The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase.

Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55PIK, a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identify to those in p85. p55PIK mRNA is expressed early during development, remains abundant in adult mouse brain and testis tissue, and is detectable in adult adipocytes and heart and kidney tissues. p55PIK forms a stable complex with p110, and it associates with IRS-1 during insulin stimulation. Moreover, the activated insulin receptor phosphorylates p55PIK in Sf9 cells, and insulin stimulates p55PIK phosphorylation in CHOIR/p55PIK cells. The unique features of p55PIK suggest that it is important in receptor signaling.     

Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution

The chemical shift assignments and secondary structure of a murine–human chimera,MH35, of leukaemia inhibitory factor (LIF), a 180-residue protein of molecular mass 20 kDa,have been determined from multidimensional heteronuclear NMR spectra acquired on auniformly 13C,15N-labelled sample. Secondary structure elements were defined on the basisof chemical shifts, NH-CH coupling constants, medium-range NOEs and the location ofslowly exchanging amide protons. The protein contains four -helices, the relativeorientations of which were determined on the basis of long-range, interhelical NOEs. The fourhelices are arranged in an up-up-down-down orientation, as found in other four-helical bundlecytokines. The overall topology of MH35-LIF is similar to that of the X-ray crystallographicstructure for murine LIF [Robinson et al. (1994) Cell, 77, 1101–1116]. Differencesbetween the X-ray structure and the solution structure are evident in the N-terminal tail, wherethe solution structure has a trans-Pro17 compared with the cis-Pro17 found in the crystalstructure and the small antiparallel -sheet encompassing residues in the N-terminus andCD loop in the crystal structure is less stable.     

Rapid and Sensitive Detection of Xanthomonas fragariae by Simple Alkaline DNA Extraction and the Polymerase Chain Reaction

Methods for DNA preparation from Xanthomonas fragariae in infected or artificially contaminated strawberry plants were compared in diagnostic assays using the polymerase chain reaction (PCR). The bacterium was detected using PCR with primers specific to a region of its hrp gene. Sensitivity of detection was 1.25 ×l 10³ CFU ml^-1 using DNA from bacterial suspensions prepared by an alkali extraction method. This was 10-fold more sensitive than DNA extraction by boiling, and was equal to that in which DNA was prepared by a more involved cetyltrimethylammonium bromide (CTAB) procedure. Sensitivity of detection from artificially contaminated strawberry tissues was 10-fold less than that from cell suspensions. The results indicated that a rapid and simple method of alkali DNA sample preparation is applicable for the sensitive and reliable detection of X. fragariae and possibly other plant pathogenic bacteria.     

Cosmid-derived markers anchoring the bovine genetic map to the physical map

The mapping strategy for the bovine genome described in this paper uses large insert clones as a tool for physical mapping and as a source of highly polymorphic microsatellites for genetic typing, and was one objective of the BovMap Project funded by the European Union (UE). Eight-three cosmid and phage clones were characterized and used to physically anchor the linkage groups defining all the bovine autosomes and the X Chromosome (Chr). By combining physical and genetic mapping, clones described in this paper have led to the identification of the linkage groups corresponding to Chr 9, 12, 16, and 25. In addition, anchored loci from this study were used to orient the linkage groups corresponding to Chr 3, 7, 8, 9, 13, 16, 18, 19, and 28 as identified in previously published maps. Comparison of the estimated size of the physical and linkage maps suggests that the genetic length of the bovine genome may be around 4000 cM. Received: 1 July 1996 / Accepted: 13 September 1996     

A novel monoantennary complex-type sugar chain found in octopus rhodopsin: occurrence of the Gal{beta}1->4Fuc group linked to the proximal N-acetylglucosaniine residue of the trimannosyl core

The N-linked sugar chains were liberated as oligosaccha-ridesfrom octopus rhodopsin by hydrazinolysis. Most of the oligosaccharideswere neutral, and separated into two major components by columnchromatography using immobilized lectins and Bio-Gel P-4. Structuralanalysis of the one major component by sequential exoglycosidasedigestion, chemical fragmentation in combination with meth-ylationanalysis revealed that it is a nonasaccharide; Man16(Gaiβ13GlcNAcβ12Man13)Manβ14GlcNAcβ14(Galβ14Fuc16)GlcNAcThis structure is quite unique in that a novel galactosylatedfucose residue is attached to the reducing terminal N-acetyl-glucosamineresidue. galactosylated Fuc N-linked sugar chain novel structure octopus rhodopsin     

Human recombinant [C22A] FK506-binding protein amide hydrogen exchange rates from mass spectrometry match and extend those from NMR.

Hydrogen/deuterium exchange behavior of human recombinant [C22A] FK506 binding protein (C22A FKBP) has been determined by protein fragmentation, combined with electrospray Fourier transform ion cyclotron resonance mass spectrometry (MS). After a specified period of H/D exchange in solution, C22A FKBP was digested by pepsin under slow exchange conditions (pH 2.4, 0 degree C), and then subjected to on-line HPLC/MS for deuterium analysis of each proteolytic peptide. The hydrogen exchange rate of each individual amide hydrogen was then determined independently by heteronuclear two-dimensional NMR on 15N-enriched C22A FKBP. A maximum entropy method (MEM) algorithm makes it possible to derive the distributions of hydrogen exchange rate constants from the MS-determined deuterium exchange-in curves in either the holoprotein or its proteolytic segments. The MEM-derived rate constant distributions of C22A FKBP and different segments of C22A FKBP are compared to the rate constants determined by NMR for individual amide protons. The rate constant distributions determined by both methods are consistent and complementary, thereby validating protein fragmentation/mass spectrometry as a reliable measure of hydrogen exchange in proteins.     

原生质体电融合酵母多倍体生理特性的研究

对原生质体电融合技术获得的11株酵母多倍体融合株,进行了一系列生理特性的分析比较．结果发现,在电融合过程中,融合株的细胞体积及DNA含量并不随着核倍性呈线性递增．而是有其特殊的变化规律。当糖化酵母sta1、sta2和sta3在细胞核中各自纯合时,明显表现出表达的剂量效应。其中sta1、sta2纯合的融合株．在YEPS培养中,GA分泌可被淀粉大量诱导,表现出一定的二次生长特性。从分析结果推测,在亲株5301-14D及HU-TY—1A中可能有对GA分泌不利的因素。     

Mast cell tryptase and asthma

Recent physiological and pharmacological studies have indicated the potential importance of tryptase, the major protein component in mast cells, in inflammatory diseases (especially asthma). Being released at inflammatory sites after the activation of mast cells, tryptase is capable of causing bronchohyperresponsiveness and infiltration of eosinophils, neutrophils, etc. in animal airways. The mechanisms by which tryptase causes bronchoconstriction involve probably the potentiation of other chemical mediators such as histamine, production of bradykinin via the hydrolysis of kininogen, and cleavage of the bronchodilating peptides VIP (vasoactive intestinal peptide) and PHM (peptide histidine-methionine). Tryptase has also been found to be a potent mitogen in vitro for airway smooth muscle cells and epithelial cells, implying its role in the hyperplasia of the asthmatic airways. The experimental data providing evidence for the above roles of tryptase are summarized in the present review, as well as the effects of tryptase inhibition in animal asthma models. The potential strategies for the development of anti-asthmatic agents based on the inhibition of tryptase are discussed.