IS3 peptide-formed ion channels in rat skeletal muscle cell membranes

A 22-mer peptide, identical to the primary sequence of domain I segment 3 (IS3) of rat brain sodium channel I, was synthesized. With the patch clamp cell-attached technique, single channel currents could be recorded from the patches of cultured rat myotube membranes when the patches were held at hyperpolarized potentials and the electrode solution contained NaCl and 1 microM IS3, indicating that IS3 incorporated into the membranes and formed ion channels. The single channel conductances of IS3 channels were distributed heterogeneously, but mainly in the range of 10-25 pS. There was a tendency that the mean open time and open probability of IS3 channels increased and the mean close time decreased with the increasing of hyperpolarized membrane potentials. IS3 channels are highly selective for Na+ and Li+ but not for Cl- and K+, similar to the authentic Na+ channels.     

去甲肾上腺素介导低氧引起家兔颈动脉体神经电活动增加

在30只家兔颈动脉体-窦神经(CSN)标本上, 记录了窦神经中39个对低氧反应敏感的化学感受性单位由去甲肾上腺素(NA)及其拮抗剂引起的反应。结果如下 (1)以低氧的改良台氏液(MTS)灌流标本时, 19个单位放电频率由0.13±0.06增至0.25±0.12 imp/s (P<0.001); (2)在灌流液中加入去甲肾上腺素(10     

High yield bio-oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides

The use of renewable energy sources is becoming increasingly necessary to mitigate global warming. Recently much research has been focused on identifying suitable biomass species, which can provide high-energy outputs, to replace conventional fossil fuels. This paper reports an approach for increasing the yield of bio-oil production from fast pyrolysis after manipulating the metabolic pathway in microalgae through heterotrophic growth. The yield of bio-oil (57.9%) produced from heterotrophic Chlorella protothecoides cells was 3.4 times higher than from autotrophic cells by fast pyrolysis. The bio-oil was characterized by a much lower oxygen content, with a higher heating value (41 MJ kg(-1)), a lower density (0.92 kg l(-1)), and lower viscosity (0.02 Pas) compared to those of bio-oil from autotrophic cells and wood. These properties are comparable to fossil oil. The research could contribute to the creation of a system to produce energy from microalgae, and also could have great commercial potential for liquid fuel production.     

Sequence and structure determinants for the self-aggregation of recombinant polypeptides modeled after human elastin

Elastin is a polymeric structural protein that imparts the physical properties of extensibility and elastic recoil to tissues. The mechanism of assembly of the tropoelastin monomer into the elastin polymer probably involves extrinsic protein factors but is also related to an intrinsic capacity of elastin for ordered assembly through a process of hydrophobic self-aggregation or coacervation. Using a series of simple recombinant polypeptides based on elastin sequences and mimicking the unusual alternating domain structure of native elastin, we have investigated the influence of sequence motifs and domain structures on the propensity of these polypeptides for coacervation. The number of hydrophobic domains, their context in the alternating domain structure of elastin, and the specific nature of the hydrophobic domains included in the polypeptides all had major effects on self-aggregation. Surprisingly, in polypeptides with the same number of domains, propensity for coacervation was inversely related to the mean Kyte-Doolittle hydropathy of the polypeptide. Point mutations designed to increase the conformational flexibility of hydrophobic domains had the unexpected effect of suppressing coacervation and promoting formation of amyloid-like fibers. Such simple polypeptides provide a useful model system for understanding the relationship between sequence, structure, and mechanism of assembly of polymeric elastin.     

The autosomal dominant hypophosphatemic rickets R176Q mutation in fibroblast growth factor 23 resists proteolytic cleavage and enhances in vivo biological potency

Missense mutations in fibroblast growth factor 23 (FGF23) are the cause of autosomal dominant hypophosphatemic rickets (ADHR). The mutations (R176Q, R179W, and R179Q) replace Arg residues within a subtilisin-like proprotein convertase (SPC) cleavage site (RXXR motif), leading to protease resistance of FGF23. The goals of this study were to examine in vivo the biological potency of the R176Q mutant FGF23 form and to characterize alterations in homeostatic mechanisms that give rise to the phenotypic presentation of this disorder. For this, wild type and R176Q mutant FGF23 were overexpressed in the intact animals using a tumor-bearing nude mouse system. At comparable circulating levels, the mutant form was more potent in inducing hypophosphatemia, in decreasing circulating concentrations of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and in causing rickets and osteomalacia in these animals compared with wild type FGF23. Parameters of calcium homeostasis were also altered, leading to secondary hyperparathyroidism and parathyroid gland hyperplasia. However, the raised circulating levels of parathyroid hormone were ineffective in normalizing the reduced 1,25(OH)(2)D(3) levels by increasing renal expression of 25(OH)D(3)-1alpha-hydroxylase (Cyp40) to promote its synthesis and by decreasing that of 25(OH)D(3)-24-hydroxylase (Cyp24) to prevent its catabolism. The findings provide direct in vivo evidence that missense mutations from ADHR kindreds are gain-of-function mutations that retain and increase the protein's biological potency. Moreover, for the first time, they define a potential role for FGF23 in dissociating parathyroid hormone actions on mineral fluxes and on vitamin D metabolism at the level of the kidney.     

Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery

This study was undertaken to demonstrate the role of the RhoA/Rho kinase pathway in endothelin-1 (ET-1)-induced contraction of the rabbit basilar artery. Isometric tension and Western blot were used to examine ET-1-induced contraction and RhoA activation. The upstream effect on ET-1-induced RhoA activity was determined by using ET(A) and ET(B) receptor antagonists, protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol-3 kinase inhibitors. The downstream effect of ET-1-induced contraction and RhoA activity was studied in the presence of the Rho kinase inhibitor Y-27632. The effect of Rho kinase inhibitor on ET-1-induced myosin light chain (MLC) phosphorylation was investigated by using urea-glycerol-PAGE immunoblotting. We found 1) ET-1 increased RhoA activity (membrane binding RhoA) in a concentration-dependent manner; 2) ET(A), but not ET(B), receptor antagonist abolished the effect of ET-1 on RhoA activation; 3) phosphodylinositol-3 kinase inhibitor, but not PKC and tyrosine kinase inhibitors, reduced ET-1-induced RhoA activation; 4) Rho kinase inhibitor Y-27632 (10 microM) inhibited ET-1-induced contraction; and 5) ET-1 increased the level of MLC phosphorylation. Rho kinase inhibitor Y-27632 reduced the effect of ET-1 on MLC phosphorylation. This study demonstrated that RhoA/Rho kinase activation is involved in ET-1-induced contraction in the rabbit basilar artery. Phosphodylinositol-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.     

Interplay between integrins and FLK-1 in shear stress-induced signaling

Blood flow can modulate vascular cell functions. We studied interactions between integrins and Flk-1 in transducing the mechanical shear stress due to flow. This application of a step shear stress caused Flk-1. Casitas B-lineage lymphoma (Cbl) activation (Flk-1. Cbl association, tyrosine phosphorylation of the Cbl-bound Flk-1, and tyrosine phosphorylation of Cbl) in bovine aortic endothelial cells (BAECs). The activation of integrins by plating BAECs on vitronectin or fibronectin also induced this Flk-1. Cbl activation. The shear-induced Flk-1. Cbl activation was blocked by inhibitory antibodies for alphavbeta3- or beta1-integrin, suggesting that it is mediated by integrins. Inhibition of Flk-1 by SU1498 also abolished this shear-induced Flk-1. Cbl activation. In contrast to the requirement of integrins for Flk-1. Cbl activation, the Flk-1 blocker SU1498 had no detectable effect on the shear-induced integrin activation, suggesting that integrins and Flk-1 play sequential roles in the signal transduction hierarchy induced by shear stress. Integrins are essential for the mechanical activation of Flk-1 by shear stress but not for the chemical activation of Flk-1 by VEGF.     

VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding

Co-expression of NRP1 and (VEGFR-2) KDR on the surface of endothelial cells (EC) enhances VEGF165 binding to KDR and EC chemotaxis in response to VEGF165. Overexpression of NRP1 by prostate tumor cells in vivo results in increased tumor angiogenesis and growth. We investigated the molecular mechanisms underlying NRP1-mediated angiogenesis by analyzing the association of NRP1 and KDR. An intracellular complex containing NRP1 and KDR was immunoprecipitated from EC by anti-NRP1 antibodies only in the presence of VEGF165. In contrast, VEGF121, which does not bind to NRP1, did not support complex formation. Complexes containing VEGF165, NRP1, and KDR were also formed in an intercellular fashion by co-culture of EC expressing KDR only, with cells expressing NRP1 only, for example, breast carcinoma cells. VEGF165 also mediated the binding of a soluble NRP1 dimer to cells expressing KDR only, confirming the formation of such complexes. Furthermore, the formation of complexes containing KDR and NRP1 markedly increased 125I-VEGF165 binding to KDR. Our results suggest that formation of a ternary complex of VEGF165, KDR, and NRP1 potentiates VEGF165 binding to KDR. These complexes are formed on the surface of EC and in a juxtacrine manner via association of tumor cell NRP1 and EC KDR.