IR-inducible clusterin gene expression: a protein with potential roles in ionizing radiation-induced adaptive responses, genomic instability, and bystander effects

Clusterin (CLU) plays numerous roles in mammalian cells after stress. A review of the recent literature strongly suggests potential roles for CLU proteins in low dose ionizing radiation (IR)-inducible adaptive responses, bystander effects, and delayed death and genomic instability. Its most striking and evident feature is the inducibility of the CLU promoter after low, as well as high, doses of IR. Two major forms of CLU, secreted (sCLU) and nuclear (nCLU), possess opposite functions in cellular responses to IR: sCLU is cytoprotective, whereas nCLU (a byproduct of alternative splicing) is a pro-death factor. Recent studies from our laboratory and others demonstrated that down-regulation of sCLU by specific siRNA increased cytotoxic responses to chemotherapy and IR. sCLU was induced after low non-toxic doses of IR (0.02-0.5 Gy) in human cultured cells and in mice in vivo. The low dose inducibility of this survival protein suggests a possible role for sCLU in radiation adaptive responses, characterized by increased cell radioresistance after exposure to low adapting IR doses. Although it is still unclear whether the adaptive response is beneficial or not to cells, survival of damaged cells after IR may lead to genomic instability in the descendants of surviving cells. Recent studies indicate a link between sCLU accumulation and cancer incidence, as well as aging, supporting involvement of the protein in the development of genomic instability. Secreted after IR, sCLU may also alter intracellular communication due to its ability to bind cell surface receptors, such as the TGF-beta receptors (types I and II). This interference with signaling pathways may contribute to IR-induced bystander effects. We hypothesize that activation of the TGF-beta signaling pathway, which often occurs after IR exposure, can in turn activate the CLU promoter. TGF-beta and IR-inducible de novo synthesized sCLU may then bind the TGF-beta receptors and suppress downstream growth arrest signaling. This complicated negative feedback regulation most certainly depends on the cellular microenvironment, but undoubtedly represents a potential link between IR-induced adaptive responses, genomic instability and bystander effects. Further elucidation of clusterin protein functions in IR responses are clearly warranted.     

Angular variances for internal bond rotations of side chains in GXG-based tripeptides derived from (13)C-NMR relaxation measurements: Implications to protein folding

The study of backbone and side-chain internal motions in proteins and peptides is crucial to having a better understanding of protein/peptide "structure" and to characterizing unfolded and partially folded states of proteins and peptides. To achieve this, however, requires establishing a baseline for internal motions and motional restrictions for all residues in the fully, solvent-exposed "unfolded state." GXG-based tripeptides are the simpliest peptides where residue X is fully solvent exposed in the context of an actual peptide. In this study, a series of GXG-based tripeptides has been synthesized with X being varied to include all twenty common amino acid residues. Proton-coupled and -decoupled (13)C-nmr relaxation measurements have been performed on these twenty tripeptides and various motional models (Lipari-Szabo model free approach, rotational anisotropic diffusion, rotational fluctuations within a potential well, rotational jump model) have been used to analyze relaxation data for derivation of angular variances and motional correlation times for backbone and side-chain chi(1) and chi(2) bonds and methyl group rotations. At 298 K, backbone motional correlation times range from about 50 to 85 ps, whereas side-chain motional correlation times show a much broader spread from about 18 to 80 ps. Angular variances for backbone phi,psi bond rotations range from 11 degrees to 23 degrees and those for side chains vary from 5 degrees to 24 degrees for chi(1) bond rotations and from 5 degrees to 27 degrees for chi(2) bond rotations. Even in these peptide models of the "unfolded state," side-chain angular variances can be as restricted as those for backbone and beta-branched (valine, threonine, and isoleucine) and aromatic side chains display the most restricted motions probably due to steric hinderence with backbone atoms. Comparison with motional data on residues in partially folded, beta-sheet-forming peptides indicates that side-chain motions of at least hydrophobic residues are less restricted in the partially folded state, suggesting that an increase in side-chain conformational entropy may help drive early-stage protein folding. Copyright 1999 John Wiley & Sons, Inc.     

Hydroxyl groups in the (beta)beta sandwich of metallo-beta-lactamases favor enzyme activity: a computational protein design study

Metallo-beta-lactamases challenge antimicrobial therapies by their ability to hydrolyze and inactivate a broad spectrum of beta-lactam antibiotics. The potential of these enzymes to acquire enhanced catalytic efficiency through mutation is of great concern. Here, we explore the potential of computational protein design to predict mutants of the imipenemase IMP-1 that modulate the catalytic efficiency of the enzyme against a range of substrates. Focusing on the four amino acid positions 69, 121, 218, and 262, we carried out a number of design calculations. Two mutant enzymes were predicted: the single mutant S262A and the double mutant F218Y-S262A. Compared to IMP-1, the single mutant (S262A) results in the loss of a hydroxyl group and the double mutant (F218Y-S262A) results in a hydroxyl transfer from position 262 to position 218. The presence of both hydroxyl groups at positions 218 and 262 was tested by examining the mutant F218Y. Kinetic constants of IMP-1, the two computationally designed mutants (S262A and F218Y-S262A), and the hydroxyl addition mutant (F218Y) were determined with seven substrates. Catalytic efficiencies are highest for the enzyme with both hydroxyl groups (F218Y) and lowest for the enzyme lacking both hydroxyl groups (S262A). The catalytic efficiencies of the two enzymes with one hydroxyl group each are intermediate, with the F218Y-S262A double mutant exhibiting enhanced hydrolysis of nitrocefin, cephalothin, and cefotaxime relative to IMP-1.     

Accurate and Rapid Identification of the Burkholderia pseudomallei Near-Neighbour,Burkholderia ubonensis,Using Real-Time PCR

Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown’s medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown’s agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown’s-positive colonies that are not B. pseudomallei.     

人肺腺癌细胞分化相关基因cDNAs的克隆

在用10^-5 mol/L全反式维甲酸(RA)诱导人肺腺癌细胞系GLC-82分化的基础上,以M13噬菌粒pSPORT1为载体,应用定向克隆技术,分别构建了未经RA诱导和RA诱导1d及4d细胞的3个cDNA文库.以含重组子的诱导文库单链DNA为靶标(Target)同未诱导文库的cDNA驱除子(Driver)进行消减杂交,富集RA特异性单链DNA,将富集的单链DNA回复为双链后转化感受态菌,建立细胞诱导分化过程中活化表达基因的cDNA消减文库,得到124个cDNA消减克隆.经同源性分析和与文库总cDNA作Southern印迹杂交,进而与RA诱导前后细胞的RNA作Northern印迹杂交,筛选出2个(RA5,RA28)诱导后呈早期瞬时表达和1个(RA42)呈早期并持续表达的cDNA克隆,cDNA全长分别为1.8,1.5和0.7kb.序列测定及初步功能分析结果表明,RA5,RA28和RA42这3个首次报道的序列,可能是人肺腺癌细胞分化相关基因的cDNA克隆.     

Aerobic exercise alone results in clinically significant weight loss for men and women: Midwest exercise trial 2

Exercise is recommended by public health agencies for weight management; however, the role of exercise is generally considered secondary to energy restriction. Few studies exist that have verified completion of exercise, measured the energy expenditure of exercise, and prescribed exercise with equivalent energy expenditure across individuals and genders.

Objective:

The objective of this study was to evaluate aerobic exercise, without energy restriction, on weight loss in sedentary overweight and obese men and women.

Design and Methods:

This investigation was a randomized, controlled, efficacy trial in 141 overweight and obese participants (body mass index, 31.0 ± 4.6 kg/m²; age 22.6 ± 3.9 years). Participants were randomized (2:2:1 ratio) to exercise at either 400 kcal/session or 600 kcal/session or to a nonexercise control. Exercise was supervised, 5 days/week, for 10 months. All participants were instructed to maintain usual ad libitum diets. Because of the efficacy design, completion of ≥90% of exercise sessions was an a priori definition of per protocol, and these participants were included in the analysis.

Results:

Weight loss from baseline to 10 months for the 400 and 600 kcal/session groups was 3.9 ± 4.9 kg (4.3%) and 5.2 ± 5.6 kg (5.7%), respectively, compared with weight gain for controls of 0.5 ± 3.5 kg (0.5%) (P < 0.05). Differences for weight loss from baseline to 10 months between the exercise groups and differences between men and women within groups were not statistically significant.

Conclusions:

Supervised exercise, with equivalent energy expenditure, results in clinically significant weight loss with no significant difference between men and women.     

Molecular investigations of a locally acquired case of melioidosis in Southern AZ, USA

Melioidosis is caused by Burkholderia pseudomallei, a Gram-negative bacillus, primarily found in soils in Southeast Asia and northern Australia. A recent case of melioidosis in non-endemic Arizona was determined to be the result of locally acquired infection, as the patient had no travel history to endemic regions and no previous history of disease. Diagnosis of the case was confirmed through multiple microbiologic and molecular techniques. To enhance the epidemiological analysis, we conducted several molecular genotyping procedures, including multi-locus sequence typing, SNP-profiling, and whole genome sequence typing. Each technique has different molecular epidemiologic advantages, all of which provided evidence that the infecting strain was most similar to those found in Southeast Asia, possibly originating in, or around, Malaysia. Advancements in new typing technologies provide genotyping resolution not previously available to public health investigators, allowing for more accurate source identification.     

Changes in the reproductive function and developmental phenotypes in mice following intramuscular injection of an activin betaA-expressing plasmid

Background  

The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model.     

Validated predictive modelling of the environmental resistome

Multi-drug-resistant bacteria pose a significant threat to public health. The role of the environment in the overall rise in antibiotic-resistant infections and risk to humans is largely unknown. This study aimed to evaluate drivers of antibiotic-resistance levels across the River Thames catchment, model key biotic, spatial and chemical variables and produce predictive models for future risk assessment. Sediment samples from 13 sites across the River Thames basin were taken at four time points across 2011 and 2012. Samples were analysed for class 1 integron prevalence and enumeration of third-generation cephalosporin-resistant bacteria. Class 1 integron prevalence was validated as a molecular marker of antibiotic resistance; levels of resistance showed significant geospatial and temporal variation. The main explanatory variables of resistance levels at each sample site were the number, proximity, size and type of surrounding wastewater-treatment plants. Model 1 revealed treatment plants accounted for 49.5% of the variance in resistance levels. Other contributing factors were extent of different surrounding land cover types (for example, Neutral Grassland), temporal patterns and prior rainfall; when modelling all variables the resulting model (Model 2) could explain 82.9% of variations in resistance levels in the whole catchment. Chemical analyses correlated with key indicators of treatment plant effluent and a model (Model 3) was generated based on water quality parameters (contaminant and macro- and micro-nutrient levels). Model 2 was beta tested on independent sites and explained over 78% of the variation in integron prevalence showing a significant predictive ability. We believe all models in this study are highly useful tools for informing and prioritising mitigation strategies to reduce the environmental resistome.