Expression of functionally relevant cell surface markers in dibutyltin-exposed human natural killer cells

Butyltin (BT) compounds are known for their worldwide contamination. Dibutyltin (DBT) is used as a stabilizer in plastic products, and as a deworming agent in poultry. Poultry products have been shown to contain measurable levels of DBT. Drinking water has also been reported to contain BTs due to leaching from PVC pipes. We, and others, have found measurable levels of DBT in human blood. BTs appear to increase the risk of cancer and other viral infections in exposed individuals. In previous studies we have shown that the tumor killing function of natural killer (NK) lymphocytes was greatly diminished after as little as a 1 h exposure to DBT and the inhibition continued even after removal of the compound. We also showed that there was a significant decrease in NK cell lysis of K562 target cells after an exposure to 1.5 microM DBT for 24 h. This 24 h exposure also decreased the ability of NK cells to bind to tumor cells. Loss of binding function was not seen when NK cells were exposed to 5-10 microM DBT for 1 h. However, NK cells exposed to 5 microM DBT for 1 h and then incubated in DBT-free media for 24, 48, or 96 h, showed a significant loss of tumor-binding function within 24 h. The effects of DBT exposure on seven cell surface molecules that are involved in NK-cell interactions with target cells were investigated. The results indicated that the exposure of NK cells to 1.5 microM DBT for 24 h decreased the expression of CD2, CD11a, CD16, CD11c. There was no decrease in expression of any of the markers studied when NK cells were exposed to 5 microM DBT for 1 h, consistent with the fact that a 1-h exposure had no effect on the ability of NK cells to bind tumor cells. However, when NK cells were exposed to 5 microM DBT for 1 h followed by 24, 48 or 96 h incubations in DBT-free media there was decreased expression of several of the cells surface molecules with the most dramatic decreases being in CD16 and CD56.     

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations.The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.     

Ligand-induced lysosomal epidermal growth factor receptor (EGFR) degradation is preceded by proteasome-dependent EGFR de-ubiquitination

Studies on the differential routing of internalized epidermal growth factor receptors (EGFRs) induced by EGF, TGF alpha, and the superagonist EGF-TGF alpha chimera E4T suggested a correlation between receptor recycling and their mitogenic potency. EGFR sorting to lysosomes depends on its kinase domain and its ubiquitination by Cbl proteins. Proteasomes have also been proposed to regulate EGFR degradation, but the underlying mechanism remains obscure. Here we evaluated EGFR activation, Cbl recruitment, EGFR ubiquitination and degradation in response to EGF, TGF alpha, and E4T. We also determined the fate of activated EGFRs and Cbl proteins by using v-ATPase (bafilomycin A1) and proteasome (lactacystin) inhibitors. Our results demonstrate that E4T and TGF alpha provoke decreased Cbl recruitment, EGFR ubiquitination and EGFR degradation compared with EGF. Furthermore, bafilomycin treatment blocks EGFR but not c-Cbl degradation. In contrast, lactacystin treatment blocks EGF-induced c-Cbl degradation but does not block EGFR degradation, even though lactacystin causes a minor delay in EGFR degradation. Surprisingly, even though bafilomycin completely blocks EGFR degradation, it does not prevent EGFR de-ubiquitination upon prolonged EGF stimulation. Strikingly, when combined with bafilomycin, lactacystin treatment stabilizes the ubiquitinated EGFR and prevents its de-ubiquitination. We conclude that the enhanced EGFR recycling that has been observed in HER-14 cells following TGF alpha or E4T stimulation correlates with decreased EGFR ubiquitination and EGFR degradation, and that proteasomal activity is required for de-ubiquitination of the EGFR prior to its lysosomal degradation.     

Glutamine synthetase from the marine cyanobacteria Prochlorococcus spp: characterization,phylogeny and response to nutrient limitation

The regulation of glutamine synthetase (EC 6.3.1.2) from Prochlorococcus was previously shown to exhibit unusual features: it is not upregulated by nitrogen starvation and it is not inactivated by darkness (El Alaoui et al. (2001) Appl Environ Microbiol 67: 2202-2207). These are probably caused by adaptations to oligotrophic environments, as confirmed in this work by the marked decrease in the enzymatic activity when cultures were subjected to iron or phosphorus starvation. In order to further understand the adaptive features of ammonium assimilation in this cyanobacterium, glutamine synthetase was purified from two Prochlorococcus strains: PCC 9511 (high-light adapted) and SS120 (low-light adapted). We obtained approximately 100-fold purified samples of glutamine synthetase electrophoretically homogeneous, with a yield of approximately 30%. The estimated molecular mass of the subunits was roughly the same for both strains: 48.3 kDa. The apparent Km constants for the biosynthetic activity were 0.30 mM for ammonium, 1.29 mM for glutamate and 1.35 mM for ATP; the optimum pH was 8.0. Optimal temperature was surprisingly high (55 degrees C). Phylogenetic analysis of glnA from three Prochlorococcus strains (MED4, MIT9313 and SS120) showed they group closely with marine Synechococcus isolates, in good agreement with other studies based on 16 S RNA sequences. All of our results suggest that the structure and kinetics of glutamine synthetase in Prochlorococcus have not been significantly modified during the evolution within the cyanobacterial radiation, in sharp contrast with its regulatory properties.     

Role of heme oxygenases in sepsis-induced diaphragmatic contractile dysfunction and oxidative stress

Heme oxygenases (HOs), essential enzymes for heme metabolism, play an important role in the defense against oxidative stress. In this study, we evaluated the expression and functional significance of HO-1 and HO-2 in the ventilatory muscles of normal rats and rats injected with bacterial lipopolysaccharide (LPS). Both HO-1 and HO-2 proteins were detected inside ventilatory and limb muscle fibers of normal rats. Diaphragmatic HO-1 and HO-2 expressions rose significantly within 1 and 12 h of LPS injection, respectively. Inhibition of the activity of inducible nitric oxide synthase (iNOS) in rats and absence of this isoform in iNOS(-/-) mice did alter sepsis-induced regulation of muscle HOs. Systemic inhibition of HO activity with chromium mesoporphyrin IX enhanced muscle protein oxidation and hydroxynonenal formation in both normal and septic rats. Moreover, in vitro diaphragmatic force generation declined substantially in response to HO inhibition both in normal and septic rats. We conclude that both HO-1 and HO-2 proteins play an important role in the regulation of muscle contractility and in the defense against sepsis-induced oxidative stress.     

Acoustic niche partitioning among seven cuckoo species in a forest in eastern China

Acoustic communication among birds plays an important role in attracting mates and defending territories. For the successful transmission of songs, individuals of different species often avoid singing at the same time to reduce acoustic interference from background noise and overlapping signals from heterospecifics. Such behavioural acoustic niche partitioning may occur especially among closely related species due to their ecological similarities. In this study, we recorded bird sounds in a subtropical forest in China in May–June 2019 and detected seven cuckoo species. Extracting characteristics of the cuckoo calls, we found that only four of the 21 pairs of species overlapped in frequency range, and 19 pairs were classified accurately using a linear discriminant analysis classifier based on their features. The remaining two species pairs could be separated based on temporal or spatial distribution patterns. We also analysed the temporal distribution patterns and overlap time of the calls, finding that the seven species exhibit partitioning in at least one of three acoustic dimensions (site, frequency, activity time). We conclude that the seven sympatric cuckoo species were strongly partitioned in acoustic signal space and minimally masked each other's signals.     

Nitric oxide delays oocyte aging

Nitric oxide (NO) is a ubiquitous signaling molecule that plays a crucial role in oocyte maturation and embryo development. However, its role in oocyte aging is unclear. To examine how NO affects oocyte aging, we retrieved young and relatively old mouse oocytes and exposed them to increasing concentrations of NO donor S-nitroso acetyl penicillamine (SNAP). Aging related phenomena of ooplasmic microtubule dynamics (OMD), cortical granule (CG) exocytosis, zona pellucida (ZP) hardening, and spindle/chromatin integrity were studied at each SNAP concentration using fluorescence immunocytochemistry and confocal microscopy and compared with respective unexposed controls. Exposure of both young and old oocytes to NO resulted in a significant diminution in OMD and ZP dissolution time, whereas spontaneous CG loss decreased in old NO exposed oocytes compared to controls (P < 0.001 for all). Furthermore, NO exposure decreased the rate of spindle abnormalities in oocytes compared to unexposed controls. Interestingly, in old oocytes, the positive influence of NO was attenuated beyond 0.23 microM/min and disappeared at 0.46 microM/min NO. Overall, a significant dose-response relationship was noted between NO exposure and markers of aging with between 50 and 100 microM SNAP (0.11-0.23 microM/min NO, P < 0.0001). Collectively, our results demonstrate for the first time that exposure to NO delays oocyte aging and improves the integrity of the microtubular spindle apparatus in young and old oocytes.     

Synthesis and characterization of a photo-cross-linked biodegradable elastomer

The synthesis and characterization of a photocurable biodegradable elastomer as a potential biomaterial for the delivery of thermosensitive drugs are described. The elastomer was prepared from UV initiated cross-linking of an acrylated star-poly(epsilon-caprolactone-co-D,L-lactide) prepolymer. The influence of the molecular weight of the acrylated prepolymer on the final elastomer mechanical and thermal properties was determined. The glass-transition temperature of the elastomers was independent of the prepolymer molecular weight and was from -6 to -8 degrees C. The Young's modulus and stress at break of the elastomers was proportional to the inverse of the prepolymer molecular weight, while the strain at break increased in a linear fashion with the prepolymer molecular weight. Over a degradation period of 12 weeks in phosphate buffered saline, the elastomers exhibited little mass loss, appreciable mechanical strength loss, and little dimensional or strain at break change.     

The Impact of Myeloperoxidase and Activated Macrophages on Metaphase II Mouse Oocyte Quality

Myeloperoxidase (MPO), an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl), a damaging reactive oxygen species (ROS) utilizing hydrogen peroxide (H₂O₂) and chloride (Cl^-). Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1) Incubation with a catalytic amount of MPO (40 nM) for different incubation periods in the presence of 100 mM Cl^- with and without H₂O₂ and with and without melatonin (100 μM), at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells); 2) Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 10⁶ cells/ml) in the absence and presence of melatonin (200 μM), an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200); 3) Untreated oocytes incubated for 4 hrs as controls (n = 73/64). Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes.