Two-pore domain K+ channels-molecular sensors

Two-pore domain K(+) (K2P) channels have been cloned from a variety of species and tissues. They have been characterised biophysically as a 'background' K(+)-selective conductance and are gated by pH, stretch, heat, coupling to G-proteins and anaesthetics. Whilst their precise physiological function is unknown, they are likely to represent an increasingly important family of membrane proteins.     

Green tea constituent epigallocatechin-3-gallate inhibits angiogenic differentiation of human endothelial cells

Several independent research studies have shown that consumption of green tea reduces the development of cancer in many animal models. Epidemiological observations, though inconclusive, are suggesting that green tea consumption may also reduce the risk of some cancers in humans. These anti-carcinogenic effects of green tea have been attributed to its constituent polyphenols. Angiogenesis is a crucial step in the growth and metastasis of cancers. We have investigated the effect of the major polyphenolic constituent of green tea, epigallocatechin-3-gallate (EGCG), on the tube formation of human umbilical vein endothelial cells (HUVEC) on matrigel. Tube formation was inhibited by treatment both prior to plating and after plating endothelial cells on matrigel. EGCG treatment also was found to reduce the migration of endothelial cells in matrigel plug model. The role of matrix metalloproteinases (MMP) has been shown to play an important role during angiogenesis. Zymography was performed to determine if EGCG had any effect on MMPs. Zymographs of EGCG-treated culture supernatants modulated the gelatinolytic activities of secreted proteinases indicating that EGCG may be exerting its inhibitory effect by regulating proteinases. These findings suggest that EGCG acts as an angiogenesis inhibitor by modulating protease activity during endothelial morphogenesis.     

A discrete domain of the human TrkB receptor defines the binding sites for BDNF and NT-4

TrkB is a member of the Trk family of tyrosine kinase receptors. In vivo, the extracellular region of TrkB is known to bind, with high affinity, the neurotrophin protein brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). We describe the expression and purification of the second Ig-like domain of human TrkB (TrkBIg(2)) and show, using surface plasmon resonance, that this domain is sufficient to bind BDNF and NT-4 with subnanomolar affinity. BDNF and NT-4 may have therapeutic implications for a variety of neurodegenerative diseases. The specificity of binding of the neurotrophins to their receptor TrkB is therefore of interest. We examine the specificity of TrkBIg(2) for all the neurotrophins, and use our molecular model of the BDNF-TrkBIg(2) complex to examine the residues involved in binding. It is hoped that the understanding of specific interactions will allow design of small molecule neurotrophin mimetics.     

Cooperativity and flexibility of cystic fibrosis transmembrane conductance regulator transmembrane segments participate in membrane localization of a charged residue

Polytopic protein topology is established in the endoplasmic reticulum (ER) by sequence determinants encoded throughout the nascent polypeptide. Here we characterize 12 topogenic determinants in the cystic fibrosis transmembrane conductance regulator, and identify a novel mechanism by which a charged residue is positioned within the plane of the lipid bilayer. During cystic fibrosis transmembrane conductance regulator biogenesis, topology of the C-terminal transmembrane domain (TMs 7-12) is directed by alternating signal (TMs 7, 9, and 11) and stop transfer (TMs 8, 10, and 12) sequences. Unlike conventional stop transfer sequences, however, TM8 is unable to independently terminate translocation due to the presence of a single charged residue, Asp(924), within the TM segment. Instead, TM8 stop transfer activity is specifically dependent on TM7, which functions both to initiate translocation and to compensate for the charged residue within TM8. Moreover, even in the presence of TM7, the N terminus of TM8 extends significantly into the ER lumen, suggesting a high degree of flexibility in establishing TM8 transmembrane boundaries. These studies demonstrate that signal sequences can markedly influence stop transfer behavior and indicate that ER translocation machinery simultaneously integrates information from multiple topogenic determinants as they are presented in rapid succession during polytopic protein biogenesis.     

Meniscofemoral ligaments--structural and material properties

The meniscofemoral ligaments (MFLs) of 28 human cadaveric knees were studied to determine their incidence, structural and material properties. Using the Race–Amis casting method for measurement, the mean cross-sectional area for the anterior MFL (aMFL) was 14.7 mm² (±14.8 mm²) whilst that of the posterior MFL (pMFL) was 20.9 mm² (±11.6 mm²). The ligaments were isolated and tensile tested in a materials testing machine. The mean loads to failure were 300.5 N (±155.0 N) for the aMFL and 302.5 N (±157.9 N) for the pMFL, with elastic moduli of 281 (±239 MPa) and 227 MPa (±128 MPa), respectively. These significant anatomical and material properties suggest a function for the MFL in the biomechanics of the knee, and should be borne in mind when considering hypotheses on MFL function. Such hypotheses include roles for the ligaments in knee stability and guiding meniscal motion.     

The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is gamma-tubulin dependent

gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.     

Wnt signaling promotes oncogenic transformation by inhibiting c-Myc-induced apoptosis

Aberrant activation of the Wnt/beta-catenin signaling pathway is associated with numerous human cancers and often correlates with the overexpression or amplification of the c-myc oncogene. Paradoxical to the cellular transformation potential of c-Myc is its ability to also induce apoptosis. Using an inducible c-MycER expression system, we found that Wnt/beta-catenin signaling suppressed apoptosis by inhibiting c-Myc-induced release of cytochrome c and caspase activation. Both cyclooxygenase 2 and WISP-1 were identified as effectors of the Wnt-mediated antiapoptotic signal. Soft agar assays showed that neither c-Myc nor Wnt-1 alone was sufficient to induce cellular transformation, but that Wnt and c-Myc coordinated in inducing transformation. Furthermore, coexpression of Wnt-1 and c-Myc induced high-frequency and rapid tumor growth in nude mice. Extensive apoptotic bodies were characteristic of c-Myc-induced tumors, but not tumors induced by coactivation of c-Myc and Wnt-1, indicating that the antiapoptotic function of Wnt-1 plays a critical role in the synergetic action between c-Myc and Wnt-1. These results elucidate the molecular mechanisms by which Wnt/beta-catenin inhibits apoptosis and provide new insight into Wnt signaling-mediated oncogenesis.     

AMID: autonomous modeler of intragenic duplication

Intragenic duplication is an evolutionary process where segments of a gene become duplicated. While there has been much research into whole-gene or domain duplication, there have been very few studies of non-tandem intragenic duplication. The identification of intragenically replicated sequences may provide insight into the evolution of proteins, helping to link sequence data with structure and function. This paper describes a tool for autonomously modelling intragenic duplication. AMID provides: identification of modularly repetitive genes; an algorithm for identifying repeated modules; and a scoring system for evaluating the modules' similarity. An evaluation of the algorithms and use cases are presented.     

Paradoxical effects of chemicals in the diet on health

In 1992, Block et al. published a summary of 200 epidemiological investigations which indicated that a diet that was high in fruit and vegetables cut cancer risks approximately in half. These investigations used conventionally farmed produce that contained traces of synthetic pesticides and mycotoxins as well as an estimated 10,000 secondary products (i.e. natural pesticides). Dietary consumption of fruits and vegetables also reduces risks of cardiovascular disease, cataracts and brain dysfunction. Before genetic manipulation is undertaken to elevate or diminish any individual constituent of fruits and vegetables, the contribution of each of these constituents to health must be better understood, as in many cases their effects on health can be paradoxical.