Activated protein C resistance caused by Arg506Gln mutation in factor Va.
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Factor Va↗
Biomedical subjects
X Xu
Publications and source records attributed to X Xu.
One-disulfide intermediates of apamin exhibit native-like structure.
The conformations of three peptide models of the one-disulfide and fully reduced forms of apamin were characterized by using nuclear magnetic resonance (NMR) spectroscopy. Apa-2 contains the native disulfide bond between Cys3 and Cys15 in apamin with the other two cysteines replaced by alanines. Apa-1 contains the native disulfide bond between Cys1 and Cys11. Apa-S has all cysteines replaced with serines, mimicking fully reduced apamin. Comparing NOESY cross peaks and coupling constants for amide protons in the peptide analogs with those in native apamin indicates that a significant portion of Apa-2 possesses native-like structural elements of apamin in addition to some random coil conformations. Apa-1 contains a short helical structure from Ala9 to Arg13, corresponding to the N-terminal portion of the alpha-helix observed in the native structure, along with some local and probably flexible secondary structures corresponding to the reverse turn region in native apamin. A larger portion of Apa-1 exists in the form of random coil conformations compared to Apa-2. Apa-S displays mainly random coil conformations with some localized helical structures from Glu7 to Arg14 which are similar to the "nascent helices" proposed by Wright et al. [Wright, P. E., Dyson, H. J., & Lerner, R. A. (1988) Biochemistry 27, 7167-7175]. Formation of the first disulfide bond in apamin seems to be important in the folding process by stabilizing native-like structure, presumably by reducing the conformational freedom and initiating formation of structure.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Increased methotrexate resistance and dhfr gene amplification as a consequence of induced Ha-ras expression in NIH 3T3 cells.
Oncogene activation and loss of tumor suppressor genes are known to play a role in tumor initiation as well as its progression. The potential roles of these genes in perturbation of genome stability has become a major interest. To better understand the relationship between expression of an oncogene and genetic instability, we have studied a cell line expressing an activated human Ha-ras under the control of bacterial lactose operon regulatory elements for changes in methotrexate resistance and dihydrofolate reductase (dhfr) gene amplification following mutant Ha-ras induction. In these cells mutant Ha-ras is directed by an inducible SV40 promoter containing a bacterial lac operator sequence which is repressed due to constitutive expression of bacterial lac repressor gene. The expression of this Ha-ras is specifically induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside (IPTG), a lactose analogue, to the culture medium. During single-step methotrexate selection, these cells showed an increased frequency of methotrexate resistance in the presence of IPTG. More than 60% of the methotrexate-resistant colonies showed a 2-6-fold amplification of the dhfr gene. One clone with rearranged dhfr had about 100-fold amplification of the gene. The increased capacity to amplify DNA in response to mutant Ha-ras induction was not locus specific since cells also displayed an increased frequency of resistance to N-(phosphonacetyl)-L-aspartic acid in the presence of ITPG. Four of the methotrexate-resistant clones with amplified dhfr gene were cultured further in the presence or absence of IPTG and subsequently compared for their ability to grow in soft agar as a measure of transformation. In medium containing methotrexate but no IPTG, the clones were unable to grow in soft agar, indicating that methotrexate resistance due to gene amplification is separable from transformation.
3T3 Cells↗
Depletion of intracellular Ca2+ stores activates nitric-oxide synthase to generate cGMP and regulate Ca2+ influx.
The mechanism of activation of the agonist-stimulated Ca2+ entry pathway in the plasma membrane is not known. To determine the role of nitric-oxide synthase (NOS) and cGMP in the regulation of this pathway, we used intact and streptolysin O (SLO)-permeable pancreatic acini and measured the relationship between Ca2+ release from internal stores, the NO metabolic pathway, generation of cGMP, and activation of Ca2+ entry. We found that agonist- or thapsigargin (Tg)-activated Ca2+ entry is inhibited by L-NA, a specific inhibitor of NOS, and by LY83583, an inhibitor of guanylyl cyclase. Inhibition of Ca2+ entry by inhibition of NOS was reversed by the NO releasing molecules NO2- and sodium nitroprusside (SNP) and by Bt2cGMP. Inhibition of Ca2+ entry by inhibition of guanylyl cyclase was reversed by Bt2cGMP, but not by the NO releasing agents. The use of L-NA-treated cells and different concentrations of SNP revealed that cGMP has a dual effect on Ca2+ entry. Increasing cGMP up to 10-fold above control activated Ca2+ entry. Further increase in cGMP up to 80-fold above control inhibited Ca2+ entry in a concentration-dependent manner. Measurement of cellular cGMP in intact cells showed that carbachol, Tg, and NO2- increased cGMP to similar levels. The effects of carbachol and Tg were inhibited by L-NA and LY83586, whereas the effect of NO2- was inhibited only by LY83583. SLO-permeabilized cells were shown to be agonist-competent in that the agonist induced Ca2+ release from the inositol 1,4,5-trisphosphate (IP3) pool and activated a NO-dependent generation of cGMP. These cells were used to study the regulation of NOS by Ca2+ and by Ca2+ content of the internal stores. When internal stores were maintained loaded with Ca2+, increasing medium [Ca2+] up to 2.5 microM only modestly increased NOS activity. In contrast, the depletion of Ca2+ from internal stores markedly increased NOS activity independent of medium [Ca2+]. Thus, NOS senses both cytosolic [Ca2+]i and internal store Ca2+ load. We propose that activation of Ca2+ entry involves an agonist-mediated Ca2+ release from internal stores which activates a cellular pool of NOS to generate cGMP, which then modulates Ca2+ entry pathway in the plasma membrane. This mechanism can explain the capacitative nature of Ca2+ entry. The biphasic effect of cGMP provides the cells with a negative feedback mechanism which inhibits Ca2+ entry during periods of high cell [Ca2+]i. This could allow oscillatory behavior of Ca2+ entry.
Amino Acid Oxidoreductases↗
Risk factors for oesophageal cancer in northeast China.
A hospital-based case-control study of oesophageal cancer was carried out in the Heilongjiang Province, a low-risk area for oesophageal cancer in China. From May 1985 to May 1989, 196 histologically confirmed cases and 392 controls with other (non-neoplastic) diseases were personally interviewed in the wards of 5 major hospitals. Information was obtained about usual consumption in the early 1980s of 32 major contributors to the diet in the province, socio-demographic status, smoking and alcohol consumption. Odds ratios (OR) were obtained from logistic regression models, and confounding was controlled by means of multivariate models. Smoking and alcohol consumption were major risk factors for oesophageal cancer in this population. Smokers of handmade cigarettes exhibited a particularly high risk. A near multiplicative synergism was found between smoking and alcohol consumption. There was a significant inverse dose-risk trend for combined consumption of vegetables and fruits; a 300-g increase per day lowered risk by 35%. Vitamin C intake was negatively associated with risk; a 100-mg increase per day lowered risk by 39%. Our data suggest a modifying effect of vitamin C and beta-carotene on risk associated with smoking, but the power of analyses was low. Salt, salt-preserved foods and pickled vegetables were not associated with increased risk. High temperature of meals and drinks was a strong risk indicator in this population. The strength of tea and overall tea consumption were independent determinants of the risk.
Adult↗
Probing the role of histidine-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase by site-specific mutagenesis.
In the X-ray structure of Escherichia coli alkaline phosphatase at 2.0-A resolution, His-372 was found only 3.8 A away from the zinc and forms a hydrogen-bonding interaction with Asp-327, a bidentate ligand of the zinc at the M1 site. However, His-372 does not directly interact with the zinc atom at the M1 site. In order to investigate the role of the side chain of His-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase, site-directed mutagenesis was used to convert His-372 to alanine. The fact that the His-372-->Ala enzyme has similar zinc binding affinity as the wild-type enzyme indicates that His-372 is not involved in zinc binding at the M1 site. However, the altered kinetic behavior of the mutant enzyme compared to the wild-type enzyme suggests that the imidazole ring of His-372 plays an indirect role in the catalytic mechanism of the enzyme. The hydrolysis activity of the His-372-->Ala enzyme at pH 8.0 is 10-fold lower than that of the wild-type enzyme. In the presence of a phosphate acceptor at pH 8.0, the mutant enzyme is approximately 80% as active as the wild-type enzyme. Therefore, the His-372-->Ala mutation selectively enhances the transphosphorylation activity of the enzyme. The His-372-->Ala enzyme also exhibits 4- and 30-fold decreases in Km as compared to the wild-type enzyme in 0.1 M MOPS buffer and 1.0 M Tris, buffer at pH 8.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Alanine↗
Promoter of the POU-M1/SGF-3 gene involved in the expression of Bombyx silk genes.
To characterize the transcription regulation of the POU-M1/SGF-3 gene, we have cloned a genomic DNA fragment encompassing the whole coding region and its flanking sequences. This gene does not contain any intron. The 5'-flanking region of the gene contains several interesting motifs, such as homeodomain-binding motifs, sequences resembling the transcriptional factor Sp1-binding site, and TGTTT motifs, but lacks some of the typical transcriptional regulatory sequences, such as TATA and CCAAT boxes. Transcriptional analysis of a series of deletion mutants of the gene in the nuclear extracts prepared from the middle silk gland of 2-day-old fifth instar larvae revealed the presence of multiple cis-regulatory elements located both upstream and downstream of the initiation site. One of these elements, the homeodomain-binding element, was identified to mediate negative regulation. By mobility shift assay using the POU-M1 specific antibodies, we found that this negative element interacts with the POU-M1/SGF-3. Transcription analysis in vitro using templates mutagenized in the PB region and one of the POU-M1 antibodies indicated that the PB region is an autoregulatory element responsible for SGF-3-dependent transcriptional repression.
Animals↗
Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site.
Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.
Alanine↗
Schistosoma mansoni: fractionation and characterization of the glycocalyx and glycogen-like material from cercariae.
The glycocalyx (GCX) that covers schistosomal cercariae is a complex molecule that has immunomodulating properties. Here, we purified milligram amounts of GCX using Anguilla lectin which binds to the GCX covering the cercarial body and tail. Typically, 10 million cercariae were extracted with phenol, dialyzed, and chromatographed on a Sepharose 2B-CL column. An average of 39 mg of total carbohydrate eluted near the void volume from which 31 mg of glycogen-like material was further separated by lectin affinity chromatography. Its identity was established by compositional analysis, sensitivity to amylase digestion, and its nuclear magnetic resonance spectrum. The lectin-bound GCX was eluted with 0.1 M fucose with a final yield of 5.3 mg carbohydrate. Fucose composed 40% of the total GCX carbohydrate with lesser but approximately equal amounts of galactose, glucosamine, and galactosamine present. NMR data indicated that the amino sugars were N-acetylated. Glucose was also present but in varying amounts in different preparations of GCX. Oligosaccharides were released from GCX by hydrazinolysis and separated by electrophoresis after reductive amination to 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). Bands comigrating with standards containing 11, 12, 16, and 17 sugar residues were detected. Thus, the GCX is a complex structure composed of oligosaccharides, probably linked to a peptide.
Amino Sugars↗
Isolation and characterization of sulfite mutants of Saccharomyces cerevisiae.
Sulfite-resistant and sulfite-sensitive mutants of Saccharomyces cerevisiae were isolated and characterized. Genetic analysis indicated that one and four genes were responsible for the resistant and sensitive responses, respectively, and suggested that defects in methionine and cysteine metabolism were not involved. Some resistant alleles, all of which were dominant, conferred greater resistance than others. Mutations conferring sensitivity were recessive and one co-segregated with impaired respiration. Two of the sensitive mutants exhibited cross-sensitivity to other metabolic inhibitors: sulfometuron methyl, cycloheximide, oligomycin, and antimycin A. A 50% glutathione deficiency in one sensitive mutant was not sufficient in itself to account for its sensitivity. Screening of other relevant mutants revealed that relative to wild-type, met8 and a thioredoxin null mutant are sensitive, and met3 and met14 mutants are not. Reduced production of extracellular acetaldehyde, a compound that detoxifies sulfite, was observed in three of the four sensitive mutants. However, acetaldehyde was also underproduced in the resistant mutant. Because sulfite is a reducing agent, cells were tested for coincident sensitivity or resistance to ascorbate, selenite, dithiothreitol, nitrite, thiosulfate, reduced glutathione, and cysteine. No consistent pattern of responses to these agents emerged, suggesting that the response to sulfite is not a simple function of redox potential.
Acetaldehyde↗
Characterization of the ATP-inhibited K+ current in canine coronary smooth muscle cells.
Intracellular adenosine triphosphate (ATP)-inhibited K+ currents (IK, ATP) in canine coronary artery smooth muscle cells were characterized in the whole-cell configuration using the suction pipette method. Cells dialysed internally with solutions containing 5 mM ATP (ATPi) showed little detectable whole-cell current at potentials more negative than -30 mV. However, cells dialysed with ATPi-free solutions developed a time- and voltage-independent current which reached a maximum of 132 +/- 25 pA at -40 mV about 10 min following patch rupture. After "run-up", the current showed little "run-down". Concentration-dependent inhibition by ATPi yielded an inhibition constant (Ki) of 350 microM and a Hill coefficient of 2.3. In ATPi-free solutions, the large current at -40 mV was reduced by glibenclamide with a Ki of 20 nM and a Hill coefficient of 0.95. Conversely, in 1 mM ATPi solutions, the small current at -40 mV was increased by P-1075 from 8 +/- 2 pA to 143 +/- 33 pA, with a dissociation constant (Kd) of 0.16 microM and a Hill coefficient of 1.7. The effect of P-1075 was antagonized by glibenclamide. Maximal current density elicited by either ATPi depletion or external application of the channel opener P-1075 was similar with slope conductances of 81 +/- 10 pS/pF and 76 +/- 13 pS/pF respectively in the potential range of -90 to -40 mV. External Ca2+ had no effect on this current. Finally, in 1 mM ATPi, 20 and 50 microM adenosine increased the current slope conductance by 36 +/- 15% and 73 +/- 10% respectively between -90 to -40 mV. The IK, ATP, although very small in these cells, was extremely effective in causing membrane potential hyperpolarization.
Adenosine↗
Prolonged inhibition of brain nitric oxide synthase by short-term systemic administration of nitro-L-arginine methyl ester.
We studied the dose-response characteristics and the temporal profile of inhibition of brain nitric oxide (NO) synthase (NOS) elicited by i.v. administration of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME). L-NAME was administered i.v. in awake rats equipped with a venous cannula. L-NAME was injected in cumulative doses of 5, 10, 20 and 40 mg/kg and rats were sacrificed 30 min after the last dose. NOS catalytic activity was assayed in forebrain cytosol as the conversion of [3H]L-arginine into [3H]L-citrulline. L-NAME attenuated brain NOS activity in a dose-dependent manner but enzyme activity could not be inhibited by more than approximately 50%. After a single 20 mg/kg injection of L-NAME the inhibition of brain NOS activity was time dependent and reached a stable level at 2 hrs (52% of vehicle). Inhibition after a single injection was still present at 96 hrs, albeit to a lower magnitude. We conclude that intravenous administration of L-NAME in rats at concentrations commonly used in physiological experiments leads to a dose and time-dependent but partial inhibition of brain NOS catalytic activity. The finding that the inhibition persists for several days after a single administration is consistent with the hypothesis that nitro-L-arginine, the active principle of L-NAME, binds to NOS irreversibly.
Amino Acid Oxidoreductases↗
Study on the role of mast cells in the development of liver fibrosis during diethylnitrosamine (DEN) -induced hepatocarcinogenesis in rats.
The mast cells (MC) and their roles in the development of liver fibrosis during experimental hepatocarcinogenesis in rats have been studied immunohistochemically and electron microscopically. Pronounced MC hyperplasia occurred after beginning of the treatment of DEN. The vast majority of mast cells in fibrotic livers was present in thickened fibrous septa. Intracellular or pericytial collagen type IV and laminin staining were found in mast cells, collagen types I, III and fibronectin were negative. Mast cells mainly distributed in proximity to basement membrane and the electron microscopical observation revealed a close topographic relationship between mast cells and fibroblasts. The fibroblasts phagocytized the granules released by mast cells and thus were activated, showing enhanced formation of collagenous fibrils. The mast cells may be derived from blood circulation or replicated in situ. Those findings indicated that mast cells take part in the process of fibrosis at least by two ways: (1) It directly synthesizes the basement membrane components; (2) It stimulates the fibroblasts or other types of cells which have the potential functions in fibrogenesis by releasing granule mediators or cytokines acting upon the fibrosis process.
Animals↗
Phencyclidine-induced behavioral sensitization.
Chronic administration of a psychomotor stimulant has been shown to produce progressively enhanced effects, a phenomenon called "reverse tolerance" or sensitization. The present study reexamined the effects of the daily injection of phencyclidine on locomotor activity and stereotypy in rats, and investigated whether drug-environment conditioning was necessary for the development of behavioral sensitization and whether (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,1 0-imine hydrogen maleate (MK-801, dizocilpine) blocked behavioral sensitization to phencyclidine. Female Sprague-Dawley rats were used. Locomotor activity and stereotypy were measured automatically with the Digiscan system. The results confirmed an earlier finding that four daily injections of phencyclidine induced sensitization to both locomotor activity and stereotypy. The development of behavioral sensitization did not require drug-environment conditioning. Moreover, MK-801 did not block behavioral sensitization to phencyclidine. The results of the present study suggest that the neuronal mechanisms underlying sensitization to phencyclidine are different from those underlying sensitization to amphetamine and cocaine.
Animals↗
Persistence of the SV40 early region without expression in permissive simian cells.
SV40 containing recombinant vectors were introduced into permissive simian, non-permissive rodent and semi-permissive human cell lines, and assayed for transformation. All mouse and human cell clones expressed T-antigen (T-Ag) and were morphologically transformed when they contained only the wt T-Ag gene (E-SV40) or the entire wt viral genome with an interrupted late region. However, of 63 simian clones with these recombinant vectors, none became morphologically transformed and T-Ag containing cells were rare or absent. Nearly all simian cell lines made either no detectable early SV40 RNA or only small amounts of viral RNA but contained viral DNA restriction fragments similar to those in the original recombinant vectors. Functional T-Ag genes were recoverable from several cell clones and used to regenerate infectious virus. Hence, T-Ag gene expression had been suppressed. We found two conditions where T-Ag expression was activated. In a BSC-1 cell line containing E-SV40 DNA, subsequent introduction of a vector with a functional viral late coding region (L-SV40) resulted in the appearance of T-Ag and transformation. These findings suggest that L-SV40 sequences activate or enhance T-Ag expression and that this activation requires a functional Vpl gene. We found also, that vectors with E-SV40 DNA from the bipartite variant EL-SV40 consistently transformed simian CV-1 cells. Transformation was shown to be effected by the multiple alterations present in the regulatory region of this variant.
Animals↗
Asymmetric cross-sensitization to the locomotor stimulant effects of phencyclidine and MK-801.
Chronic administration of a psychomotor stimulant has been shown to produce progressively enhanced effects, a phenomenon called "reverse tolerance" or sensitization. Sensitization which develops to the psychomotor stimulant effect of a drug generalizes to drugs with similar neurochemical mechanisms of action, a phenomenon called cross-sensitization. The present study compared the psychomotor stimulant effects of phencyclidine and MK-801, examined the effects of the daily injection of phencyclidine and MK-801 on locomotor activity and investigated whether reciprocal cross-sensitization occurred between phencyclidine and MK-801. Adult female Sprague-Dawley rats were used. Their locomotor activity was measured automatically for a 2 h period following drug injection. Phencyclidine and MK-801 both increased locomotor activity. Four daily injections of phencyclidine in a dose of 3.2 mg/kg i.p., or MK-801 in a dose of 0.32 mg/kg i.p., produced sensitization to locomotor activity. Moreover, MK-801 sensitized rats showed cross-sensitization to phencyclidine. However, phencyclidine sensitized rats did not show cross-sensitization to MK-801. This finding suggests that there are significant differences in the neurochemical mechanisms underlying phencyclidine-induced and MK-801-induced sensitization. Phencyclidine sensitization may not be mediated by NMDA receptors.
Animals↗
Establishing a resuscitation model in rabbits with closed-thoracic cardiopulmonary by-pass.
This study aims to establish an animal model of resuscitation in rabbits by using closed-thoracic cardiopulmonary by-pass (CTCPB). The rabbits were randomly divided into four groups according to cardiac arrest times which were 8, 10, 12, and 15 min. Neurologic outcome and blood lactate were determined within 150 min after resuscitation. It was indicated that the rabbits' neurologic functions were progressively injured with prolonged cardiac arrest time. The threshold of circulatory arrest time that induced a vegetative state in the rabbits was between 10 and 12 min. There were no significant differences in cardiac resuscitability among the four groups, as was so for plasma lactate, although it increased significantly from the control levels. The establishment of a small-animal model of resuscitation by using CTCPB, and the problems in dealing with it are also described and discussed in detail in this paper. Our experience indicated that this is a simple, convenient, and economical animal model for the study of resuscitation.
Animals↗
Estrogen receptor-mediated direct stimulation of colon cancer cell growth in vitro.
In vivo and epidemiological data suggest a mitogenic role for estrogens (E) in colon cancer. The presence of estrogen receptor (ER) and ER mRNA in colonic epithelium and colon cancer cells, make it necessary to explore the possible direct effects of E on colon cancer growth. In this study, a 15-mer oligodoxynucleotide (oligo) antisense to the region of the translation start codon of estrogen receptor mRNA inhibited ER expression in a mouse colon cancer cell line (MC-26), as determined by receptor binding assay. Antisense oligo also decreased ER mRNA levels in MC-26 cells. The growth-stimulatory effect of E was abolished by antisense oligo treatment, demonstrating that the ER is directly involved in the regulation of colon cancer cell growth.
Adenocarcinoma↗