共查询到20条相似文献,搜索用时 15 毫秒
1.
Heparinase I from Flavobacterium heparinum has important uses for elucidating the complex sequence heterogeneity of heparin-like glycosaminoglycans (HLGAGs). Understanding the biological function of HLGAGs has been impaired by the limited methods for analysis of pure or mixed oligosaccharide fragments. Here, we use methodologies involving MS and capillary electrophoresis to investigate the sequence of events during heparinase I depolymerization of HLGAGs. In an initial step, heparinase I preferentially cleaves exolytically at the nonreducing terminal linkage of the HLGAG chain, although it also cleaves internal linkages at a detectable rate. In a second step, heparinase I has a strong preference for cleaving the same substrate molecule processively, i.e., to cleave the next site toward the reducing end of the HLGAG chain. Computer simulation showed that the experimental results presented here from analysis of oligosaccharide degradation were consistent with literature data for degradation of polymeric HLGAG by heparinase I. This study presents direct evidence for a predominantly exolytic and processive mechanism of depolymerization of HLGAG by heparinase I. 相似文献
2.
AJ Rhomberg Z Shriver K Biemann R Sasisekharan 《Canadian Metallurgical Quarterly》1998,95(21):12232-12237
Heparin-like glycosaminoglycans, acidic complex polysaccharides present on cell surfaces and in the extracellular matrix, regulate important physiological processes such as anticoagulation and angiogenesis. Heparin-like glycosaminoglycan degrading enzymes or heparinases are powerful tools that have enabled the elucidation of important biological properties of heparin-like glycosaminoglycans in vitro and in vivo. With an overall goal of developing an approach to sequence heparin-like glycosaminoglycans using the heparinases, we recently have elaborated a mass spectrometry methodology to elucidate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase I. In this study, we investigate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase II, which possesses the broadest known substrate specificity of the heparinases. We show here that heparinase II cleaves heparin-like glycosaminoglycans endolytically in a nonrandom manner. In addition, we show that heparinase II has two distinct active sites and provide evidence that one of the active sites is heparinase I-like, cleaving at hexosamine-sulfated iduronate linkages, whereas the other is presumably heparinase III-like, cleaving at hexosamine-glucuronate linkages. Elucidation of the mechanism of depolymerization of heparin-like glycosaminoglycans by the heparinases and mutant heparinases could pave the way to the development of much needed methods to sequence heparin-like glycosaminoglycans. 相似文献
3.
We retrospectively reviewed 11 patients with culture-proven Acanthamoeba keratitis who presented at the National Taiwan University Hospital between 1989 and 1996. We assessed predisposing factors, initial diagnosis, clinical presentation, treatment, and outcome. A history of contact lens-wear, poor contact lens hygiene, intractable eye pain, and ring infiltrates in the cornea were the most prominent characteristics and clinical manifestations. Acanthamoeba keratitis was often misdiagnosed, with herpetic keratitis (7/11) being the most common initial diagnosis from referring hospitals. These patients were usually treated on the basis of the inaccurate diagnosis for more than 1 month (range 1-8 mo) before referral. All patients ultimately received penetrating keratoplasty because of poor response to delayed medical treatment. We suggest that inadequate contact lens hygiene may be important in Acanthamoeba keratitis. This condition is often misdiagnosed and, as early diagnosis is a major factor for successful medical treatment in such patients, awareness in clinical practice is critical. 相似文献
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M Jücker K McKenna AJ da Silva CE Rudd RA Feldman 《Canadian Metallurgical Quarterly》1997,272(4):2104-2109
The c-fps/fes proto-oncogene encodes a 92-kDa protein-tyrosine kinase that is expressed at high levels in macrophages. We have previously shown that overexpression of c-fps/fes in a CSF-1-dependent macrophage cell line (BAC1.2F5) partially released these cells from their factor dependence and that this correlated with the tyrosine phosphorylation of a subset of proteins in a tissue-specific manner. We have now identified one of the macrophage substrates of Fes as the crk-associated substrate (Cas) and a second substrate as a 130-kDa protein that has been previously described as a T cell activation-dependent substrate and is unrelated to Cas. Both of these proteins, which have optimal consensus sequences for phosphorylation by Fes, were tightly associated with this kinase through its SH2 domain, suggesting that they were direct substrates of Fes. Remarkably, when the Fes SH2 domain was used as an affinity reagent to identify potential substrates of endogenous Fes in control BAC1.2F5 cells, the phosphotyrosyl proteins that were recognized were the same as those that were specifically phosphorylated when Fes was overexpressed in the same cells. We conclude that the substrates we identified may be structurally related or identical to the physiological targets of this kinase in macrophages. The known functions of Cas and p130 suggest that Fes kinase may play a role in signaling triggered by cell adhesion and cell-cell interactions during immune responses of macrophages. 相似文献
6.
Some time ago, it was found that attachment of hydrophilic polyoxyethylene chains to various hydrophobic phenols and alcohols gave water-soluble products which, although inactive in vitro, influenced and experimental tuberculous infection. With short chains the infection was suppressed, and with long chains it was promoted. Later work concentrated on Macrocyclon (short chain) and HOC-60 (long chain), both derived from a hydrophobic, polyphenolic calixarene. Growth of Mycobacterium tuberculosis inside macrophages (M phi) was inhibited by Macrocyclon and stimulated by HOC-60. Also, triglyceride lipase from M phi extracts and an extracellular phospholipase were inhibited by Macrocyclon and stimulated by HOC-60. This suggestion of a mechanism has been strengthened by the finding that M phi cultivated in monolayers and treated with Macrocyclon showed accumulation of lipid and little formation of fatty acid after incubation of killed cells. With HOC-60, lipid was depleted and much fatty acid was found. 相似文献
7.
Wingless (Wg) treatment of Drosophila wing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in vitro assays for Wg protein. Previously analyzed stocks of Drosophila Schneider S2 cells could not respond to added Wg, because they lack the Wg receptor, Dfrizzled-2. However, we found that a line of S2 cells obtained from another source express Dfrizzled-2 and Dfrizzled-1. Thus, we designated this cell line as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and DE-cadherin protein levels and by hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induced the same changes in Dsh, Arm, and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells was phosphorylated as a consequence of expression of Dfrizzled-2 or mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as sensitive to Wg as are clone 8 cells but can grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of Wg signaling. 相似文献
8.
KM Bhat 《Canadian Metallurgical Quarterly》1998,95(7):1027-1036
The Drosophila Frizzled (Fz) and Frizzled2 (DFz2) proteins function as receptors for Wingless (Wg) in tissue culture cells. While previous results indicate that loss of function for fz has tissue polarity defects, loss-of-function effects of Dfz2 are not known. Here, we have examined the requirements of fz and Dfz2 during neurogenesis. Our results indicate that both Fz and DFz2 function in Wg signaling, and loss of either of the two affects the same subset of neuroblasts as those affected by loss of wg. While these defects are partially penetrant in embryos lacking either fz or Dfz2, the penetrance is significantly enhanced in embryos lacking both. Since the penetrance of the CNS phenotypes is not complete in double mutants, additional components that allow some degree of Wg signaling must exist in vivo. 相似文献
9.
D McHugh 《Canadian Metallurgical Quarterly》1997,94(15):8006-8009
The Annelida, which includes the polychaetes and the clitellates, has long held the taxonomic rank of phylum. The unsegmented, mud-dwelling echiuran spoon worms and the gutless, deep-sea pogonophoran tube worms (including vestimentiferans) share several embryological and morphological features with annelids, but each group also has been considered as a separate metazoan phylum based on the unique characters each group displays. Phylogenetic analyses of DNA sequences from the nuclear gene elongation factor-1alpha place echiurans and pogonophorans within the Annelida. This result, indicating the derived loss of segmentation in echiurans, has profound implications for our understanding of the evolution of metazoan body plans and challenges the traditional view of the phylum-level diversity and evolutionary relationships of protostome worms. 相似文献
10.
Mouse embryos homozygous for the Gpi1-sa-m1H null allele (abbreviated to m) of glucose phosphate isomerase (GPI) die early in development. A histological study was undertaken to identify the earliest abnormalities attributable to the absence of this glycolytic enzyme. Two groups of embryos were produced and examined histologically from E6.5 to E9.5 days. Experimental embryos were produced by crossing heterozygous Gpi1-sa/m females with heterozygous Gpi1-sb/m males and compared with control embryos produced by crossing heterozygous Gpi1-sb/Gpi1-sb males. The first sign of abnormality attributable to homozygous m/m embryos appeared at 7.5 days when 32.2% of the embryos in the experimental group were histologically abnormal or retarded, compared to 8.3% in the control group. The putative homozygous m/m embryos had a range of abnormalities, but consistently the egg cylinder failed to be divided into the three cavities characteristic of normal 7.5-day embryos. This suggests that a deficiency in extraembryonic mesoderm formation resulted in the failure to form the amnion or chorionic mesoderm. At 8.5 and 9.5 days the abnormal embryos from the experimental cross had progressed little further. It is suggested that in the absence of GPI, energy production is impaired so that the embryo fails to develop beyond the egg cylinder stage and gastrulation has begun completed. Developmental failure may occur before gastrulation or once gastrulation has begun and produced some mesoderm. It is concluded that glucose phosphate isomerase and presumably, therefore, glycolysis is needed for normal gastrulation of mouse embryos. 相似文献
11.
GO Cory L MacCarthy-Morrogh S Banin I Gout PM Brickell RJ Levinsky C Kinnon RC Lovering 《Canadian Metallurgical Quarterly》1996,157(9):3791-3795
Wiskott-Aldrich syndrome is an X-linked combined immunodeficiency affecting cells of several different hemopoietic lineages. The Wiskott-Aldrich syndrome protein (WASP), which has no homology with any other known protein families, is rich in proline motifs known to contribute to Src homology 3 binding sites. However, its function has not been determined. The Tec family of cytoplasmic tyrosine kinases, which include Btk (the X-linked agammaglobulinemia gene), Itk, and Tec, is thought to be involved in lymphoid cell signaling pathways. In this work, we show binding of WASP to the Src homology 3 domains of Btk, Itk, Tec, Grb2, and phospholipase C-gamma, which suggests a function for WASP in lymphoid cell signaling. 相似文献
12.
The expression of poliovirus 2BC protein in yeast and mammalian cells leads to a number of metabolic and morphological alterations, such as growth inhibition, intracellular membrane proliferation, blockade of the exocytic pathway, and enhanced membrane permeability. Yeast cells that express poliovirus 2BC in an inducible manner were used to identify the regions of 2BC implicated in the modifications of these cellular functions. Several 2BC deletion mutants were generated to define the minimal portion of 2BC required to alter these activities. Additional deletion mutants that were obtained by random mutagenesis followed by selection in yeast cells provided new insights into the structure and mechanism of action of 2BC. The activity responsible for membrane proliferation is located in 2C, while the activities responsible for membrane permeabilization and inhibition of the exocytic pathway are located in 2B. Several regions of 2B and 2C required for the different functions of 2BC were identified. Thus, the integrity of the N termini of both 2B and 2C is necessary for 2BC-induced cytotoxicity. It is also possible to separate the different cellular alterations provoked by 2BC by the use of several 2BC variants. Deletion of amino acids 52 to 65 in 2B generates a 2BC deletion variant, 2bC deltaAvrII, that still blocks yeast growth but is unable to enhance membrane permeability or to inhibit the exocytic pathway. On the other hand, 2Bcl28*.32b and 2Bcl28*.3c, which contain only 73 and 77 amino acids of 2B, interfere with yeast division and enhance membrane permeability but affect the exocytic pathway only weakly and do not induce membrane proliferation. Our findings indicate that Saccharomyces cerevisiae represents a useful model system to analyze the functions of poliovirus 2BC and show the feasibility of separating the activities assigned to this protein. 相似文献
13.
H Fukaki J Wysocka-Diller T Kato H Fujisawa PN Benfey M Tasaka 《Canadian Metallurgical Quarterly》1998,14(4):425-430
The hepatic clearance of ONO-5046 (N-[2-[4-(2,2-dimethylpropionyloxy)phenylsulphonylamino]benz oyl]aminoacetic acid), a low-molecular-weight neutrophil elastase inhibitor, has been investigated in rats and in the rat perfused liver. This ester was easily hydrolysed to its inactive metabolite EI-601 (N-[2-[(4-hydroxyphenyl)sulphonylamino]benzoyl]aminoacetic acid) in liver homogenate and in erythrocytes suspension in-vitro. On the other hand, it was stable in biological media such as plasma and whole blood, which contain plasma proteins. Scatchard plot analysis of ONO-5046 binding to bovine serum albumin (BSA) in-vitro indicated that the association constant (K) and number of binding sites (n) were 6.91 x 10(4) (M(-1)) and 4.33, respectively. Thus, ONO-5046 (100 microM) would bind to plasma proteins to an extent >99% at physiological plasma-protein concentrations. The total plasma clearance of ONO-5046 in rats was constant (approximately 9 mL min(-1) kg(-1)) under different steady-state plasma concentrations (5-50 microM) a value equivalent to the hepatic clearance. In the rat perfused liver, the hepatic extraction ratio of ONO-5046 was significantly reduced by adding BSA to the dosing solution. Thus, the relatively low hepatic clearance of ONO-5046, which has an ester linkage in its structure and is naturally susceptible to enzymatic hydrolysis, was found to be because of the extremely high protein-binding of the compound. 相似文献
14.
MS Moore J DeZazzo AY Luk T Tully CM Singh U Heberlein 《Canadian Metallurgical Quarterly》1998,93(6):997-1007
Upon exposure to ethanol, Drosophila display behaviors that are similar to ethanol intoxication in rodents and humans. Using an inebriometer to measure ethanol-induced loss of postural control, we identified cheapdate, a mutant with enhanced sensitivity to ethanol. Genetic and molecular analyses revealed that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, we find that enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol. Taken together, our results provide functional evidence for the involvement of the cAMP signal transduction pathway in the behavioral response to intoxicating levels of ethanol. 相似文献
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16.
ADP-ribosyl cyclase catalyzes the synthesis of two structurally and functionally different Ca2+ releasing molecules, cyclic ADP-ribose (cADPR) from beta-NAD and nicotinic acid-adenine dinucleotide phosphate (NAADP) from beta-NADP. Their Ca2+-mobilizing effects in ascidian oocytes were characterized in connection with that induced by inositol 1,4,5-trisphosphate (InsP3). Fertilization of the oocyte is accompanied by a decrease in the oocyte Ca2+ current and an increase in membrane capacitance due to the addition of membrane to the cell surface. Both of these electrical changes could be induced by perfusion, through a patch pipette, of nanomolar concentrations of cADPR or its precursor, beta-NAD, into unfertilized oocytes. The changes induced by beta-NAD showed a distinctive delay consistent with its enzymatic conversion to cADPR. The cADPR-induced changes were inhibited by preloading the oocytes with a Ca2+ chelator, indicating the effects were due to Ca2+ release induced by cADPR. Consistently, ryanodine (at high concentration) or 8-amino-cADPR, a specific antagonist of cADPR, but not heparin, inhibited the cADPR-induced changes. Both inhibitors likewise blocked the membrane insertion that normally occurred at fertilization consistent with it being mediated by a ryanodine receptor. The effects of NAADP were different from those of cADPR. Although NAADP induced a similar decrease in the Ca2+ current, no membrane insertion occurred. Moreover, pretreatment of the oocytes with NAADP inhibited the post-fertilization Ca2+ oscillation while cADPR did not. A similar Ca2+ oscillation could be artificially induced by perfusing into the oocytes a high concentration of InsP3 and NAADP could likewise inhibit such an InsP3-induced oscillation. This work shows that three independent Ca2+ signaling pathways are present in the oocytes and that each is involved in mediating distinct changes associated with fertilization. The results are consistent with a hierarchical organization of Ca2+ stores in the oocyte. 相似文献
17.
Limited proteolysis of the Mn-stabilizing protein (MSP) from the thermophilic cyanobacterium Synechococcus elongatus with chymotrypsin, trypsin or lysylendopeptidase that yielded four major polypeptides of 26 kDa, 22 kDa, 15 kDa and 11 kDa on denaturing gel electrophoresis resulted in total loss of the binding capacity of the protein to PSII complexes. Analyses of electrophoretic patterns and amino-terminal sequences of the proteolytic products revealed that the three proteases specifically cleaved the protein at a site between Phe156 and Gly163 or between Arg184 and Ser191. Site-directed mutagenesis was used to construct two mutant MSPs that had a nick between Phe156 and Leu157, a chymotrypsin-cleavage site, and Met before Leu157 or in place of Leu157. The two mutant proteins failed to bind to PSII complexes, although they largely retained ordered secondary structure and comigrated with the wild-type proteins in non-denaturing gel electrophoresis. The loss of the protein binding can be ascribed to introduction of a nick because a mutant protein that had Met in place of Leu157 but no nick was able to specifically bind to the functional site of PSII complexes and restore the oxygen-evolving activity as effectively as the wild-type protein. In contrast, a mutant MSP with Met inserted between Phe156 and Leu157 bound only weakly and non-specifically to PSII complexes and failed to reactivate oxygen evolution. Thus, the binding of the protein to the functional site of the PSII complex was highly sensitive to a small structural change that was caused by cleavage or insertion of a single amino acid residue between Phe156 and Leu157. The results suggest that the Phe156-Gly163 and Arg184-Ser191 sequences of the cyanobacterial MSP are regions for interaction with PSII complexes. 相似文献
18.
To investigate the role of cyclic adenosine monophosphate (cAMP) in penile erection in relation to the effect of prostaglandin E1 (PGE1) male adult New Zealand white rabbits were utilized as a model to study intracavernous pressure (ICP) in vivo. After intracavernous injection of PGE1 (0.2-1.6 micrograms/kg) and 8-bromocyclic adenosine monophosphate (8-Br-cAMP, 0.5-1.5 mg/kg), both drugs raised the ICP in a dose-dependent manner. The increased ICPs induced by PGE1 and 8-Br-cAMP were 33.4 +/- 8.12 and 24.1 +/- 4.9 mm Hg, respectively (p < 0.05, paired Student's t test). Administrations of cyclic adenosine monophosphothioate, Rp-isomer (cAMP antagonist, 0.02-0.08 mumol/kg) prior to PGE1 injections inhibited the effect of PGE1 in vivo in a dose-dependent manner. The systemic blood pressures and heart rates in rabbits were unchanged during all the intracavernous injections. The corpus cavernosal tissues isolated from rabbits were studied for the cAMP contents after incubation of different doses of PGE1 in vitro. The cAMP contents were also elevated in a manner parallel with the increases in PGE1 concentrations (3-9 microM). We conclude: (1) the feasibility of intracavernous injection of vasoactive drugs is similar to that in man, thus the rabbit can be used as a suitable alternative for the studies of penile erection, and (2) cAMP is mediated in PGE1-induced relaxation of the rabbit corpus cavernosum, and the cAMP system only participates partially in penile erection. 相似文献
19.
Ventral structures in the central nervous system are patterned by signals emanating from the underlying mesoderm as well as originating within the neuroectoderm. Mutations in the zebrafish, Danio rerio, are proving instrumental in dissecting these midline signals. The cyclops mutation leads to a loss of medial floor plate and to severe deficits in ventral forebrain development, leading to cyclopia. Here, we report that the cyclops locus encodes the nodal-related protein Ndr2, a member of the transforming growth factor type beta superfamily of factors. The evidence includes identification of a missense mutation in the initiation codon and rescue of the cyclops phenotype by expression of ndr2 RNA, here renamed "cyclops." Thus, in interaction with other molecules implicated in these processes such as sonic hedgehog and one-eyed-pinhead, cyclops is required for ventral midline patterning of the embryonic central nervous system. 相似文献
20.
Cell surface glycosaminoglycans are not obligatory for Plasmodium berghei sporozoite invasion in vitro 总被引:1,自引:0,他引:1
The malaria circumsporozoite (CS) protein binds to glycosaminoglycan chains from heparan sulfate proteoglycans present on the basolateral surface of hepatocytes and hepatoma cells in vitro. When injected into mice, CS protein is rapidly cleared from the blood circulation by hepatocytes. The binding region for the HSPGs is the evolutionarily conserved region II-plus of the CS protein. Here we have asked whether the presence of glycosaminoglycans on the plasma membrane of target cells is required for sporozoite invasion in vitro. Two types of target cells were used: HepG2 cells, which are permissive for Plasmodium berghei sporozoite development into mature exoerythrocytic forms, and CHO cells, in which the intracellular development of the parasites is arrested early after penetration. The invasion of mutant CHO cells expressing undersulfated glycosaminoglycans or no glycosaminoglycans was only inhibited 41-49% or 24-32%, respectively, in comparison to invasion of CHO-K1 cells. Previous cleavage of HepG2 surface membrane glycosaminoglycans with heparinase or heparitinase had no significant inhibitory effect on subsequent P. berghei sporozoite invasion and EEF development in these cells, although the glycosaminoglycan lyase treatments removed over 80% of CS binding sites from the cell surface. These results suggest that although the presence of glycosaminoglycans on the target cell surface enhances sporozoite invasion, glycosaminoglycans are not required for sporozoite penetration or the development of exoerythrocytic forms in vitro. 相似文献