Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides

Protein TrwC is the conjugative relaxase responsible for DNA processing in plasmid R388 bacterial conjugation. TrwC has two catalytic tyrosines, Y18 and Y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. Suicide substrates containing a 3'-S-phosphorothiolate linkage at the cleavage site displaced TrwC reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. Two distinct covalent TrwC-oligonucleotide complexes could be separated from noncovalently bound protein by SDS-PAGE. As observed by mass spectrometry, one complex contained a single, cleaved oligonucleotide bound to Y18, whereas the other contained two cleaved oligonucleotides, bound to Y18 and Y26. Analysis of the cleavage reaction using suicide substrates and Y18F or Y26F mutants showed that efficient Y26 cleavage only occurs after Y18 cleavage. Strand-transfer reactions carried out with the isolated Y18-DNA complex allowed the assignment of specific roles to each tyrosine. Thus, only Y18 was used for initiation. Y26 was specifically used in the second transesterification that leads to strand transfer, thus catalyzing the termination reaction that occurs in the recipient cell.     

Direct and indirect effects of additions of chromophoric dissolved organic matter on zooplankton during large‐scale mesocosm experiments in an oligotrophic lake

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1,3-Disubstituted-imidazo[1,5-a]pyrazines as insulin-like growth-factor-I receptor (IGF-IR) inhibitors

A series of novel 8-amino-1,3-disubstituted-imidazo[1,5-a]pyrazines was designed and synthesized as IGF-IR inhibitors.     

Autonomic neural control and implications for remote medical monitoring in space

Long-duration space travel or extended stays on the moon or Mars will pose new challenges for maintaining and monitoring the health status of astronauts. Remote medical monitoring systems will need to be developed for a number of applications, including providing decision support for care-givers in the event of traumatic injury in space. The focus of this brief review is to introduce potential methods of monitoring astronaut status remotely from simple ECG recordings.     

CONDOR: a database resource of developmentally associated conserved non-coding elements

Background  

Comparative genomics is currently one of the most popular approaches to study the regulatory architecture of vertebrate genomes. Fish-mammal genomic comparisons have proved powerful in identifying conserved non-coding elements likely to be distal cis-regulatory modules such as enhancers, silencers or insulators that control the expression of genes involved in the regulation of early development. The scientific community is showing increasing interest in characterizing the function, evolution and language of these sequences. Despite this, there remains little in the way of user-friendly access to a large dataset of such elements in conjunction with the analysis and the visualization tools needed to study them.     

Failure of low-velocity swimming to enhance recovery from exhaustive exercise in largemouth bass (Micropterus salmoides)

This study was intended to discover whether forcing largemouth bass (Micropterus salmoides) to swim at 0.5 body lengths/second following exercise would expedite recovery relative to fish recovered in static water. Exercise resulted in a suite of physiological disturbances for largemouth bass that included a depletion of anaerobic energy stores, an accumulation of lactate, and increased cardiac output. At 1 h following exercise, exhaustively exercised largemouth bass forced to swim exhibited expedited recovery relative to fish in static water, evidenced by lower concentrations of lactate in white muscle, elevated concentrations of phosphocreatine in white muscle, and reduced concentrations of glucose in plasma. By 4 h postexercise, largemouth bass forced to swim during recovery exhibited signs of physiological disturbance that were absent in fish recovered in static water. These signs of disturbance included a loss of osmotically active particles from plasma, elevated lactate in plasma, reductions of phospocreatine in white muscle, and increased cardiac output. These results are discussed in relation to the body of work with salmonid fishes showing physiological benefits to recovering fish in flowing water.     

Physiological and behavioral consequences of long-term artificial selection for vulnerability to recreational angling in a teleost fish

Few studies have examined the physiological and behavioral consequences of fisheries-induced selection. We evaluated how four generations of artificial truncation selection for vulnerability to recreational angling (i.e., stocks selected for high and low vulnerability [HVF and LVF, respectively]) affected cardiovascular physiology and parental care behavior in the teleost fish largemouth bass Micropterus salmoides. Where possible, we compared artificially selected fish to control fish (CF) collected from the wild. Although, compared to control fish, resting cardiac activity was approximately 18% lower for LVF and approximately 20% higher for HVF, maximal values did not vary among treatments. As a result, the HVF had less cardiac scope than either LVF or CF. Recovery rates after exercise were similar for HVF and CF but slower for LVF. When engaged in parental care activities, nesting male HVF were captured more easily than male LVF. During parental care, HVF also had higher turning rates and pectoral and caudal fin beat rates, increased vigilance against predators, and higher in situ swimming speeds. Energetics simulations indicated that to achieve the same level of growth, the disparity in metabolic rates would require HVF to consume approximately 40% more food than LVF. Selection for angling vulnerability resulted in clear differences in physiological and energetic attributes. Not only is vulnerability to angling a heritable trait, but high vulnerability covaries with factors including higher metabolic rates, reduced metabolic scope, and increased parental care activity. Despite these energetically costly differences, HVF and LVF of the same age were of similar size, suggesting that heightened food consumption in HVF compensated for added costs in experimental ponds. Ultimately, angling vulnerability appears to be a complex interaction of numerous factors leading to selection for very different phenotypes. If HVF are selectively harvested from a population, the remaining fish in that population may be less effective in providing parental care, potentially reducing reproductive output. The strong angling pressure in many freshwater systems, and therefore the potential for this to occur in the wild, necessitate management approaches that recognize the potential evolutionary consequences of angling.     

Quantitative proteomics analysis reveals that proteins differentially expressed in chronic pancreatitis are also frequently involved in pancreatic cancer

The effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early stage, a difficult challenge. One major obstacle in the development of diagnostic biomarkers of early pancreatic cancer has been the dual expression of potential biomarkers in both chronic pancreatitis and cancer. To better understand the limitations of potential protein biomarkers, we used ICAT technology and tandem mass spectrometry-based proteomics to systematically study protein expression in chronic pancreatitis. Among the 116 differentially expressed proteins identified in chronic pancreatitis, most biological processes were responses to wounding and inflammation, a finding consistent with the underlining inflammation and tissue repair associated with chronic pancreatitis. Furthermore 40% of the differentially expressed proteins identified in chronic pancreatitis have been implicated previously in pancreatic cancer, suggesting some commonality in protein expression between these two diseases. Biological network analysis further identified c-MYC as a common prominent regulatory protein in pancreatic cancer and chronic pancreatitis. Lastly five proteins were selected for validation by Western blot and immunohistochemistry. Annexin A2 and insulin-like growth factor-binding protein 2 were overexpressed in cancer but not in chronic pancreatitis, making them promising biomarker candidates for pancreatic cancer. In addition, our study validated that cathepsin D, integrin beta1, and plasminogen were overexpressed in both pancreatic cancer and chronic pancreatitis. The positive involvement of these proteins in chronic pancreatitis and pancreatic cancer will potentially lower the specificity of these proteins as biomarker candidates for pancreatic cancer. Altogether our study provides some insights into the molecular events in chronic pancreatitis that may lead to diverse strategies for diagnosis and treatment of these diseases.     

ERbeta shifts from mitochondria to nucleus during estrogen-induced neoplastic transformation of human breast epithelial cells and is involved in estrogen-induced synthesis of mitochondrial respiratory chain proteins

Both estrogen receptors (ER) alpha (ERalpha) and beta (ERbeta) are localized in the nucleus, plasma membrane, and mitochondria, where they mediate the different physiological effects of estrogens. It has been observed that the relative subcellular localization of ERs is altered in several cancer cells. We have demonstrated that MCF-10F cells, the immortal and non-tumorigenic human breast epithelial cells (HBEC) that are ERalpha-negative and ERbeta-positive, are transformed in vitro by 17beta-estradiol (E(2)), generating highly invasive cells that are tumorigenic in severe combined immunodeficient mice. E(2)-transformed MCF-10F (trMCF) cells exhibit progressive loss of ductulogenesis, invasive (bsMCF) and tumorigenic (caMCF) phenotypes. Immunolocalization of ERbeta by confocal fluorescent microscopy and electron microscopy revealed that ERbeta is predominantly localized in mitochondria of MCF-10F and trMCF cells. Silencing ERbeta expression with ERbeta-specific small interference RNA (siRNA-ERbeta) markedly diminishes both nuclear and mitochondrial ERbeta in MCF-10F cells. The ERbeta shifts from its predominant localization in the mitochondria of MCF-10F and trMCF cells to the nucleus of bsMCF cells, becoming predominantly nuclear in caMCF cells. Furthermore, we demonstrated that the mitochondrial ERbeta in MCF-10F cells is involved in E(2)-induced expression of mitochondrial DNA (mtDNA)-encoded respiratory chain (MRC) proteins. This is the first report of an association of changes in the subcellular localization of ERbeta with various stages of E(2)-induced transformation of HBEC and a functional role of mitochondrial ERbeta in mediating E(2)-induced MRC protein synthesis. Our findings provide a new insight into one of the potential roles of ERbeta in human breast cancer.