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951.
Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position.
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L. Szilk J. Moitra C. Vinson 《Protein science : a publication of the Protein Society》1997,6(6):1273-1283
Using a dimeric bZIP protein, we have designed a leucine zipper that becomes more stable after a serine in the e position is phosphorylated by protein kinase A (delta delta GP = -1.4 kcal mol-1 dimer-1 or -0.7 kcal mol-1 residue-1). Mutagenesis studies indicate that three arginines form a network of inter-helical (i,i' + 5; i, i' + 2) and intra-helical (i, i + 4) attractive interactions with the phosphorylated serine. When the arginines are replaced with lysines, the stabilizing effect of serine phosphorylation is reduced (delta delta GP = -0.5 kcal mol-1 dimer-1). The hydrophobic interface of the leucine zipper needs a glycine in the d position to obtain an increase in stability after phosphorylation. The phosphorylated protein binds DNA with a 15-fold higher affinity. Using a transient transfection assay, we document a PKA dependent four-fold activation of a reporter gene. Phosphorylation of a threonine in the same e position decreases the stability by delta delta GP = +1.2 kcal mol-1 dimer-1. We present circular dichroism (CD) thermal denaturations of 15 bZIP proteins before and after phosphorylation. These data provide insights into the structural determinants that result in stabilization of a coiled coil by phosphorylation. 相似文献
952.
Akira Uchimura Toshiyuki Shimizu Masahiro Morita Hitomi Ueno Kazuhiro Motoki Hideaki Fukushima Takenori Natori Yasuhiko Koezuka 《Bioorganic & medicinal chemistry》1997,5(12):2245-2249
We compared the immunostimulatory effects of chemically synthesized α-galactosylceramides (α-GalCers), α-glucosylceramides (α-GluCers), 6″-monoglycosylated α-GalCer and 6″- or 4″-monoglycosylated α-GluCer and made the following observations: (1) the length of the fatty acid side chain in the ceramide portions greatly affects the immunostimulatory effects of α-GalCers and α-GluCers; (2) the configuration of the 4″-hydroxyl group of the inner pyranose moiety plays an important role in the immunostimulatory effects of monoglycosylated α-
-pyranosylceramides; (3) the free 4″-hydroxyl group of the inner pyranose of monoglycosylated α-
-pyranosylceramides plays a more important role in their immunostimulatory effects than the free 6″-hydroxyl group. 相似文献
953.
954.
EB病毒BNLF-1基因的分子生物学研究进展 总被引:2,自引:0,他引:2
位于EB病毒基因组U5-TR区内的BNLF-1基因,其转译产物为潜伏膜蛋白(latent membrane protein1, LMP-1),由于LMP-1可以导致细胞转化并在EB病毒致癌过程中具有重要作用,因而成为近年来EB病毒分子生物学及相关肿瘤如人鼻咽癌、伯基特淋巴瘤、何杰金氏病等疾病病因发病学研究的热点,并取得了一批有重要意义的成果,文章从BNLF-1的基因结构及表达调控, LMP蛋白的结构及生化功能, LMP-1的生物学功能和LMP-1研究进行评述. 相似文献
955.
Kenji Hara Henneke Pangkey Kiyoshi Osatomi Keiko Yatsuda Atsushi Hagiwara Katsuyasu Tachibana Tadashi Ishihara 《Hydrobiologia》1997,358(1-3):89-94
We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl2 and MnCl_2. 相似文献
956.
H.C. Chan S.H. Law P.S. Leung L.X.M. Fu P.Y.D. Wong 《The Journal of membrane biology》1997,156(3):241-249
The β-adrenergic (cAMP-dependent) regulation of Cl− conductance is defective in cystic fibrosis (CF). The present study explored alternative regulation of anion secretion in
CF pancreatic ductal cells (CFPAC-1) by angiotensin II (AII) using the short-circuit current (I
SC
) technique. An increase in I
SC
could be induced in CFPAC-1 cells by basolateral or apical application of AII in a concentration-dependent manner (EC50 at 3 μm and 100 nm, respectively). Angiotensin receptor subtypes were identified using specific antagonists, losartan and PD123177, for AT1 and AT2 receptors, respectively. It was found that losartan (1 μm) could completely inhibit the AII-induced I
SC
, whereas, PD123177 exerted insignificant effect on the I
SC
, indicating predominant involvement of AT1 receptors. The presence of AT1 receptors in CFPAC-1 cells was also demonstrated by immunohistochemical studies using specific antibodies against AT1 receptors. Confocal microscopic study demonstrated a rise in intracellular Ca2+ upon stimulation by AII indicating a role of intracellular Ca2+ in mediating the AII response. Depletion of intracellular but not extracellular pool of Ca2+ diminished the AII-induced I
SC
. Treatment of the monolayers with a Cl− channel blocker, DIDS, markedly reduced the I
SC
, indicating that a large portion of the AII-activated I
SC
was Cl−-dependent. AII-induced I
SC
was also observed in monolayers whose basolateral membranes had been permeabilized by nystatin, suggesting that the I
SC
was mediated by apical Cl− channels. Our study indicates an AT1-mediated Ca2+-dependent regulatory mechanism for anion secretion in CF pancreatic duct cells which may be important for the physiology
and pathophysiology of the pancreas.
Received: 17 June 1996/Revised: 14 November 1996 相似文献
957.
We here report on studies on the frog skin epithelium to identify the nature of its excretory H+ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry
using V-ATPase-directed antibodies. Bafilomycin A1 (10 μm) blocked H+ excretion (69 ± 8% inhibition) and therefore Na+ absorption (61 ± 17% inhibition after 60 min application, n= 6) in open-circuited skins bathed on their apical side with a 1 mm Na2SO4 solution, ``low-Na+ conditions' under which H+ and Na+ fluxes are coupled 1:1. The electrogenic outward H+ current measured in absence of Na+ transport (in the presence of 50 μm amiloride) was also blocked by 10 μm bafilomycin A1 or 5 μm concanamycin A. In contrast, no effects were found on the large and dominant Na+ transport (short-circuit current), which develops with apical solutions containing 115 mm Na+ (``high-Na+ conditions'), demonstrating a specific action on H+ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit
E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds
to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane
extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody
labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations
now deliver conclusive evidence that H+ excretion is mediated by a V-ATPase being the electrogenic H+ pump in frog skin.
Received: 21 May 1996/Revised: 24 December 1996 相似文献
958.
Ayako Yamamoto Tetsuo Hashimoto Emiko Asaga Masami Hasegawa Nobuichi Goto 《Journal of molecular evolution》1997,44(1):98-105
Major parts of amino-acid-coding regions of elongation factor (EF)-1α and EF-2 in Trichomonas tenax were amplified by PCR from total genomic DNA and the products were cloned into a plasmid vector, pGEM-T. The three clones
from each of the products of the EF-1α and EF-2 were isolated and sequenced. The insert DNAs of the clones containing EF-1α
coding regions were each 1,185 bp long with the same nucleotide sequence and contained 53.1% of G + C nucleotides. Those of
the clones containing EF-2 coding regions had two different sequences; one was 2,283 bp long and the other was 2,286 bp long,
and their G + C contents were 52.5 and 52.9%, respectively. The copy numbers of the EF-1α and EF-2 gene per chromosome were
estimated as four and two, respectively.
The deduced amino acid sequences obtained by the conceptual translation were 395 residues from EF-1α and 761 and 762 residues
from the EF-2s. The sequences were aligned with the other eukaryotic and archaebacterial EF-1αs and EF-2s, respectively.
The phylogenetic position of T. tenax was inferred by the maximum likelihood (ML) method using the EF-1α and EF-2 data sets. The EF-1α analysis suggested that
three mitochondrion-lacking protozoa, Glugea plecoglossi, Giardia lamblia, and T. tenax, respectively, diverge in this order in the very early phase of eukaryotic evolution. The EF-2 analysis also supported the
divergence of T. tenax to be immediately next to G. lamblia.
Received: 15 February 1996 / Accepted: 28 June 1996 相似文献
959.
W. H. H. Krueger G. E. Gonye D. L. Madison K. E. Murray M. Kumar N. Spoerel S. E. Pfeiffer 《Journal of neurochemistry》1997,69(4):1343-1355
Abstract: Although the myelin membrane contains only a small set of major proteins, more sensitive assays indicate the presence of a plethora of uncharacterized proteins. We have used an antibody perturbation approach to reversibly block the differentiation of prooligodendroblasts into myelinating cells, and, in combination with a differential screening procedure, identified novel mRNAs that are activated during this period. One cDNA, TPO1, recognizes a 5.5-kb mRNA that is strongly up-regulated in oligodendrocytes after release of the differentiation block and that is expressed at high levels in brain tissue during active myelination. This cDNA represents at least two mRNAs differing from each other in their 5'-termini. The TPO1 cDNA contains an open reading frame of 1,380 bp, encoding a protein of 51.8 kDa with a predicted pl of 9.1 that contains two regions homologous to nonclassic zinc finger motifs. Subcellular localization studies suggest the enriched presence of TPO1 in spherical structures along the major cytoplasmic processes of oligodendrocytes. TPO1, along with homologues expressed in testis, placenta, and PC12 cells, form a novel family of proteins with multiple hydrophobic domains possibly serving as membrane spanning regions. We postulate that in oligodendrocytes, TPO1 encodes a protein factor involved in myelin biogenesis. 相似文献
960.
Jörg B. Schulz Dirk Bremen John C. Reed Jürgen Lommatzsch Shinichi Takayama Ullrich Wüllner Peter-A. Löschmann Thomas Klockgether Michael Weller 《Journal of neurochemistry》1997,69(5):2075-2086
Abstract: Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N -acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert -butyl-α-phenylnitrone, and the antioxidant, N -acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone. 相似文献