Dynamics of the DNA repair proteins WRN and BLM in the nucleoplasm and nucleoli

We have investigated the mobility of two EGFP-tagged DNA repair proteins, WRN and BLM. In particular, we focused on the dynamics in two locations, the nucleoli and the nucleoplasm. We found that both WRN and BLM use a “DNA-scanning” mechanism, with rapid binding–unbinding to DNA resulting in effective diffusion. In the nucleoplasm WRN and BLM have effective diffusion coefficients of 1.62 and 1.34 μm²/s, respectively. Likewise, the dynamics in the nucleoli are also best described by effective diffusion, but with diffusion coefficients a factor of ten lower than in the nucleoplasm. From this large reduction in diffusion coefficient we were able to classify WRN and BLM as DNA damage scanners. In addition to WRN and BLM we also classified other DNA damage proteins and found they all fall into one of two categories. Either they are scanners, similar to WRN and BLM, with very low diffusion coefficients, suggesting a scanning mechanism, or they are almost freely diffusing, suggesting that they interact with DNA only after initiation of a DNA damage response.     

Environmental factors regulate the effects of roach Rutilus rutilus and pike Esox lucius on perch Perca fluviatilis populations in small boreal forest lakes

In this study of 18 small boreal forest lakes, the effects of abiotic and biotic factors (roach Rutilus rutilus and pike Esox lucius) on various population variables of perch Perca fluviatilis were examined. As a single variable, the gillnet catch per unit effort (CPUE) of R. rutilus was negatively related to the mean mass of small (< 200 mm) and the growth rate of young (1–2 years) P. fluviatilis. The mean mass of large (≥ 200 mm) P. fluviatilis was the highest at intermediate CPUE of R. rutilus. Redundancy analysis including environmental factors and P. fluviatilis population variables suggested that ‘predation–productivity–humus' gradient affected P. fluviatilis populations by decreasing the CPUE and mean mass of small individuals but increasing these variables of large individuals. The CPUE of R. rutilus and lake area had a negative effect on small and a positive effect on large P. fluviatilis growth rate. In small boreal forest lakes, P. fluviatilis populations are affected by the partially opposite forces of competition by R. rutilus and predation by E. lucius, and the intensity of these interactions is regulated by several environmental factors.     

Tick-susceptible Bos taurus cattle display an increased cellular response at the site of larval Rhipicephalus (Boophilus) microplus attachment, compared with tick-resistant Bos indicus cattle

Cattle demonstrate divergent and heritable phenotypes of resistance and susceptibility to infestation with the cattle tick Rhipicephalus (Boophilus) microplus. Bos indicus cattle are generally more resistant to tick infestation than Bos taurus breeds although large variations in resistance can occur within subspecies and within breed. Increased tick resistance has been previously associated with an intense hypersensitivity response in B. taurus breeds; however, the mechanism by which highly resistant B. indicus cattle acquire and sustain high levels of tick resistance remains to be elucidated. Using the commercially available Affymetrix microarray gene expression platform, together with histological examination of the larval attachment site, this study aimed to describe those processes responsible for high levels of tick resistance in Brahman (B. indicus) cattle that differ from those in low-resistance Holstein-Friesian (B. taurus) cattle. We found that genes involved in inflammatory processes and immune responsiveness to infestation by ticks, although up-regulated in tick-infested Holstein-Friesian cattle, were not up-regulated in Brahman cattle. In contrast, genes encoding constituents of the extracellular matrix were up-regulated in Brahmans. Furthermore, the susceptible Holstein-Friesian animals displayed a much greater cellular inflammatory response at the site of larval R. microplus attachment compared with the tick-resistant Brahman cattle.     

1H NMR metabonomics approach to the disease continuum of diabetic complications and premature death

Subtle metabolic changes precede and accompany chronic vascular complications, which are the primary causes of premature death in diabetes. To obtain a multimetabolite characterization of these high‐risk individuals, we measured proton nuclear magnetic resonance (¹H NMR) data from the serum of 613 patients with type I diabetes and a diverse spread of complications. We developed a new metabonomics framework to visualize and interpret the data and to link the metabolic profiles to the underlying diagnostic and biochemical variables. Our results indicate complex interactions between diabetic kidney disease, insulin resistance and the metabolic syndrome. We illustrate how a single ¹H NMR protocol is able to identify the polydiagnostic metabolite manifold of type I diabetes and how its alterations translate to clinical phenotypes, clustering of micro‐ and macrovascular complications, and mortality during several years of follow‐up. This work demonstrates the diffuse nature of complex vascular diseases and the limitations of single diagnostic biomarkers. However, it also promises cost‐effective solutions through high‐throughput analytics and advanced computational methods, as applied here in a case that is representative of the real clinical situation.     

Molecular dissection of 2B4 signaling: implications for signal transduction by SLAM-related receptors

2B4 is a SLAM-related receptor expressed on natural killer (NK) cells and cytotoxic T cells. It can regulate killing and gamma interferon secretion by NK cells, as well as T-cell-mediated cytotoxicity. There are conflicting data regarding the mechanism of action of 2B4. In these studies, we attempted to understand better the nature and basis of 2B4 signaling. Our studies showed that engagement of 2B4 on NK cells triggered a tyrosine phosphorylation signal implicating 2B4, Vav-1, and, to a lesser extent, SHIP-1 and c-Cbl. Structure-function analyses demonstrated that this response was defined by a series of tyrosine-based motifs in the cytoplasmic region of 2B4 and was not influenced by the extracellular or transmembrane segment of 2B4. In addition, the 2B4-induced signal was absolutely dependent on coexpression of SAP, a Src homology 2 (SH2) domain-containing adaptor associating with SLAM-related receptors and mutated in X-linked lymphoproliferative disease. It was also observed that 2B4 was detectably associated with the Src-related protein tyrosine kinase FynT in an immortalized NK cell line. Mutation of arginine 78 of SAP, a residue critical for binding of SAP to FynT, eliminated 2B4-mediated protein tyrosine phosphorylation, implying that SAP promotes 2B4 signaling most probably by recruiting FynT. Finally, despite the similarities in the signaling modalities of 2B4 and its relative SLAM, the natures of the tyrosine phosphorylation signals induced by these two receptors were found to be different. These differences were not caused by variations in the extent of binding to SAP but rather were dictated by the tyrosine-based sequences in the cytoplasmic domain of the receptors. Taken together, these data lead to a better understanding of 2B4 signaling. Furthermore, they provide firm evidence that the signals transduced by the various SLAM-related receptors are unique and that the specificity of these signals is defined by the distinctive arrays of intracytoplasmic tyrosines in the receptors.     

Efficacy of two rabeprazole/gatifloxacin-based triple therapies for Helicobacter pylori infection

OBJECTIVES: To evaluate the efficacy of two novel treatment regimens consisting of gatifloxacin (400 mg daily), amoxicillin (1 g twice daily), and rabeprazole 20 mg once (RAG20) or twice daily (RAG40) given for 7 days in the eradication of Helicobacter pylori. METHODS: Eligible patients undergoing endoscopy and having a positive rapid urease assay for H. pylori were enrolled in this open-label trial. Gastric biopsies from a random cohort of patients were cultured for H. pylori and in vitro susceptibility to gatifloxacin and amoxicillin was performed using the E-test. Compliance and side-effects were evaluated by phone calls. (14)C-urea breath tests were performed a minimum of 4 weeks after therapy and 3 weeks after any acid suppressive therapy. RESULTS: A total of 104 patients, 52 in each group (40 females and 64 males; mean age 45.7 years) were enrolled sequentially. Eradication occurred in 43 out of 52 patients in RAG20 group (both per-protocol and intention-to-treat analysis: 83%; 95% CI: 72-93%) and in 48 of 52 patients in the RAG40 group (both per-protocol and intention-to-treat analysis: 92%; 95% CI: 85-99%). Seven patients in the RAG40 group who had previously failed one or more treatment regimens for H. pylori were cured. No significant adverse effects were reported. All 50 recovered H. pylori strains were susceptible to amoxicillin and gatifloxacin in vitro. CONCLUSIONS: A 7-day regimen of gatifloxacin-rabeprazole-amoxicillin is effective eradication therapy for H. pylori. The use of rabeprazole twice daily results in superior eradication rates including cases of failed primary therapy. This new regimen is simple, well-tolerated, and may lead to higher compliance and lower costs.     

Fast determination of propranolol in urine and pharmaceutical preparations by stopped-flow and micellar-stabilized room-temperature phosphorescence: validation of the method

The stopped-flow mixing technique has been used to study the kinetic determination of propranolol by means of micellar-stabilized room-temperature phosphorescence. This mixing system diminishes the time required for the deoxygenation of micellar medium by sodium sulfite, allowing a kinetic curve that levels off within only 7s to be obtained. The phosphorescence enhancers thallium (I) nitrate, sodium dodecyl sulfate, and sodium sulfite were optimized to obtain maximum sensitivity and selectivity. A pH value of 6.54 was selected as adequate for phosphorescence development. The kinetic curves of propranolol phosphorescence were scanned at lambda(ex)=290 nm and lambda(em)=524 nm. The calibration graphs were linear for the concentration range from 25 to 400 ng mL(-1). The phosphorescence lifetime of propranolol is approximately 1210 micros. The detection limit calculated as proposed clayton was 13.53 ng mL(-1) and by applying the error propagation theory, the detection limit was 8.37 ng mL(-1). The repeatability was studied using 10 solutions of 200 ng mL(-1) of propranolol; if error propagation theory is assumed, the relative error is 1.94%. The standard deviation for a replicate sample was 4.0 ng mL(-1). This method was successfully applied to the determination of propranolol in commercial formulations and in urine. Suitable recovery values were obtained.     

Acetoclastic and hydrogenotrophic methane production and methanogenic populations in an acidic West-Siberian peat bog

Sites in the West Siberian peat bog 'Bakchar' were acidic (pH 4.2-4.8), low in nutrients, and emitted CH4 at rates of 0.2-1.5 mmol m(-2) h(-1). The vertical profile of delta13CH4 and delta13CO2 dissolved in the porewater indicated increasing isotope fractionation and thus increasing contribution of H2/CO2-dependent methanogenesis with depth. The anaerobic microbial community at 30-50 cm below the water table produced CH4 with optimum activity at 20-25 degrees C and pH 5.0-5.5 respectively. Inhibition of methanogenesis with 2-bromo-ethane sulphonate showed that acetate, phenyl acetate, phenyl propionate and caproate were important intermediates in the degradation pathway of organic matter to CH4. Further degradation of these intermediates indicated that 62-72% of the CH4 was ultimately derived from acetate, the remainder from H2/CO2. Turnover times of [2-14C]acetate were on the order of 2 days (15, 25 degrees C) and accounted for 60-65% of total CH4 production. Conversion of 14CO2 to 14CH4 accounted for 35-43% of total CH4 production. These results showed that acetoclastic and hydrogenotrophic methanogenesis operated closely at a ratio of approximately 2 : 1 irrespective of the incubation temperature (4, 15 and 25 degrees C). The composition of the archaeal community was determined in the peat samples by terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of amplified SSU rRNA gene fragments, and showed that members of Methanomicrobiaceae, Methanosarcinaceae and Rice cluster II (RC-II) were present. Other, presumably non-methanogenic archaeal clusters (group III, RC-IV, RC-V, RC-VI) were also detected. Fluorescent in situ hybridization (FISH) showed that the number of Bacteria decreased (from 24 x 10(7) to 4 x 10(7) cells per gram peat) with depth (from 5 to 55 cm below the water table), whereas the numbers of Archaea slightly increased (from 1 x 10(7) to 2 x 10(7) cells per gram peat). Methanosarcina spp. accounted for about half of the archaeal cells. Our results show that both hydrogenotrophic and acetoclastic methanogenesis are an integral part of the CH4-producing pathway in acidic peat and were represented by appropriate methanogenic populations.