Monomeric midkine induces tumor cell proliferation in the absence of cell-surface proteoglycan binding

Midkine (MK), a retinoic acid-inducible heparin-binding protein, is a mitogen which initiates a cascade of intracellular protein tyrosine phosphorylation mediated by the JAK/STAT pathway after binding to its high affinity p200(+)/MKR cell surface receptor in the G401 cell line [Ratovitski, E. A. (1998) J. Biol. Chem. 273, 3654-3660]. In this study, we determined the biophysical characteristics of purified recombinant murine MK and analyzed the requirements for ligand multimerization and cell surface proteoglycan binding for the G401 cell mitogenic activity of MK. Our studies indicate that the secreted form of MK (M = 13 kDa) exists in solution as an asymmetric monomer with a frictional coefficient of 1. 48 and a Stokes radius of 23.7 A. By constructing bead models of MK using the program AtoB and the program HYDRO to predict the hydrodynamic properties of each model, our data suggest that MK has a dumb-bell shape in solution composed of independent N- and C-terminal domains separated by an extended linker. This asymmetric MK monomer is a biologically active ligand with mitogenic activity on G401 cells in vitro. Neither heparin-induced formation of noncovalent MK multimers nor tissue transglutaminase II covalent multimerization of MK enhanced MK mitogenic activity in this system. Since neither heparin competition nor cell treatment with chondroitinase ABC or heparinase III abolished the mitogenic effects of MK on G401 cells, cell-surface proteoglycan binding by MK does not appear to be a requirement for its observed mitogenic effects. These results provide strong evidence that the MK-specific p200(+)/MKR has distinctive biochemical properties which distinguish it from the receptor tyrosine phosphatase cell-surface proteoglycan PTPzeta/RPTPbeta and support the hypothesis that the diverse biological effects of MK are mediated by multiple cell-specific signal transduction receptors.     

Glycosylations versus conformational preferences of cancer associated mucin core

Synthetic oligosaccharide vaccines based on core STn (sialyl 2-6 GalNAc) carbohydrate epitopes are being evaluated by a number of biopharmaceutical firms as potential immunotherapeutics in the treatment of mucin-expressing adenocarcinomas. The STn carbohydrate epitopes exist as discontinuous clusters, O-linked to proximal serine and threonine residues within the mucin sequence. In an effort to probe the structure and dynamics of STn carbohydrate clusters as they may exist on the cancer-associated mucin, we have used NMR spectroscopy and MD simulations to study the effect of O-glycosylation of adjacent serine residues in a repeating (Ser)n sequence. Three model peptides/glyco-peptides were studied: a serine trimer containing no carbohydrate groups ((Ser)₃ trimer); a serine trimer containing three Tn (GalNAc) carbohydrates -linked to the hydroxyls of adjacent serine sidechains ((Ser.Tn)₃ trimer); and a serine trimer containing three STn carbohydrates -linked to the hydroxyls of adjacent serine sidechains ((Ser.STn)₃ trimer). Our results demonstrate that clustering of carbohydrates shifts the conformational equilibrium of the underlying peptide backbone into a more extended and rigid state, an arrangement that could function to optimally present the clustered carbohydrate antigen to the immune system. Steric effects appear to drive these changes since an increase in the size of the attached carbohydrate (STn versus Tn) is accompanied by a stronger shift in the equilibrium toward the extended state. In addition, NMR evidence points to the formation of hydrogen bonds between the peptide backbone NH protons and the proximal GalNAc groups in the (Ser.Tn)₃ and (Ser.STn)₃ trimers. The putative peptide-sugar hydrogen bonds may also play a role in influencing the conformation of the underlying peptide backbone, as well as the orientation of the O-linked carbohydrate. The significance of these results will be discussed within the framework of developing clustered STn-based vaccines, capable of targeting the clustered STn epitopes on the cancer-associated mucin.     

Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain

Protein kinase GCN2 regulates translation in amino acid-starved cells by phosphorylating elF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNAphe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+ C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation (GCN2c-E803V) that weakens PK-C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain.     

豚鼠主动脉前庭自发性慢反应电位去极离子流的初步分析

为研究主动脉前庭自发慢反应电位的去极离充性质,利用豚鼠的离体以及心脏,常规玻璃微电极细胞内记录方法和离子通道组断剂,观测最大舒张电位（ＭＤＰ）、０相除极幅度（ＡＰＡ）、０相最大除极速度（Ｖｍａｘ）、４个自动除极速度（ＶＤＤ）、复极５０％（ＡＰＤ５０）和９０％（ＡＰＤ９０）的时间以及自发放电频率（ＲＰＦ）。结果发现：⑴０．５μｍｏｌ／Ｌ尼索地平（Ｎｉｓ）可使该慢电位的ＡＰＡ、Ｖｍａｘ、ＶＤＤ明显减小     

Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii

Two cellulase cDNAs, celB29 and celB2, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii strain SG4. The nucleotide sequences of celB2 and celB29 and the primary structures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54kDa. Analysis of the 1451bp celB2 cDNA revealed an 1164bp open reading frame coding for a 44kDa protein consisting of 388 amino acids. Both deduced proteins had a high sequence similarity in central regions containing putative catalytic domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hydrolase family 5 and were most closely related to endoglucanases from the anaerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpinomyces sp. The classification of CelB29 and CelB2 as endoglucanases was supported by enzyme assays. The cloned enzymes had high activities towards barley beta-glucan, lichenan and carboxymethylcellulose (CMC), but not Avicel, laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 showed activity against p-nitrophenyl-beta-D-cellobioside (pNP-G(2)) to p-nitrophenyl-beta-D-cellopentaoside (pNP-G(5)) but not p-nitrophenyl-beta-D-glucopyranoside (pNP-G(1)) with preferential activity against p-nitrophenyl-beta-D-cellotrioside (pNP-G(3)). Based on these results, we proposed that CelB29 and CelB2 are endoglucanases with broad substrate specificities for short- and long-chain beta-1,4-glucans.     

Rac1-induced endocytosis is associated with intracellular proteolysis during migration through a three-dimensional matrix

Transfection of Rat1 fibroblasts with an activated form of rac1 (V12rac1) stimulated cell migration in vitro compared to transfection of Rat1 fibroblasts with vector only or with dominant negative rac1 (N17rac1). To investigate the involvement of proteases in this migration, we used a novel confocal assay to evaluate the ability of the Rat1 transfectants to degrade a quenched fluorescent protein substrate (DQ-green bovine serum albumin) embedded in a three-dimensional gelatin matrix. Cleavage of the substrate results in fluorescence, thus enabling one to image extracellular and intracellular proteolysis by living cells. The Rat1 transfectants accumulated degraded substrate intracellularly. V12rac1 increased accumulation of the fluorescent product in vesicles that also labeled with the lysosomal marker LysoTracker. Treatment of the V12rac1-transfected cells with membrane-permeable inhibitors of lysosomal cysteine proteases and a membrane-permeable selective inhibitor of the cysteine protease cathepsin B significantly reduced intracellular accumulation of degraded substrate, indicating that degradation occurred intracellularly. V12rac1 stimulated uptake of dextran 70 (a marker of macropinocytosis) and polystyrene beads (markers of phagocytosis) into vesicles that also labeled for cathepsin B. Thus, stimulation of the endocytic pathways of macropinocytosis and phagocytosis by activated Rac1 may be responsible for the increased internalization and subsequent degradation of extracellular proteins.     

Numerical simulation of pulsatile flow in a compliant curved tube model of a coronary artery

The endothelial cells (ECs) lining a blood vessel wall are exposed to both the wall shear stress (WSS) of blood flow and the circumferential strain (CS) of pulsing artery wall motion. These two forces and their interaction are believed to play a role in determining remodeling of the vessel wall and development of arterial disease (atherosclerosis). This study focused on the WSS and CS dynamic behavior in a compliant model of a coronary artery taking into account the curvature of the bending artery and physiological radial wall motion. A three-dimensional finite element model with transient flow and moving boundaries was set up to simulate pulsatile flow with physiological pressure and flow wave forms characteristic of the coronary arteries. The characteristic coronary artery curvature and flow conditions applied to the simulation were: aspect ratio (lambda) = 10, diameter variation (DV) = 6 percent, mean Reynolds number (Re) = 150, and unsteadiness parameter (alpha) = 3. The results show that mean WSS is about 50 percent lower on the inside wall than the outside wall while WSS oscillation is stronger on the inside wall. The stress phase angle (SPA) between CS and WSS, which characterizes the dynamics of the mechanical force pattern applied to the endothelial cell layer, shows that CS and WSS are more out of phase in the coronaries than in any other region of the circulation (-220 deg on the outside wall, -250 deg on the inside wall). This suggests that in addition to WSS, SPA may play a role in localization of coronary atherosclerosis.     

用性信息素加农药诱杀器防治梨小食心虫的田间试验

在设计出性信息素加农药诱杀器后,以60、135、240、480个／km2诱杀器田间处理,看出以135个／km2诱杀器处理即有明显的防治效果。经在不同的虫口密度果园试验,以诱蛾量下降率、为害率和雌蛾交配率3项指标考察,该诱杀器1次挂出,在田间有效期达70～80天,取得了较理想防治效果。     

红外光谱技术在生物过程监测中的应用

在线监测化学组分的浓度对许多生物过程都是十分必要的。然而,探头需耐高温灭菌的要求和生物体系自身的复杂性给许多分析技术的在线监测带来了困难。近几年,随仪器和数据处理技术的迅速发展,应用红外光谱技术对生物过程的原位或在线监测日益广泛。本文对红外过程分析技术进行了较全面的综述,介绍了红外分析的原理、进展及在生物过程监测中的应用。