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81.
A soluble ATP-dependent enzyme which phosphorylates myo-inositol has been characterized in Dictyostelium. The myo-inositol kinase activity was partially purified from amoebae by chromatography on DEAE-Sepharose and phenyl-Sepharose columns. The product of both the partially purified activity and of a crude cytosolic fraction was myo-inositol 3-phosphate. The partially purified preparations of myo-inositol kinase (a) possessed a Km for myo-inositol of 120 microM (in the presence of 5 mM-ATP) and for ATP of 125 microM (in the presence of 1 microM-myo-inositol), (b) did not recognize allo-, epi-, muco-, neo-, scyllo-, 1 D-chiro or 1 L-chiro-inositol as substrates, (c) were competitively inhibited by three naturally occurring analogues of myo-inositol: 1 L-chiro-inositol (Ki 49.5 +/- 0.7 microM: the structural equivalent of myo-inositol, except that the D-3 hydroxy moiety is axial), D-3-deoxy-myo-inositol [Ki 103 +/- 1 microM: (-)-viburnitol], and sequoyitol (Ki 271 +/- 7 microM; unlike 1 L-chiro-inositol and D-3-deoxy-myo-inositol, this was a substrate for the kinase), and finally (d) were apparently non-competitively inhibited by myo-inositol 3-phosphate. The product of myo-inositol kinase could be detected in intact amoebae and was a substrate for the first in a series of inositol polyphosphate kinases present in Dictyostelium which ultimately yield myo-inositol hexakisphosphate. The activity of myo-inositol D-3-hydroxykinase in Dictyostelium lysates showed evidence of developmental regulation. 相似文献
82.
Spectroscopic, thermodynamic and kinetic properties of Candida nitratophila nitrate reductase.
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C J Kay M J Barber L P Solomonson D Kau A C Cannons C R Hipkin 《The Biochemical journal》1990,272(2):545-548
HL-60 cells possess a 60 kDa Ca2(+)-binding protein that is contained in a discrete subcellular compartment, referred to as calciosomes. Subcellular fractionation studies have suggested that, in HL-60 cells, this intracellular compartment is an Ins(1,4,5)P3-sensitive Ca2+ store. In order to investigate the structural relationship of the 60 kDa Ca2(+)-binding protein of HL-60 cells to other Ca2(+)-binding proteins, we have purified the protein by ammonium sulphate extraction, acid precipitation, and DEAE-cellulose and phenyl-Sepharose column chromatography. The N-terminal sequence of the protein shows 93% identity with rabbit muscle calreticulin, a recently cloned sarcoplasmic reticulum Ca2(+)-binding protein. No amino acid sequence similarity with calsequestrin was found, although the purified protein cross-reacted with anti-calsequestrin antibodies. The calreticulin-related protein of HL-60 cells might play a role as an intravesicular Ca2(+)-binding protein of an Ins(1,4,5)P3-sensitive Ca2+ store. 相似文献
83.
Kay Robinson-Beers Th. D. Sharkey R. F. Evert 《Plant biology (Stuttgart, Germany)》1990,103(4):424-429
Sink-to-source transition was studied in developing sugarcane (Saccharum interspecific variety L62–96) leaves. Fully-expanded, mature sugarcane leaves were fed 14CO2 for 20 minutes, incorporating about 617 Bq. After five hours the leaves of each plant were cut into 1-cm-length segments that were weighed and then placed in scintillation cocktail for counting. All leaves younger than the leaf fed 14CO2 imported labeled photoassimilate. Three to four leaves had both importing and non-importing regions within the blade and a distinct transition region between them. A transition region was observed in leaves which had expanded to between 30 and 90 % of final blade length. Radioactivity per gram fresh weight was calculated as a measure of sink strength. Sink strength was greatest in the youngest leaf and declined with leaf age. The results of this study indicate that 1) import of photosynthate by developing sugarcane leaves occurs over a longer span of developmental ages than in dicotyledonous leaves and 2) the actual tissue region undergoing transition within such a leaf can be resolved as narrow zone between the importing and non-importing regions. 相似文献
84.
Recombinant adenoviruses with large deletions generated by Cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo. 总被引:13,自引:6,他引:7
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In vivo gene transfer of recombinant E1-deficient adenoviruses results in early and late viral gene expression that elicits a host immune response, limiting the duration of transgene expression and the use of adenoviruses for gene therapy. The prokaryotic Cre-lox P recombination system was adapted to generate recombinant adenoviruses with extended deletions in the viral genome (referred to here as deleted viruses) in order to minimize expression of immunogenic and/or cytotoxic viral proteins. As an example, an adenovirus with a 25-kb deletion that lacked E1, E2, E3, and late gene expression with viral titers similar to those achieved with first-generation vectors and less than 0.5% contamination with E1-deficient virus was produced. Gene transfer was similar in HeLa cells, mouse hepatoma cells, and primary mouse hepatocytes in vitro and in vivo as determined by measuring reporter gene expression and DNA transfer. However, transgene expression and deleted viral DNA concentrations were not stable and declined to undetectable levels much more rapidly than those found for first-generation vectors. Intravenous administration of deleted vectors in mice resulted in no hepatocellular injury relative to that seen with first-generation vectors. The mechanism for stability of first-generation adenovirus vectors (E1a deleted) appeared to be linked in part to their ability to replicate in transduced cells in vivo and in vitro. Furthermore, the deleted vectors were stabilized in the presence of undeleted first-generation adenovirus vectors. These results have important consequences for the development of these and other nonintegrating vectors for gene therapy. 相似文献
85.
The prion protein gene is dispensable for the development of spongiform myeloencephalopathy induced by the neurovirulent Cas-Br-E murine leukemia virus.
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The Cas-Br-E murine leukemia virus (MuLV) induces paralysis in susceptible mice that is accompanied by a severe spongiform myeloencephalopathy. These neurodegenerative lesions are very similar to those observed in prion diseases. To determine whether the prion protein gene (Prn-p) product was a downstream effector of this neurovirulent MuLV, we inoculated Prn-p(-/-) knockout homozygote and control heterozygote or wild-type mice with this retrovirus. All groups developed typical paralysis and spongiform encephalopathy, and no differences in clinical or histological phenotypes were observed between these groups. These results indicate that the Cas-Br-E MuLV does not require the prion protein to induce lesions. Thus, MuLV and prion proteins may induce a very similar disease through distinct pathways, or the viral Env protein, which harbors the primary determinant of pathogenicity, may act in a common pathway but downstream of the prion protein. 相似文献
86.
Mark A. Pook Rekhaben Thakrar Bruce Pottinger Brian Harding David Porteous Veronica van Heningen John Cowell Carol Jones Sue Povey Kay E. Davies Rajesh V. Thakker 《Human genetics》1996,97(6):742-749
EagI and NotI linking libraries were prepared in the lambda vector, EMBL5, from the mouse-human somatic cell hybrid 1W1LA4.9, which contains
human chromosomes 11 and Xp as the only human component. Individual clones containing human DNA were isolated by their ability
to hybridise with total human DNA and digested with SalI and EcoRI to identify the human insert size and single-copy fragments. The mean (± SD) insert sizes of the EagI and NotI clones were 18.3 ± 3.2 kb and 16.6 ± 3.6 kb, respectively. Regional localisation of 66 clones (52 EagI, 14 NotI) was achieved using a panel of 20 somatic cell hybrids that contained different overlapping deletions of chromosomes 11
or Xp. Thirty-nine clones (36 EagI, 3 NotI) were localised to chromosome 11; 17 of these were clustered in 11q13 and another nine were clustered in 11q14–q23.1. Twenty-seven
clones (16 EagI, 11 NotI) were localised to Xp and 10 of these were clustered in Xp11. The 66 clones were assessed for seven different microsatellite
repetitive sequences; restriction fragment length polymorphisms for five clones from 11q13 were also identified. These EagI and NotI clones, which supplement those previously mapped to chromosome 11 and Xp, should facilitate the generation of more detailed
maps and the identification of genes that are associated with CpG-rich islands.
Received: 27 December 1995 / Revised: 30 January 1996 相似文献
87.
Christopher M. Hylton Kay Denyer Peter L. Keeling Ming-Tang Chang Alison M. Smith 《Planta》1996,198(2):230-237
The effects of waxy mutations on starch-granule-bound starch synthases (EC 2.4.1.18) in the developing endosperm of barley (Hordeum vulgare L.) and maize (Zea mays L.) have been investigated. Three granule-bound starch synthases in barley endosperm were identified by use of antibodies to known starch synthases, by reconstitution and assay of individual proteins from sodium dodecyl sulphate-polyacrylamide gels of granule-bound proteins, and by partial purification of proteins released by enzymic digestion of starch. These are proteins of 60, 77 and 90 kDa. Use of antibodies to known starch synthases and partial purification of proteins released by enzymic digestion of starch indicated that there may be at least four granule-bound starch synthases in maize endosperm: proteins of 59, 74, 77 and 83 kDa. Mutations at the waxy loci of both species affected only the 60- (barley) and 59-(maize) kDa isoforms. No evidence was found that other putative isoforms are altered in abundance or activity by the mutations. The contribution of our results to understanding of the starch synthase activity of intact starch granules and the mechanism of amylose synthesis is discussed.We are very grateful to Dr. Roger Ellis (SCRI, Dundee, Scotland) for the gift of barley seeds, and to Drs Roger Ellis, Alan Schulman and Cathie Martin for helpful advice and comments during the course of this work. 相似文献
88.
Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal 总被引:10,自引:9,他引:1
We have developed a new approach to the measurement of phylogenetic signal
in character state matrices called relative apparent synapomorphy analysis
(RASA). RASA provides a deterministic, statistical measure of natural
cladistic hierarchy (phylogenetic signal) in character state matrices. The
method works by determining whether a measure of the rate of increase of
cladistic similarity among pairs of taxa as a function of phenetic
similarity is greater than a null equiprobable rate of increase. Our
investigation of the utility and limitations of RASA using simulated and
bacteriophage T7 data sets indicates that the method has numerous
advantages over existing measures of signal. A first advantage is
computational efficiency. A second advantage is that RASA employs known
methods of statistical inference, providing measurable sensitivity and
power. The performance of RASA is examined under various conditions of
branching evolution as the number of characters, character states per
character, and mutations per branch length are varied. RASA appears to
provide an unbiased and reliable measure of phylogenetic signal, and the
general approach promises to be useful in the development of new techniques
that should increase the rigor and reliability of phylogenetic estimates.
相似文献
89.
90.
Michael E. Houston Cindy L. Gannon Cyril M. Kay Robert S. Hodges 《Journal of peptide science》1995,1(4):274-282
A series of 14 residue amphipathic α-helical peptides, in which the sidechains of glutamic acid and lysine have been covalently joined, was synthesized in order to determine the effect of spacing, position and orientation of these lactam bridges. It was found that although an (i, i+3) spacing would position the lactam bridge on the same face of the helix, these lactams with 18-member rings were actually helix-destabilizing regardless of position or location. On the other hand, (i, i+4) lactams with 21-member rings were helix-stabilizing but this was dependent on orientation. Glutamic acid-lysine lactams increased the helical content of the peptide when compared with their linear homologue in benign conditions (50 mM KH2PO4, 100 mM KCl, pH 7). Two Glu-Lys (i, i+4) lactams located at the N- and C-termini gave rise to a peptide with greater than 99% helical content in benign conditions. Peptides with Lys-Glu oriented lactams were random structures in benign conditions but in the presence of 50% TFE could be induced into a helical conformation. The stability of the single-stranded α-helices, as measured by thermal denaturations in 25% TFE indicated that Glu-Lys oriented lactam bridges stabilized the helical conformation relative to the linear unbridged peptide. One Glu-Lys lactam in the middle of the peptide was more effective at stabilizing helical structure than two Glu-Lys lactams positioned one at each end of the molecule. The lactams with the Lys-Glu orientation were destabilizing relative to the unbridged peptide. This study demonstrates that correct orientation and position of a lactam bridge is critical in order to design peptides with high helical content in aqueous media. 相似文献