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Biomedical subjects

D Wu

Publications and source records attributed to D Wu.

At least 19 recordsLinked to original sources
PubMed1992

Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the alpha subunits of Gq family.

High efficiency transient transfection of Cos-7 cells was previously used to establish the functional coupling between G alpha q/G alpha 11 and phospholipase C beta 1 (Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817). Here the same system was used to study the functional coupling between other guanine nucleotide-binding regulatory protein (G-protein) alpha subunits and phospholipases and to study which G alpha subunits mediate the activation of phospholipase C by the alpha 1-adrenergic receptor subtypes, alpha 1 A, alpha 1 B, and alpha 1 C. We found that G alpha 14 and G alpha 16 behaved like G alpha 11 or G alpha q, i.e. they could activate endogenous phospholipases in Cos-7 cells in the presence of AIFn. The synergistic increase in inositol phosphate release in Cos-7 cells after they were cotransfected with cDNAs encoding G alpha subunits and phospholipase C beta 1 indicates that both G alpha 16 and G alpha 14 can activate phospholipase C beta 1. The activation of phospholipase C beta 1 was restricted to members of the Gq subfamily of alpha subunits. They activated phospholipase C beta 1 but not phospholipase C gamma 1, gamma 2, or phospholipase C delta 3. The cotransfection of Cos-7 cells with cDNAs encoding three different alpha 1-adrenergic receptors and G alpha q or G alpha 11 leads to an increase in norepinephrine-dependent inositol phosphate release. This indicates that G alpha q or G alpha 11 can mediate the activation of phospholipase C by all three subtypes of alpha 1-adrenergic receptors. With the same assay system, G alpha 16 and G alpha 14 appear to be differentially involved in the activation of phospholipase C by the alpha 1-adrenergic receptors. The alpha 1 B subtype receptor gave a ligand-mediated synergistic response in the cells cotransfected with either G alpha 14 or G alpha 16. However, the alpha 1 C receptor responded in cells cotransfected with G alpha 14 but not G alpha 16, and the alpha 1 A receptor showed little synergistic response in cells transfected with either G alpha 14 or G alpha 16. The ability of the alpha 1 A and alpha 1 C receptors to activate phospholipase C through G alpha q and G alpha 11 was also demonstrated in a cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C.

The activation of heterotrimeric G proteins results in the exchange of GDP bound to the alpha-subunit for GTP and the subsequent dissociation of a complex of the beta- and gamma-subunits (G beta gamma). The alpha-subunits of different G proteins interact with a variety of effectors, but less is known about the function of the free G beta gamma complex. G beta gamma has been implicated in the activation of a cardiac potassium channel, a retinal phospholipase A2 (ref. 9) and a specific receptor kinase, and in vitro reconstitution experiments indicate that the G beta gamma complex can act with G alpha subunit to modulate the activity of different isoforms of adenylyl cyclase. Of two phospholipase activities that can be separated in extracts of HL-60 cells, purified G beta gamma is found to activate one of them. Here we report that in co-transfection assays G beta gamma subunits specifically activate the beta 2 and not the beta 1 isoform of phospholipase, which acts on phosphatidylinositol. We use transfection assays to show also that receptor-mediated release of G beta gamma from G proteins that are sensitive to pertussis toxin can result in activation of the phospholipase. This effect may be the basis of the pertussis-toxin-sensitive phospholipase C activation seen in some cell systems (reviewed in refs 13 and 14).

Adenylate Cyclase Toxin↗
PubMed1992

Metabolic effects of short-chain ceramide and glucosylceramide on sphingolipids and protein kinase C.

Recent studies have identified a potential role for glucosylceramide (GlcCer) in growth promotion and hormonal signalling. In an effort to demonstrate a growth-promoting activity of GlcCer, we prepared a GlcCer having a short-chain acid (octanoyl), in the belief that this glycolipid could be absorbed more readily and more uniformly by cultured cells. By using a mixture of two specific lecithins, dioleoylglycerophosphocholine and 1-stearoyl-2-palmitoylglycerophosphocholine, we were able to prepare dispersions containing a high molar proportion of the GlcCer and the related ceramide, octanoyl sphingosine. Unexpectedly, both sphingolipids inhibited protein and DNA synthesis in Madin-Darby canine kidney cells and produced large increases in the levels of the natural lipids, GlcCer, ceramide, free sphingosine, and an amine that may be glucosylsphingosine (GlcSph). Decreases were seen in the level of sphingomyelin and the proportion of protein kinase C in the cell membranes. The level of lactosylceramide was diminished by octanoyl GlcCer but elevated considerably by octanoyl sphingosine. Diacylglycerols were increased by the lecithins in the liposomes, but the exogenous sphingolipids had no effect. Octanoyl sphingosine labeled in the sphingoid base yielded labeled GlcCer and sphingomyelin labeled in both long-chain and very-long-chain fatty acid families, as well as the octanoyl version. The two families of ceramides, however, had relatively little radioactivity. Some of these changes are attributed to rapid hydrolysis of the added lipids with the formation, particularly from the ceramide, of sphingosine and its anabolic metabolite, GlcSph. Several observations support the idea that the octanoyl sphingosine inhibited the phosphocholinetransferase that synthesizes sphingomyelin while the octanoyl GlcCer inhibited GlcCer beta-glucosidase and GlcCer galactosyltransferase. The use of unnatural short-chain lipids in the study of cell growth and other phenomena may result in unexpected changes in related metabolites and the findings from such experiments should therefore be interpreted cautiously.

Animals↗
PubMed1992

Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide.

Proteins with relative molecular masses of 14,000 to 205,000 were separated by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using non-cross-linked linear polyacrylamide gels on both coated and uncoated fused-silica capillaries. It was determined that viscosity of the acrylamide solution was a major factor affecting column stability with linear acrylamide gels. When the viscosity of the acrylamide solution reaches 100 cP, electro-osmotically driven displacement of the gels is insignificant. Uncoated capillaries provided better resolution, stability, and reproducibility than surface coated capillaries when the concentration of linear polyacrylamide was greater than 4%. At lower gel concentrations, non-cross-linked polyacrylamide is easily displaced from the columns. A calibration plot of log molecular mass vs. mobility with non-linear polyacrylamide was linear, which indicated that resolution was equivalent to that obtained with cross-linked acrylamide. Separations with model proteins indicated that baseline resolution between protein species that vary 10% in molecular mass can be achieved.

Acrylic Resins↗
PubMed1992

A polymerase chain reaction-based method for isolation of gene-specific sequences from the interferon-alpha gene cluster.

The interferon-alpha gene is a gene family of over 20 distinct genes having 80-95% homology with one another at a nucleotide level. Because of the high homology in the gene cluster, the available interferon-alpha gene probes can hybridize to multiple bands of different size on Southern blot analysis of restricted human genomic DNA. We used the polymerase chain reaction with the primers synthesized from Alu repetitive sequence and the conserved sequences of the interferon-alpha gene cluster to generate specific probes for individual interferon-alpha genes. The amplification products were subcloned into a plasmid vector and analyzed by DNA sequencing and Southern blotting of the restricted human placental DNA. One clone, which derived from interferon-alpha 14 gene, produced a single 5.2-kb band in Southern blots of the HindIII-restricted human placental DNA. This stands in contrast to the 10 bands of different size that were detected with a cDNA for the interferon-alpha I' gene. Our results indicate that a polymerase chain reaction-based method can be used to isolate gene-specific sequences from the interferon-alpha gene cluster. Since a variety of human cancers has been found to have the complete or partial deletion of the interferon-alpha gene cluster, the gene-specific probe generated by this method may aid in determining the breakpoints in the vicinity of the gene cluster.

Animals↗
PubMed1992

Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes.

The relative specificities of members of the G alpha q family of GTP-binding proteins were tested for their ability to activate different phosphoinositide-specific phospholipase C (PI-PLC) beta isozymes. Cos-7 cells were transfected with cDNA corresponding to G alpha q, G alpha 11, G alpha 14, and G alpha 16. Most of the recombinant protein was bound to the cell membrane and these membranes were washed to elute endogenous PI-PLC activity. The membrane preparation was reconstituted with purified preparations of the PI-PLC beta isozymes and guanosine 5'-O-thiotriphosphate (GTP gamma S)-stimulated enzyme activity was measured. All four proteins of the G alpha q family were found to stimulate PI-PLC beta 1, with G alpha q and G alpha 11 being most efficient. On the other hand, G alpha 16 was found to most effectively activate PI-PLC beta 2, while G alpha q, G alpha 11, and G alpha 14 showed less stimulation. Specific anti- G alpha 16 antibody blocked the stimulation of both PI-PLC beta 1 and PI-PLC beta 2 in the enriched membrane fraction. We conclude that there is specificity in the interaction of different members of the Gq family with different PI-PLC beta effectors. This specificity may be important in generating tissue- or receptor-specific responses in vivo.

Amino Acid Sequence↗
PubMed1992

Usefulness of predischarge electrophysiologic study in predicting late outcome after surgical ablation of the accessory pathway in the Wolff-Parkinson-White syndrome.

A predischarge electrophysiologic study was performed in 113 patients with the Wolff-Parkinson-White (WPW) syndrome who had undergone surgical ablation of the accessory pathway. The study was performed 5 to 20 (mean 10 +/- 3) days after surgery. There were 82 male and 31 female patients (aged 4 to 58 years, mean 36 +/- 13). Sixty-one patients (54%) had manifest, 52 (46%) had concealed and 12 (11%) had multiple accessory pathways. All but 1 patient had atrioventricular reentrant tachycardia incorporating single or multiple accessory pathways during the control electrophysiologic study. The accessory pathways were located in the left ventricular free wall in 60% of cases, right ventricular free wall in 22%, posteroseptum in 13%, and anteroseptum in 5%. The predischarge electrophysiologic study showed that the accessory pathway was capable of anterograde and retrograde conductions in 4 patients (all with manifest WPW syndrome). Four patients showed induction of supraventricular tachycardia, including 2 with atrioventricular reentrant tachycardia, and 2 with atrioventricular nodal reentrant tachycardia. Recurrence of supraventricular tachycardia was noted in 5 patients during a follow-up of 28 +/- 26 months. Of these 5 patients, 2 had inducible and 3 had no inducible supraventricular tachycardia during the predischarge electrophysiologic study.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity.

Bacillus thuringiensis produces a variety of delta-endotoxins which bind to specific receptors in insect larval midguts. Following insertion into the membrane there is an alteration of ion flux culminating in osmotic lysis. Mutagenic oligonucleotides were used to define regions in one of these toxins involved in specificity and toxicity. One region is highly conserved among all toxins sequenced to date and many mutations resulted in loss of toxicity for three test Lepidoptera. The mutant toxins had lost the capacity to inhibit K(+)-dependent amino acid transport into larval midgut vesicles, but there was no effect on their ability to compete with wild type toxin for binding. The results are consistent with this amphiphilic helical region of the toxin being essential for toxicity. A second mutagenized region overlapped a portion of another potential amphiphilic helix. Mutations of only 2 residues, Ala-92 and Arg-93, resulted in loss of toxicity for two lepidopteran larvae but some activity remained for a third. The A92D mutant toxin competed with the wild type toxin for binding to vesicles prepared from midguts from the sensitive but not from the insensitive larvae. Decreased toxicity was also found when this mutation was transferred to two other related protoxin genes. A number of mutations of each of these residues was analyzed and selective loss of toxicity correlated with the absence of a positive charge. Despite being distal from the presumptive specificity domain, 1 or both of these residues must have an important role in the specific binding of toxins.

Amino Acid Sequence↗
PubMed1992

Presence of functionally active cytochrome P-450IIE1 in the plasma membrane of rat hepatocytes.

Recent experiments have described the presence of cytochrome P-450 and certain P-450 isozymes in the plasma membrane of rat liver. Experiments were carried out to evaluate whether cytochrome P-450IIE1 was present in the plasma membrane fraction of livers from control rats and rats treated with 4-methylpyrazole, which induces this isozyme. Using immunofluorescence, fluorescence was detected at the surface of intact hepatocytes that were initially incubated with anti-P-450IIE1 IgG, but not preimmune IgG, followed by incubating with goat antirabbit IgG conjugated with either fluorescein or rhodamine. The fluorescence appeared to be uniformly distributed across the entire surface. Intense intracellular staining could be observed when the hepatocytes were permeabilized by acetone treatment. Similar results were obtained with control hepatocytes; however, the fluorescence intensity was considerably less than that shown by the induced hepatocytes. Hepatocytes isolated from the pericentral zone of the liver acinus displayed more intense fluorescence at the surface than did hepatocytes from the periportal zone. Purified plasma membranes oxidized dimethylnitrosamine to formaldehyde at rates that were 14% to 30% that of the microsomes, which exceeds the 3% contamination of the plasma membranes by microsomes as assessed by glucose-6-phosphatase activity. Immunoblots of the plasma membranes revealed the presence of a single band, whose intensity of staining was 14% to 26% that of the microsomes. Oxidation of dimethylnitrosamine and immunoblot intensity were about twofold greater with plasma membrane fractions from 4-methylpyrazole-treated rats than controls. These results suggest the presence of inducible, functionally active P-450IIE1 in the plasma membrane, which may be of toxicological significance in view of the preferential metabolism of a variety of hepatotoxins and carcinogens and the elevated production of reactive oxygen intermediates by this isozyme.

Animals↗
PubMed1992

Nature of dual atrioventricular node pathways and the tachycardia circuit as defined by radiofrequency ablation technique.

OBJECTIVES: A comprehensive electrophysiologic study followed by selective radiofrequency ablation from three sites was performed in patients with atrioventricular (AV) node reentrant tachycardia to better delineate the nature of the tachycardia circuit. BACKGROUND: We postulated that the retrograde fast pathway is the anterior superficial group of transitional cells and the slow pathway is the compact node with its posterior input of transitional cells. Twenty-three consecutive patients were studied. In nine, the atria could be dissociated from the tachycardia by delivery of an atrial extrastimulus during tachycardia. METHODS: Radiofrequency ablation was performed with three approaches. The anterior approach was designed to interrupt the anterior superficial atrial input to the compact node, the posterior approach to interrupt the posterior atrial input to the compact node and the inferior approach to destroy the compact node itself. RESULTS: Selective ablation of the retrograde fast pathway was achieved in seven patients, six with the anterior and one with the inferior approach. Anterograde fast pathway conduction was not affected, whereas retrograde fast pathway conduction was either abolished or markedly depressed. None had induction of echoes or tachycardia after ablation. Selective ablation of the slow pathway was successful in 13 patients, 1 with anterior, 3 with posterior and 9 with inferior approaches. In these 13 patients, both anterograde and retrograde fast pathway conduction were not affected, the dual pathway physiology was abolished and the tachycardia was not inducible after ablation. Ablation of both the retrograde fast pathway and the slow pathway occurred with the inferior approach in three patients. CONCLUSIONS: We conclude that the retrograde fast pathway is likely to be the anterior superficial group of transitional cells, whereas the slow pathway is the compact node and its posterior input of transitional cells. A barrier seems to exist between the atrium and the tachycardia circuit. In a broad view of the AV node structure, the tachycardia circuit is confined to the node.

Atrial Function↗
PubMed1992

Effect of 60Co gamma-irradiation on the nonspecific cytotoxicity of alveolar macrophages in vitro.

This paper reports on the effect of radiation on the nonspecific cytotoxicity of rat alveolar macrophages (AM). AM (effector cells) of bacille Calmette-Guerin-activated Wistar rats were irradiated with 60Co gamma rays in vitro to give doses of 0, 100, 300, and 500 Gy. Three hours after irradiation, the AM were cultured with human lung adenocarcinoma AGZY83-a and HeLa target cells in 125I-deoxyuridine-containing media for 6 hr and the cytotoxicity indexes determined. The results indicated that the cytotoxicity indexes of AM against human lung adenocarcinoma cells and HeLa cells were 94.3 +/- 0.3% and 81.3 +/- 1.9%, respectively. The cytotoxicity indexes using an effector/target cell ratio (E/T) of 10 seemed to be greater than with ratios of 20 and 30. The cytotoxicity indexes of AM (7 rats), irradiated to give doses of 0, 100, 300, and 500 Gy, against adenocarcinoma cells at an E/T ratio of 10 were 87.9 +/- 8.4%, 65.4 +/- 14.1%, 47.5 +/- 17.5%, and 36.7 +/- 9.7%, respectively. The significance of the nonspecific cytotoxicity of AM in the immunological elimination of tumors and the inhibitory effect of radiation on AM cytotoxicity are discussed.

Adenocarcinoma↗
PubMed1992

Erythrocyte sodium-lithium countertransport in Chinese: its relationship to family history of hypertension.

Rates of sodium (Na+)-stimulated lithium (Li+) efflux (Na(+)-Li+ countertransport) and ouabain-sensitive Na+ efflux (Na+ pump) were determined in erythrocytes of Chinese normotensive and hypertensive subjects. Near-maximal rate of Na(+)-Li+ countertransport was found to be significantly higher in hypertensive than normotensive subjects. No significant difference was observed for the rate of Na+ pump between them. A second series of study involved normotensive subjects without and with hypertensive parent(s) (group A and B, respectively) and hypertensive subjects (group C). We found that the rate of Na(+)-Li+ countertransport in group A was significantly lower than that of group B and C, while no difference existed between group B and C. No significant difference was observed for the rate of Na+ pump among the three groups. Our results suggested that Na(+)-Li+ countertransport activity could be a genetic marker for essential hypertension in Chinese, similar to that as proposed in Caucasians.

Adult↗
PubMed1992

[DNA repair capacity measurement and possibility of heterozygote detection using nucleoid sedimentation technique among members taken from 3 xeroderma pigmentosum (XP) families].

The nucleoid sedimentation test was used to analyse the DNA repair capacity in 35 members taken from 3 xeroderma pigmentosum (XP) families after their peripheral blood lymphocytes being exposed to UV irradiation or N-methyl-N-nitro-N-nitrosoguanidine (MNNG) at a dose of 2.5 microJ/mm2 and 2 micrograms/ml respectively. The results showed that the 6 XP patients all failed to repair their damaged DNA even after 20-hour incubation at 37 degrees C. The repair ratios were 0.61 +/- 0.13 for UV and 0.44 +/- 0.15 for MNNG while those of non-related normal members usually reached 0.96 +/- 0.07 and 0.71 +/- 0.07 for both UV and MNNG after 11-hour incubation. It is interesting to note that 9 obligatory heterozygotes (parents and grandparents of XP patients) and 4 highly possible heterozygotes (blood relatives of XP patients) also showed very poor DNA repair capacity: they could not accomplish their repair process even after 20-hour incubation. It has been showed that the nucleoid sedimentation test can probably be used to pick out the heterozygotes in XP families.

Adolescent↗
PubMed1991

Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.

Two independent approaches were employed to explore the potential role of endogenous glucosylceramide or a closely related glucosphingolipid in mediating the cellular proliferation of Madin-Darby canine kidney cells. First, cultured cells were depleted of glucosphingolipids by exposure to a glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. This agent markedly inhibited cell growth and DNA synthesis in a time- and concentration-dependent manner. Second, cells were grown in the presence of conduritol B epoxide, an inhibitor of glucosylceramide beta-D-glucosidase. Exposure of cells to this inhibitor resulted in the time-dependent accumulation of glucosylceramide with a corresponding increase in cellular proliferation. Alterations in protein kinase C activity were evaluated as a potential mechanism for these effects on growth. Both membrane- and cytosol-associated protein kinase C (PKC) activity declined under conditions of glucosylceramide synthase inhibition and increased under conditions of beta-glucosidase inhibition. The changes in PKC activity were evident after DEAE-cellulose purification. Diacylglycerol levels increased in response to both glucosylceramide synthase and beta-glucosidase inhibition. Ceramide and sphingosine levels changed only in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, increasing due to lack of conversion to glucosylceramide. However, the elevation in endogenous sphingosine was probably insufficient to account for the decrease in PKC, considering the high level of diacylglycerol in the cells. These data demonstrate an association between glucosylceramide levels, PKC activity, and cell growth.

Animals↗
PubMed1991

Sequence of an operon containing a novel delta-endotoxin gene from Bacillus thuringiensis.

An insect larval toxin designated CryII is produced by several subspecies of Bacillus thuringiensis and differs from the other major delta-endotoxins in these bacteria in its size, toxicity profile and presence as part of an operon with three open reading frames (ORF). Such an operon from a novel B. thuringiensis isolate has been cloned and differs from one previously characterized in the following ways: (a) the size and number of amino acid repeats in one of the ORFs; (b) the smaller size of the CryII protoxin and the presence of a unique 110-kDa CryII-related antigen; and (c) high larvicidal activity for a particular Lepidopteran but low activity for a Dipteran. Various subclones of this operon were introduced into a plasmid-free B. thuringiensis strain and only the cryII gene was found to be necessary for protoxin accumulation.

Amino Acid Sequence↗