Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6

Ischemia-reperfusion (I/R) has critical consequences in the heart. Recent studies on the functions of I/R-activated kinases, such as p38 mitogen-activated protein kinase (MAPK), showed that I/R injury is reduced in the hearts of transgenic mice that overexpress the p38 MAPK activator MAPK kinase 6 (MKK6). This protection may be fostered by changes in the levels of many proteins not currently known to be regulated by p38. To examine this possibility, we employed the multidimensional protein identification technology MudPIT to characterize changes in levels of proteins in MKK6 transgenic mouse hearts, focusing on proteins in mitochondria, which play key roles in mediating I/R injury in the heart. Of the 386 mitochondrial proteins identified, the levels of 58 were decreased, while only 2 were increased in the MKK6 transgenic mouse hearts. Among those that were decreased were 21 mitochondrial oxidative phosphorylation complex proteins, which was unexpected because p38 is not known to mediate such decreases. Immunoblotting verified that proteins in each of the five oxidative phosphorylation complexes were reduced in MKK6 mouse hearts. On assessing functional consequences of these reductions, we found that MKK6 mouse heart mitochondria exhibited 50% lower oxidative respiration and I/R-mediated reactive oxygen species (ROS) generation, both of which are predicted consequences of decreased oxidative phosphorylation complex proteins. Thus the cardioprotection observed in MKK6 transgenic mouse hearts may be partly due to decreased electron transport, which is potentially beneficial, because damaging ROS are known to be generated by mitochondrial complexes I and III during reoxygenation.     

Comparison of Barium and Arsenic Concentrations in Well Drinking Water and in Human Body Samples and a Novel Remediation System for These Elements in Well Drinking Water

Health risk for well drinking water is a worldwide problem. Our recent studies showed increased toxicity by exposure to barium alone (≤700 µg/L) and coexposure to barium (137 µg/L) and arsenic (225 µg/L). The present edition of WHO health-based guidelines for drinking water revised in 2011 has maintained the values of arsenic (10 µg/L) and barium (700 µg/L), but not elements such as manganese, iron and zinc. Nevertheless, there have been very few studies on barium in drinking water and human samples. This study showed significant correlations between levels of arsenic and barium, but not its homologous elements (magnesium, calcium and strontium), in urine, toenail and hair samples obtained from residents of Jessore, Bangladesh. Significant correlation between levels of arsenic and barium in well drinking water and levels in human urine, toenail and hair samples were also observed. Based on these results, a high-performance and low-cost adsorbent composed of a hydrotalcite-like compound for barium and arsenic was developed. The adsorbent reduced levels of barium and arsenic from well water in Bangladesh and Vietnam to <7 µg/L within 1 min. Thus, we have showed levels of arsenic and barium in humans and propose a novel remediation system.     

Morphological,molecular and MALDI-TOF MS identification of ticks and tick-associated pathogens in Vietnam

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising and reliable tool for arthropod identification, including the identification of alcohol-preserved ticks based on extracted leg protein spectra. In this study, the legs of 361 ticks collected in Vietnam, including 251 Rhiphicephalus sanguineus s.l, 99 Rhipicephalus (Boophilus) microplus, two Amblyomma varanensis, seven Dermacentor auratus, one Dermacentor compactus and one Amblyomma sp. were submitted for MALDI-TOF MS analyses. Spectral analysis showed intra-species reproducibility and inter-species specificity and the spectra of 329 (91%) specimens were of excellent quality. The blind test of 310 spectra remaining after updating the database with 19 spectra revealed that all were correctly identified with log score values (LSV) ranging from 1.7 to 2.396 with a mean of 1.982 ± 0.142 and a median of 1.971. The DNA of several microorganisms including Anaplasma platys, Anaplasma phagocytophilum, Anaplasma marginale, Ehrlichia rustica, Babesia vogeli, Theileria sinensis, and Theileria orientalis were detected in 25 ticks. Co-infection by A. phagocytophilum and T. sinensis was found in one Rh. (B) microplus.     

Psychedelic-inspired drug discovery using an engineered biosensor

1. Download : Download high-res image (206KB)
2. Download : Download full-size image

     

An asparaginase of Aspergillus nidulans is subject to oxygen repression in addition to nitrogen metabolite repression

Summary Of five amidohydrolase activities subject to nitrogen metabolite repression in Aspergillus nidulans, l-asparaginase shows clearest evidence of also being subject to repression by atmospheric oxygen. Such oxygen repressibility is only evident under nitrogen metabolite derepressed conditions. Asparaginase levels are also considerably elevated by areA300, an altered function allele of the positive acting wide domain regulatory gene areA mediating nitrogen metabolite repression and are drastically reduced by loss of function mutations in areA. A. nidulans has two l-asparaginase enzymes and it has been shown by the use of appropriate mutants that these regulatory effects are exerted on the expression of that specified by the ahrA gene but probably not that specified by the apnA gene. Present address: (until 25 August, 1988) Department of Genetics, University of Georgia, Athens, GA 30602, USA     

Effect of prolactin and cyclic AMP on 125I-labelled low density lipoprotein uptake and metabolism by luteinized porcine granulosa cells in culture

The present study examines the effect of prolactin (PRL) and N6-2(1)-O-dibutyryladenosine 3'5'-cyclic monophosphate (cAMP) on low density lipoprotein (LDL) uptake and metabolism by luteinized porcine granulosa cells in culture. Granulosa cells from preovulatory follicles were plated with 1% serum and 1 microgram/mL of insulin for the first 48 h. Following plating (day 3) the cells were cultured in serum-free media with the same dose of insulin. The next day the medium was replaced with serum- and insulin-free medium, and to some cultures 1.23 IU/mL of human chorionic gonadotrophin (hCG) was added. On day 5 the medium was again replaced and graded amounts of PRL (0, 0.03, 0.3, and 3 micrograms/mL) were added. Following 48 h of incubation with PRL, 20 micrograms/mL of 125I-labelled LDL was added to cultures. Surface-bound, internalized, and degraded LDLs were quantitated at 12 h following addition of LDL. To examine the effect of cAMP on LDL metabolism, the cells were exposed for 24 h to cAMP (3mM) on day 6 of culture. PRL had a stimulatory effect on LDL degradation by luteinized granulosa cells. Pre-exposure of cells to hCG augmented the stimulatory effect of PRL. Addition of cAMP also enhanced LDL degradation by luteinized granulosa cells. Both PRL and cAMP increased surface binding of LDL in cells pre-exposed to hCG, but there was no effect on internalization. The increase in cell surface binding of LDL with PRL and cAMP was less than their effect on LDL degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipase C-�� Regulates Epidermal Morphogenesis in Caenorhabditis elegans

Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP₃)-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP₃ production is stimulated is unknown. IP₃ is produced by the action of phospholipase C (PLC). We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-ε produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP₃ receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-γ and EGL-8/PLC-β can compensate for reduced PLC-1 activity. Our work places PLC-ε into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-ε.     

Repair of oxidative guanine damage in plasmid DNA by indoles involves proton transfer between complementary bases

We have used the single electron oxidizing agent (SCN)(2)(*)(-) (generated by gamma-irradiation of aqueous thiocyanate) to produce guanyl radicals in plasmid DNA. The stable product(s) formed from these radicals can be detected after conversion with a base excision repair endonuclease to single strand breaks. The yield of enzyme-induced breaks is decreased by the presence during irradiation of indole compounds. Rate constants for the reduction of DNA guanyl radicals by these indoles can be calculated from the concentration dependence of the attenuation in the yield of enzyme sensitive sites. Indoles bearing electron-donating groups (methoxy or methyl) appear to react at the diffusion-controlled rate, but those bearing electron-withdrawing groups (cyano or nitro) are significantly less reactive. At physiological pH values, the reduction of a DNA guanyl radical involves the transfer of a proton as well as an electron. Comparison of the kinetic results with literature thermodynamic data suggests that the source of this proton is the complementary base-paired cytosine.     

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis

For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine‐tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5′‐ and 3′‐end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications.