A short motif in Kir6.1 consisting of four phosphorylation repeats underlies the vascular KATP channel inhibition by protein kinase C

Vascular ATP-sensitive K(+) channels are inhibited by multiple vasoconstricting hormones via the protein kinase C (PKC) pathway. However, the molecular substrates for PKC phosphorylation remain unknown. To identify the PKC sites, Kir6.1/SUR2B and Kir6.2/SUR2B were expressed in HEK293 cells. Following channel activation by pinacidil, the catalytic fragment of PKC inhibited the Kir6.1/SUR2B currents but not the Kir6.2/SUR2B currents. Phorbol 12-myristate 13-acetate (a PKC activator) had similar effects. Using Kir6.1-Kir6.2 chimeras, two critical protein domains for the PKC-dependent channel inhibition were identified. The proximal N terminus of Kir6.1 was necessary for channel inhibition. Because there was no PKC phosphorylation site in the N-terminal region, our results suggest its potential involvement in channel gating. The distal C terminus of Kir6.1 was crucial where there are several consensus PKC sites. Mutation of Ser-354, Ser-379, Ser-385, Ser-391, or Ser-397 to nonphosphorylatable alanine reduced PKC inhibition moderately but significantly. Combined mutations of these residues had greater effects. The channel inhibition was almost completely abolished when 5 of them were jointly mutated. In vitro phosphorylation assay showed that 4 of the serine residues were necessary for the PKC-dependent (32)P incorporation into the distal C-terminal peptides. Thus, a motif containing four phosphorylation repeats is identified in the Kir6.1 subunit underlying the PKC-dependent inhibition of the Kir6.1/SUR2B channel. The presence of the phosphorylation motif in Kir6.1, but not in its close relative Kir6.2, suggests that the vascular K(ATP) channel may have undergone evolutionary optimization, allowing it to be regulated by a variety of vasoconstricting hormones and neurotransmitters.     

Human vasoactive hormone adrenomedullin and its binding protein rescue experimental animals from shock

We recently discovered that vascular responsiveness to adrenomedullin (AM), a vasoactive hormone, decreases after hemorrhage, which is markedly improved by the addition of its binding protein AMBP-1. One obstacle hampering the development of AM/AMBP-1 as resuscitation agents in trauma victims is the potential immunogenicity of rat proteins in humans. Although less potent than rat AM, human AM has been shown to increase organ perfusion in rats. We therefore hypothesized that administration of human AM/AMBP-1 improves organ function and survival after severe blood loss in rats. To test this, male Sprague-Dawley rats were bled to and maintained at an MAP of 40 mmHg for 90 min. They were then resuscitated with an equal volume of shed blood in the form of Ringer's lactate (i.e., low-volume resuscitation) over 60 min. At 15 min after the beginning of resuscitation, human AM/AMBP-1 (12/40 or 48/160 microg/kg BW) were administered intravenously over 45 min. Various pathophysiological parameters were measured 4h after resuscitation. In additional groups of animals, a 12-day survival study was conducted. Our result showed that tissue injury as evidenced by increased levels of transaminases, lactate, and creatinine, was present at 4h after hemorrhage and resuscitation. Moreover, pro-inflammatory cytokines TNF-alpha and IL-6 were also significantly elevated. Administration of AM/AMBP-1 markedly attenuated tissue injury, reduced cytokine levels, and improved the survival rate from 29% (vehicle) to 62% (low-dose) or 70% (high-dose). However, neither human AM alone nor human AMBP-1 alone prevented the significant increase in ALT, AST, lactate and creatinine at 4h after the completion of hemorrhage and resuscitation. Moreover, the half-life of human AM and human AMBP-1 in rats was 35.8 min and 1.68 h, respectively. Thus, administration of human AM/AMBP-1 may be a useful approach for attenuating organ injury, and reducing mortality after hemorrhagic shock.     

Tea polyphenols, their biological effects and potential molecular targets

Tea is the most popular beverage in the world, second only to water. Tea contains an infusion of the leaves from the Camellia sinensis plant rich in polyphenolic compounds known as catechins, the most abundant of which is (-)-EGCG. Although tea has been consumed for centuries, it has only recently been studied extensively as a health-promoting beverage that may act to prevent a number of chronic diseases and cancers. The results of several investigations indicate that green tea consumption may be of modest benefit in reducing the plasma concentration of cholesterol and preventing atherosclerosis. Additionally, the cancer-preventive effects of green tea are widely supported by results from epidemiological, cell culture, animal and clinical studies. In vitro cell culture studies show that tea polyphenols potently induce apoptotic cell death and cell cycle arrest in tumor cells but not in their normal cell counterparts. Green tea polyphenols were shown to affect several biological pathways, including growth factor-mediated pathway, the mitogen-activated protein (MAP) kinase-dependent pathway, and ubiquitin/proteasome degradation pathways. Various animal studies have revealed that treatment with green tea inhibits tumor incidence and multiplicity in different organ sites such as skin, lung, liver, stomach, mammary gland and colon. Recently, phase I and II clinical trials have been conducted to explore the anticancer effects of green tea in humans. A major challenge of cancer prevention is to integrate new molecular findings into clinical practice. Therefore, identification of more molecular targets and biomarkers for tea polyphenols is essential for improving the design of green tea trials and will greatly assist in a better understanding of the mechanisms underlying its anti-cancer activity.     

Robust exponential stability of markovian jumping neural networks with time-varying delay

This paper considers the robust stability of a class of neural networks with Markovian jumping parameters and time-varying delay. By employing a new Lyapunov-Krasovskii functional, a sufficient condition for the global exponential stability of the delayed Markovian jumping neural networks is established. The proposed condition is also extended to the uncertain cases, which are shown to be the improvement and extension of the existing ones. Finally, the validity of the results are illustrated by an example.     

Vanadium Uptake by Biomass of <Emphasis Type="Italic">Coprinus comatus</Emphasis> and Their Effect on Hyperglycemic Mice

The Vanadium uptake by Coprinus comatus and their co-effect on hyperglycemic mice were studied. By fermentation and AAS analysis, the optimal concentration of vanadium in medium was 0.4%, and the content of vanadium accumulated in the mycelia was 3,528.0 microg/g. At the concentration of 0.4%, the vanadium-associated toxicity was reduced, and its anti-diabetic effects were maintained.     

A chemical lipid modification of recombinant preS antigen to study the mechanism of HBV attachment to the host cell

Surface antigen preS of Hepatitis B virus plays fundamental roles in mediating receptor recognition and virus internalization. Myristoylation at N-terminal Gly(2) residue of preS is essential for viral attachment and infectivity. A number of myristoylated proteins have been shown to undergo a conformational change (myristoyl switch) that alters their affinity to cell membrane. However, there is little knowledge about what effect this fatty acylation contributes in virus-host cell interaction. Here we demonstrated a new method for lipid modification of recombinant preS protein at N-terminal residue 2 with alkylating chemicals. Modified preS was able to inhibit HBV penetrating into HepG2 cells with an increased efficiency compared to unmodified form. Flow cytometric analysis indicated that lipid modification enhanced the binding affinity of preS to hepatocytes, but not resulting from hydrophobic interaction. CD analysis further revealed a conformational change of modified preS in the presence of membrane mimetics. These findings imply that the conformation transition induced by fatty acylation is important for efficient attachment of virus to cell receptors, and this method of chemical lipid modification provides a basis for designing therapeutic inhibitors to Hepatitis B virus.     

Optimization of Vi capsular polysaccharide production during growth of Salmonella enterica serotype Typhi Ty2 in a bioreactor

Vi capsular polysaccharide is synthesized during growth of Salmonella typhi Ty2 and is spontaneously released from the bacterial cells into the culture medium during culture. Vi production was dependent on cell growth and the greater the cell mass the greater the production of Vi. Using fed batch culture to optimize bacterial growth resulted is an increase in cell mass and consequently Vi production. The yield of Vi obtained in fed batch culture was 415 mg l⁻¹, which was over three times that, obtained in batch culture. A proportion of the Vi remained cell associated in the form of a capsule and at least part of this was released from the bacterial surface by sonication. The size of the Vi polysaccharide produced was consistently high and did not change during the different phases of bacterial growth. The synthesis of Vi was also dependent upon the media components and the fermentation conditions. The presence of high concentrations of glucose at the beginning of growth inhibited the production of Vi, particularly during the stationary phase. At a concentration of 400 mM sodium phosphate the synthesis of Vi was strongly inhibited.     

The matrix protein CCN1 (CYR61) promotes proliferation, migration and tube formation of endothelial progenitor cells

Neovascularization and re-endothelialization relies on circulating endothelial progenitor cells (EPCs), but their recruitment and angiogenic roles are subjected to regulation by the vascular microenvironment, which remains largely unknown. The present study was designed to investigate the effects of mature ECs and matrix protein CCN1 on the properties of EPCs. In a coculture system, effects of ECs on proliferation, migration and participation in tube-like formation of EPCs were evaluated, and functional assays were employed to identify the exact role of CCN1 in EPCs vitality and function. We demonstrated that ECs, as an indispensable part of the cellular milieu, significantly promoted the proliferation, migration and tube formation activities of EPCs, and more importantly, CCN1 was potentially involved in such effects of ECs. Expression of CCN1 in EPCs was significantly increased by serum, VEGF, ECs-cocultivation and ECs conditioned medium. Moreover, Ad-CCN1-mediated overexpression of CCN1 directly enhanced migration and tube formation of EPCs, whereas silencing of endogenous CCN1 in EPCs inhibits cell functions. Furthermore, CCN1 induced the expressions of chemokines and growth factors, such as MCP-1 and VEGF, suggesting a complex interaction between those proangiogenic factors. Our data suggest that matrix protein CCN1 may play an important role in microenvironment-mediated biological properties of EPCs.