Novel regulation of protein kinase C-η

Protein kinase C (PKC) is the receptor for tumor promoting phorbol esters, which are potent activators of conventional and novel PKCs, but persistent treatment with phorbol esters leads to downregulation of these PKCs. However, PKCη, a novel PKC isozyme, resists downregulation by tumor-promoting phorbol esters, but little is known about how PKCη level is regulated. Phosphorylation and dephosphorylation play an important role in regulating activity and stability of PKCs. In the present study, we have investigated the molecular mechanism of PKCη regulation. Several PKC activators, including phorbol 12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate and indolactam V caused upregulation of PKCη, whereas the general PKC inhibitor Gö 6983, but not the conventional PKC inhibitor Gö 6976 led to the downregulation of PKCη. Upregulation of PKCη was associated with an increase in phosphorylation of PKCη. Silencing of phosphoinositide-dependent kinase-1, which phosphorylates PKCη at the activation loop, failed to prevent PKC activator-induced upregulation of PKCη. Knockdown of PKCε but not PKCα inhibited PKC activator-induced upregulation of PKCη. Thus, our results suggest that the regulation of PKCη is unique and PKCε is required for the PKC activator-induced upregulation of PKCη.     

The endothelium: influencing vascular smooth muscle in many ways

The endothelium, although only a single layer of cells lining the vascular and lymphatic systems, contributes in multiple ways to vascular homeostasis. Subsequent to the 1980 report by Robert Furchgott and John Zawadzki, there has been a phenomenal increase in our knowledge concerning the signalling molecules and pathways that regulate endothelial - vascular smooth muscle communication. It is now recognised that the endothelium is not only an important source of nitric oxide (NO), but also numerous other signalling molecules, including the putative endothelium-derived hyperpolarizing factor (EDHF), prostacyclin (PGI(2)), and hydrogen peroxide (H(2)O(2)), which have both vasodilator and vasoconstrictor properties. In addition, the endothelium, either via transferred chemical mediators, such as NO and PGI(2), and (or) low-resistance electrical coupling through myoendothelial gap junctions, modulates flow-mediated vasodilatation as well as influencing mitogenic activity, platelet aggregation, and neutrophil adhesion. Disruption of endothelial function is an early indicator of the development of vascular disease, and thus an important area for further research and identification of potentially new therapeutic targets. This review focuses on the signalling pathways that regulate endothelial - vascular smooth muscle communication and the mechanisms that initiate endothelial dysfunction, particularly with respect to diabetic vascular disease.     

Inhibition of hepatitis C virus NS5A by fluoro-olefin based γ-turn mimetics

The HCV non-structural protein NS5A has been established as a viable target for the development of direct acting antiviral therapy. From computational modeling studies strong intra-molecular hydrogen bonds were found to be a common structural moiety within known NS5A inhibitors that have low pico-molar replicon potency. Efforts to reproduce these γ-turn-like substructures provided a novel NS5A inhibitor based on a fluoro-olefin isostere. This fluoro-olefin containing inhibitor exhibited picomolar activity (EC(50)=79 pM) against HCV genotype 1b replicon without measurable cytotoxicity. This level of activity is comparable to the natural peptide-based inhibitors currently under clinic evaluation, and demonstrates that a peptidomimetic approach can serve as a useful strategy to produce potent and structurally unique inhibitors of HCV NS5A.     

Detoxification potential and expression analysis of eutypine reducing aldehyde reductase (VrALR) during progressive drought and recovery in Vigna radiata (L.) Wilczek roots

Generation of reactive oxygen species (ROS) in plants is an inevitable consequence of adverse environmental cues and the ability to detoxify deleterious by-products of ROS-mediated oxidation reactions reflect an important defence strategy to combat abiotic stress. Here, we have cloned the eutypine reducing aldehyde reductase gene (VrALR) from Vigna radiata (L.) Wilczek roots. We have expressed and purified the VrALR protein and analyzed its enzyme kinetic parameters and catalytic efficiency with three different substrates to confirm its identity. The functional characterization of this enzyme was unravelled through heterologous expression of the gene in Escherichia coli BL21 and an oxidative stress-sensitive Saccharomyces cerevisiae mutant strain, W3O3-1-A. Finally, the endogenous VrALR enzyme activity and the mRNA expression patterns of the VrALR gene in the roots of V. radiata in response to progressive drought stress in vivo was studied to correlate the ROS-detoxifying role of this important enzyme under the influence of progressive drought stress. Our results, for the first time, demonstrate that eutypine reducing VrALR provides varying degree of stress tolerance in bacteria, yeast systems and also plays a promising protective role against oxidative stress in V. radiata roots during gradual water deprivation. The present study provides an unequivocal evidence to understand the crucial role of aldehyde reductase ROS-detoxifying system which is highly essential for developing stress tolerance in economically important crop plants.     

Neighborhood influences on perceived social support among parents: findings from the project on human development in Chicago neighborhoods

Background

Social support is frequently linked to positive parenting behavior. Similarly, studies increasingly show a link between neighborhood residential environment and positive parenting behavior. However, less is known about how the residential environment influences parental social support. To address this gap, we examine the relationship between neighborhood concentrated disadvantage and collective efficacy and the level and change in parental caregiver perceptions of non-familial social support.

Methodology/Principal Findings

The data for this study came from three data sources, the Project on Human Development in Chicago Neighborhoods (PHDCN) Study''s Longitudinal Cohort Survey of caregivers and their offspring, a Community Survey of adult residents in these same neighborhoods and the 1990 Census. Social support is measured at Wave 1 and Wave 3 and neighborhood characteristics are measured at Wave 1. Multilevel linear regression models are fit. The results show that neighborhood collective efficacy is a significant (ß = .04; SE = .02; p = .03), predictor of the positive change in perceived social support over a 7 year period, however, not of the level of social support, adjusting for key compositional variables and neighborhood concentrated disadvantage. In contrast concentrated neighborhood disadvantage is not a significant predictor of either the level or change in social support.

Conclusion

Our finding suggests that neighborhood collective efficacy may be important for inducing the perception of support from friends in parental caregivers over time.     

Requirement for a uroplakin 3a-like protein in the development of zebrafish pronephric tubule epithelial cell function, morphogenesis, and polarity

Uroplakin (UP)3a is critical for urinary tract development and function; however, its role in these processes is unknown. We examined the function of the UP3a-like protein Upk3l, which was expressed at the apical surfaces of the epithelial cells that line the pronephric tubules (PTs) of the zebrafish pronephros. Embryos treated with upk3l-targeted morpholinos showed decreased pronephros function, which was attributed to defects in PT epithelial cell morphogenesis and polarization including: loss of an apical brush border and associated phospho-ERM proteins, apical redistribution of the basolateral Na(+)/K(+)-ATPase, and altered or diminished expression of the apical polarity complex proteins Prkcz (atypical protein kinase C zeta) and Pard3 (Par3). Upk3l missing its C-terminal cytoplasmic domain or containing mutations in conserved tyrosine or proline residues did not rescue, or only partially rescued the effects of Upk3l depletion. Our studies indicate that Upk3l promotes epithelial polarization and morphogenesis, likely by forming or stimulating interactions with cytoplasmic signaling or polarity proteins, and that defects in this process may underlie the pathology observed in UP3a knockout mice or patients with renal abnormalities that result from altered UP3a expression.     

Importance of intracellular pH in determining the uptake and efficacy of the weakly basic chemotherapeutic drug, doxorubicin

Low extracellular pH (pH(e)), that is characteristic of many tumours, tends to reduce the uptake of weakly basic drugs, such as doxorubicin, thereby conferring a degree of physiological resistance to chemotherapy. It has been assumed, from pH-partition theory, that the effect of intracellular pH (pH(i)) is symmetrically opposite, although this has not been tested experimentally. Doxorubicin uptake into colon HCT116 cells was measured using the drug's intrinsic fluorescence under conditions that alter pH(i) and pH(e) or pH(i) alone. Acutely, doxorubicin influx across the cell-membrane correlates with the trans-membrane pH-gradient (facilitated at alkaline pH(e) and acidic pH(i)). However, the protonated molecule is not completely membrane-impermeant and, therefore, overall drug uptake is less pH(e)-sensitive than expected from pH-partitioning. Once inside cells, doxorubicin associates with slowly-releasing nuclear binding sites. The occupancy of these sites increases with pH(i), such that steady-state drug uptake can be greater with alkaline cytoplasm, in contradiction to pH-partition theory. Measurements of cell proliferation demonstrate that doxorubicin efficacy is enhanced at alkaline pH(i) and that pH-partition theory is inadequate to account for this. The limitations in the predictive power of pH-partition theory arise because it only accounts for the pH(i)/pH(e)-sensitivity of drug entry into cells but not the drug's subsequent interactions that, independently, show pH(i)-dependence. In summary, doxorubicin uptake into cells is favoured by high pH(e) and high pH(i). This modified formalism should be taken into account when designing manoeuvres aimed at increasing doxorubicin efficacy.     

Exploring the role of BCHE in the onset of Diabetes, Obesity and Neurological Disorders

Diabetes, Obesity and Neurological disturbances, most often show co-occurrence. There has been an extensive research in this domain, but the exact mechanism underlying the co-occurrence of the three conditions is still an enigma. The current paper is an approach to establish the role of Butyryl cholinesterase (BCHE) in Diabetes, Obesity and Neurological disorders by performing a comparative analysis with Neuroligin (NLGN2) a protein belonging to the same family. BCHE has its role in glucose regulation, Lipid metabolism and nerve signaling. Emphasis is laid on BCHE's diverse functions whose impediment affects the above mentioned metabolic pathways. Insilco techniques were employed to analyze the sequence, structural and functional similarities of the two proteins. A point mutation is focused which is common to both BCHE and Neuroligin. The mutation occurs at the homologous position in both the proteins making them deficient. This affects the three metabolic pathways leading to the respective disorders. The work describes the pathway that describes the role of BCHE in the onset of obesity mediated diabetes. The pathway further explains the association between Diabetes, Obesity and neurological disturbances.