sphingosine kinase (SPHK1) inhibited basal and TGF-beta-stimulated COL1A2 promoter activity. These results suggest that endogenous ceramide, but not sphingosine or sphingosine 1-phosphate, is a positive regulator of collagen gene expression. Mechanistically, we demonstrate that Smad3 is a target of YSR2. TGF-beta-induced Smad3 phosphorylation was elevated in the presence of YSR2. Cotransfection of YSR2 with wild-type Smad3, but not with the phosphorylation-deficient mutant of Smad3 (Smad3A), resulted in a dramatic increase of COL1A2 promoter activity. In conclusion, this study demonstrates a direct role for the endogenous sphingolipid mediators in regulating the TGF-beta signaling pathway.     

The immunosuppressive activity of peptide fragments of vaccinia virus C10L protein and a hypothesis on the role of this protein in the viral invasion

Our previous studies revealed that the 143-148 fragment of interleukin-1 receptor antagonist (IL-1 Ra) molecule with a Val-Thr-Lys-Phe-Tyr-Phe (VTKFYF) sequence inhibits the interleukin-1 (IL-1) interaction with its cellular receptor. The Val-Thr-Arg-Phe-Tyr-Phe (VTRFYF) sequence of the 322-327 fragment of the C-terminal domain of vaccinia virus protein related to the C10L vaccinia gene shows a very high homology to the 143-148 IL-1 Ra fragment, suggesting a similar inhibitory activity. To test this suggestion, we investigated the inhibitory activity of a series of synthetic peptides derived from 316 to 327 fragment of C10L on the interaction of IL-1 with its receptor. We also tested the peptides for their influence on the humoral and cellular immune response. The results indicate that biological activities of the C10L fragments are similar to those obtained for respective fragments of IL-1 Ra. The C-terminal domain of C10L protein can be easily folded into spatial structure similar to the crystallographic one of IL-1 Ra. Based on the crystallographic structure of IL-1 Ra, we constructed a 3-D model of the C10L protein. According to the model, the Val(322)-Asn(328) sequence is localized on the surface of the molecule and, therefore, it may be involved in the interactions with receptors. Our results indicate that the C10L viral protein can play an important role in vaccinia virus evasion of the host immune system. It may consist in the blockade of IL-1 receptors by the C10L protein, a homologue of the IL-1 Ra.     

Stress responses and replication of plasmids in bacterial cells

Plasmids, DNA (or rarely RNA) molecules which replicate in cells autonomously (independently of chromosomes) as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage lambda that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.     

New type of snRNP containing nuclear bodies in plant cells

In larch (Larix decidua Mill.) microspores a new type of nuclear bodies has been found which are an element of the spatial organization of the splicing system in plant cell. These are bizonal bodies, ultrastructurally differentiated into a coiled part and a dense part. Using immunocytochemistry and in situ hybridization at the EM level, the coiled part of the bizonal body was found to contain snRNA including U2 snRNA, Sm proteins and nucleolar proteins of the agyrophilic type and fibrillarin. The dense part contains Sm proteins but lacks snRNA. Such a separation of macromolecules related to splicing occurring within the bizonal bodies microspore is striking by the similarity of these bodies to amphibian oocyte snurposomes. The occurrence in plant cells, beside widely known coiled bodies (CBs), also of other nuclear bodies related to splicing proves that in plants similarly as for animals the differentiation among domains containing elements of the splicing system occurs.