Biomedical subjects

D Wu

Publications and source records attributed to D Wu.

At least 415 records · Page 23Linked to original sources

PubMed1995

The impact of heavy metals from environmental tobacco smoke on indoor air quality as determined by Compton suppression neutron activation analysis.

The method of instrumental neutron activation analysis (NAA) has been improved for air filter samples in the determination of low level heavy metals in indoor air. By using the techniques of epithermal neutron irradiation in conjunction with Compton suppression, the detection limits of cadmium, arsenic and antimony measurements have been dramatically reduced to 2 ng for Cd, 0.2 ng for As, and 0.03 ng for Sb. The determination of these heavy metals in particulate material generated from cigarette smoking in indoor environments has been conducted. Other elements, Br, Cl, Na, K, Zn were also found at elevated levels.

Air Pollution, Indoor↗

PubMed1995

Activation of rat choline acetyltransferase by limited proteolysis.

In the past, purification of choline acetyltransferase (ChAT, EC 2.3.1.6.), the enzyme responsible for the biosynthesis of the neurotransmitter acetylcholine, has yielded fragmented species of the enzyme. The nature and possible function of these forms of ChAT are not well understood. Using a bacterial expression system, recombinant rat ChAT in its active form has been purified to homogeneity. The purified enzyme was found to be activated to >25-fold when assayed at low ionic strength and >5-fold when assayed at high ionic strength by limited proteolysis with either trypsin or chymotrypsin, but not with proteinase K. The activated ChAT shows an increased Km for both substrates, diminished sensitivity to salt activation and a pH optimum that is shifted approximately 1 pH unit. On a denaturing SDS-polyacrylamide gel, the activated ChAT is composed of three to four polypeptides; however, it migrates as an intact 68-k-Da protein species on gel filtration. In order to delineate the site of cleavage by proteolysis, the newly generated fragments have been subjected to N-terminal sequencing. By comparing cleavage sites between trypsin and chymotrypsin, the putative activation sites were identified.

Amino Acid Sequence↗

PubMed1995

Selective coupling of beta 2-adrenergic receptor to hematopoietic-specific G proteins.

The coupling of the beta 2-adrenergic receptor (AR) to the alpha subunits of the Gq class of G proteins was investigated in a cotransfection system. COS-7 cells cotransfected with the beta 2-AR cDNA and the G alpha 15 or G alpha 16 cDNA showed marked norepinephrine-induced increases in accumulation of inositol phosphates in a concentration-dependent manner. However, cells cotransfected with the cDNA encoding G alpha q, G alpha 11, or G alpha 14 instead of G alpha 16 gave no ligand-dependent activation of phospholipase C (PLC). The facts that the beta-AR agonist isoprenaline can also induce activation of PLC in cells coexpressing beta 2-AR and G alpha 16 and that the beta 2-AR-specific antagonist propranolol can block norepinephrine-induced activation of PLC in these cotransfected cells further indicate that it is the beta 2-AR that mediates the activation of phospholipase C in these cotransfected cells. To test the possibility of involvement of G beta gamma, a G beta gamma antagonist, G gamma 3 mutant with substitution of a Ser residue for the C-terminal Cys residue, was used because this protein, when expressed in COS-7 cells, can inhibit only G beta gamma-mediated but not G alpha-mediated activation of PLC. The result that the G gamma 3 mutant could not inhibit beta 2-adrenergic receptor-mediated activation of PLC in cells cotransfected with the G alpha 16 cDNA suggests that G beta gamma is unlikely to be a major mediator of beta 2-adrenergic receptor-induced activation of PLC. Thus, we conclude that the beta 2-adrenergic receptor can specifically couple to G alpha 15 and G alpha 16, but not to G alpha q, G alpha 11, or G alpha 14 to activate PLC.

Animals↗

PubMed1995

Different alpha 1-adrenergic receptor sequences required for activating different G alpha subunits of Gq class of G proteins.

In order to understand the specific interactions between receptors and guanine nucleotide-binding regulatory protein (G proteins), we attempted to delineate the alpha 1 B-adrenergic receptor sequences involved in activation of the alpha subunits of the Gq class of G proteins. A number of specific mutations were introduced into the third inner loop of the receptor, and the mutants were tested for their abilities to activate different G alpha subunits of the Gq class. Our results indicate that the receptor sequences required for activating G alpha q/11, G alpha 14, or G alpha 16 are different. The sequence extending from residues Lys240 to His252 is required for activation of G alpha q/11, but not for activation of G alpha 14 or G alpha 16. Two segments in the third loop of the receptor are required for activation of G alpha 14: one is located at the N terminus of the loop ending at residue Asn226, and the other is located at the C terminus of the loop starting from residue Ser278. The latter contains a BBXXB motif, which is apparently critical for G alpha 14 coupling, but not for G alpha 16 or G alpha q/11 coupling. Furthermore, the three amino acids stretch (Tyr217 to Val219) included in the N-terminal segment is not only required for G alpha 14 coupling, but also for G alpha q/11 coupling. It may be involved to some extent in G alpha 16 coupling as well.

Amino Acid Sequence↗

PubMed1995

Novel Saccharomyces cerevisiae gene, MRK1, encoding a putative protein kinase with similarity to mammalian glycogen synthase kinase-3 and Drosophila Zeste-White3/Shaggy.

A Saccharomyces cerevisiae gene was identified that would encode a protein similar to the mammalian protein kinase glycogen synthase kinase-3 (GSK-3) and the Drosophila Zeste-White3/Shaggy gene product. The open reading frame predicts a 375 amino acid polypeptide with a putative protein kinase domain that displays 70% and 39% identity, respectively, to two known yeast proteins, Mds1p and Mck1p. The new gene, designated MRK1 (Mds1p Related Kinase), is located on chromosome IV. Disruption of MRK1 was not lethal and did not elicit any alteration in glycogen accumulation. In addition, an mck1 mds1 mrk1 triple disruptant was viable.

Amino Acid Sequence↗

PubMed1995

The N terminus of phosducin is involved in binding of beta gamma subunits of G protein.

Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland. It binds to the beta gamma subunits of guanine nucleotide-binding proteins (G proteins) (G beta gamma) and may regulate G-protein function. In this study, the ability of specific regions of phosducin to bind G beta gamma was characterized. A series of deletion mutants were made in bovine phosducin. They were tested in cotransfection assays for their ability to inhibit G beta gamma-mediated phospholipase C beta 2 isoform activation. Overexpression of the N-terminal half of phosducin showed inhibition, whereas overexpression of the C-terminal half did not. The first 63 amino acid residues were required for inhibition. A tryptophan-to-valine substitution at residue 29, which is part of a well conserved 11-amino acid sequence, severely impaired phosducin inhibitory function. Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-G beta gamma interaction in vitro. The N-terminal 63-amino acid fragment was able to bind to G beta gamma. In contrast, the C-terminal half failed to bind to G beta gamma. The substitution mutants showed little or no binding. Furthermore, direct measurements of interaction between G beta gamma and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residue.

Amino Acid Sequence↗

PubMed1995

Na+ transport processes in isolated guinea pig nasal gland acinar cells.

In the dispersed acinar cells of the submucosal nasal gland in the guinea pig, intracellular Na+ concentration ([Na+]i) was measured with a microfluorimetric imaging method and the cytosolic indicator dye, sodium-binding benzofuran isophthalate, under HCO3(-)-free conditions. In the unstimulated condition, the [Na+]i was averaged to 12.8 +/- 5.2 mM. Addition of 100 microM ouabain or removal of external K+ caused an increase in [Na+]i. Replacement of external Cl- with NO3- or addition of 0.5 mM furosemide reversibly decreased the [Na+]i. The recovery process from the reduced [Na+]i was inhibited by removal of either K+ or Cl- in the bath solution. These findings indicate the presence of a continuous influx of Na+ coupled with K+ and Cl- movement. Application of acetylcholine (ACh, 1 microM) caused an increase in [Na+]i by about 15-20 mM, which was completely inhibited by addition of 10 microM atropine. Increased cytosolic Na+ induced by ACh was extruded by the Na(+)-K+ pump. Removal of external Cl- and addition of 50 microM dimethylamiloride inhibited ACh-induced increase in [Na+]i by about 66% and 19%, respectively. In both unstimulated and stimulated state, Na(+)-K+ pump, Na-K-Cl cotransport, and Na(+)-H+ exchange play a critical role in maintaining intracellular electrolyte environment and in controlling a continuous secretion of nasal fluids.

Acetylcholine↗

PubMed1995

Improved production of the insecticidal CryIVD protein in Bacillus thuringiensis using cryIA(c) promoters to express the gene for an associated 20-kDa protein.

Previous studies have shown that a 20-kDa protein enhances production of the insecticidal CytA and CryIVA proteins of Bacillus thuringiensis in Escherichia coli as well as CytA production and crystal formation in B. thuringiensis. To determine whether the 20-kDa protein could enhance CryIVD production, an expression vector was constructed with the 20-kDa open-reading frame under control of cryIA(c) promoters and the cryIVD gene under control of its own promoter. Acrystalliferous cells of B. thuringiensis transformed with this plasmid, designated pWF53, produced large bitrapezoidal CryIVD crystals that averaged 1.3 x 0.92 x 0.31 microns, approximately fivefold larger in volume than wild-type CryIVD crystals, and 1.7 fold the volume of crystals produced using the cryIVD operon, which contains the cryIVD gene and the gene for the 20-kDa protein. These results demonstrate that the 20-kDa protein significantly improves net synthesis of CryIVD and promotes CryIVD crystal formation. Improved production of proteins as diverse as CryIVD and CytA by the 20-kDa protein indicates this protein may be useful in facilitating the production of other proteins.

Bacillus thuringiensis↗

PubMed1995

Cellular mechanisms in activation of Na-K-Cl cotransport in nasal gland acinar cells of guinea pigs.

The cellular regulation mechanism of Na-K-Cl cotransport was studied in dispersed acinar cells of the guinea pig nasal gland by a microfluorimetric imaging method using the Na(+)-sensitive dye sodium-binding benzofuran isophthalate. Addition of 1 micron acetylcholine (ACh) induced an immediate increase in intracellular Na+ concentration ([Na+]i) by 36.7 +/- 9.9 mM, which was almost completely abolished by the addition of atropine. The increased [Na+]i after cholinergic stimulation was due to the external (Cl-)-dependent cotransport system (about 80% of the total Na+ influx) and the dimethyl amiloride-sensitive (Na+)-H+ exchange system (of about 20%). The ACh-induced increase in [Na+]i was dependent on extracellular Ca2+ and was prevented by pretreatment with 8-(N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate or O-O'-bis(2-aminophenyl)ethyleneglycol-N, N, N', N'-tetraacetic acid tetraacetoxymethylester. Addition of 1 microns ionomycin mimicked the ACh-induced increase in [Na+]i which was dependent on external Cl-. Moreover, both a calmodulin antagonist trifluoperazine and a myosin light chain kinase inhibitor ML-7 reduced the ACh-induced response in [Na+]i. However, the following treatment did not affect the basal [Na+]i nor the ACh-induced increase in [Na+]i: (i) addition of dibutyryl cAMP, 8-Br-cGMP, or phorbol 12-myristate 13-acetate, (ii) pretreatment of protein kinase inhibitors, H-89, H-8, H-7 or chelerythrine, (iii) prevention of cytosolic Cl- efflux by the addition of diphenylamine-2-carboxylic acid or, (iv) prevention of cytosolic K+ efflux by the addition of charybdotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1995

Multiplane transesophageal echocardiography in the diagnosis of congenital coronary artery fistula.

The purpose of this study was to examine the advantages of multiplane transesophageal echocardiography in the diagnosis of congenital coronary artery fistula, specifically in depicting the origin, the course, and the drainage site. Seven consecutive patients ranging in age from 20 to 72 years with a suspected coronary artery fistula underwent conventional transthoracic and multiplane transesophageal echocardiographic studies between March 1993 and July 1994. When a coronary artery fistula was noted, the origin, the course, and the drainage site were carefully searched for. All patients then underwent a cardiac catheterization with the performance of coronary angiography. A large right coronary artery fistula was detected in three patients; one of them had a drainage to the posterior left ventricle, one to the lateral right ventricle, and the other to the medial aspect of the right ventricle just below the insertion of the septal leaflet of the tricuspid valve. A small coronary artery fistula arising from the left coronary artery was noted in four patients, two from the left anterior descending artery and the other two from the left circumflex artery. Three of these four patients had a drainage to the main pulmonary artery and one to the left ventricle. The drainage site was clearly depicted in all seven patients, whereas the origin and the course were precisely defined in five patients by using multiplane transesophageal echocardiographic examination. The multiplane transesophageal echocardiography provides a panoramic view of the coronary artery and the fistulous vessel with a precise definition of the origin, the course, and the drainage site of the fistula. Therefore it is the noninvasive diagnostic mode of choice.

Adult↗

PubMed1995

A combined casein-free-nicotinamide diet prevents diabetes in the NOD mouse with minimum insulitis.

We have previously shown that diabetes in the NOD mouse can be prevented if mice are placed from weaning on an infant formula diet in which the protein source is replaced with casein hydrolysate (Pregestimil) or soy protein (Prosobee), or if 1% nicotinamide is given in the drinking water. Nicotinamide somewhat suppresses insulitis but the hydrolysed casein formula does not. In this study, Prosobee was given concurrently with oral nicotinamide from weaning and their effects on the development of insulitis and diabetes measured. These effects were also assessed in mice given Prosobee alone from conception (day -20) or from weaning. Unlike the earlier experiments, a marked suppression of insulitis was observed when the diets and nicotinamide were given concurrently (mean insulitis scores +95% confidence intervals (back transformed): day 40 = 0.4% [0.03, 1.17] vs. 12.5% [2.52, 28.40] and at day 90 = 8.8% [3.65, 15.68] vs. 48.1% [33.89, 62.49], P = 0.0001). A similar suppression was observed on day 90 with Pregestimil combined with nicotinamide 7.3% [3.88, 11.70] vs. 43.8% [32.59, 55.35] (P = 0.0001). Qualitatively, introduction of Prosobee from conception appeared to elicit a greater degree of suppression of insulitis than when introduced from day 21. Insulitis lesions were examined immunohistochemically for CD4, CD8 and MAC-1 cells. The proportion of these cells was not different for any regime despite the great differences in total number of inflammatory cells in and around the islets of mice fed the combined diet. All the three dietary treatments (Prosobee from day -20, Prosobee from day 21, Prosobee+nicotinamide from day 21) resulted in substantial protection from diabetes in mice followed until 250 days. We conclude that the complete prevention of diabetes in the NOD mouse fed a casein-free diet together with nicotinamide is accompanied by marked inhibition of insulitis, which is not seen when either dietary agent is introduced alone. The somewhat greater suppression of insulitis in mice given the soy diet from conception compared to those fed from day 21 may indicate that even maternal diet during gestation may influence diabetes outcome in the offspring.

Animals↗

PubMed1995

A cell type-specific silencer in the human choline acetyltransferase gene requiring two distinct and interactive E boxes.

We have previously reported that cholinergic neuron-specific expression of the human choline acetyltransferase gene is mediated by two co-operative silencers. We have now localized the proximal silencer to the region from nucleotide -2195 to -2409, which contains two distinct E boxes (CACCTG and CATGTG). Deletion or mutation of either of these E boxes results in a loss of silencer activity. There are specific nuclear proteins in adrenergic cells which bind to each of the two E boxes. However, nuclear proteins from cholinergic cells only bind the 5' E box not the 3' E box. It is this interaction which appears to be the cause of the inactivity of this silencer in these cells.

Base Sequence↗

PubMed1995

Progression to complete atrioventricular block in a patient with bundle branch re-entry tachycardia.

A 38-year-old man with no significant structural heart disease suffered from one episode of wide QRS tachycardia. The electrocardiogram showed a PR interval of 0.20 second and a QRS duration of 0.10 second. His bundle recording revealed an HV interval of 90-100 ms. The tachycardia was inducible with programmed stimulation and displayed a QRS morphology of complete left bundle branch block. It was characterized by an atrioventricular dissociation, a cycle length of 280 ms, and an H deflection preceding each QRS complex. Pacing from the right ventricular apex at a cycle length of 270 ms entrained the tachycardia, while at a cycle length of 260 ms, the tachycardia was terminated. Four years later, the patient presented with complete atrioventricular block with a wide QRS escape rhythm. An electrophysiologic study conducted while he was in 1:1 atrioventricular conduction showed an HV interval of 100 ms. Second-degree infrahisian block developed at an atrial paced cycle length of 700 ms. There was no induction of tachycardia with programmed stimulation before or after isoproterenol. The patient was treated with an implantation of a permanent pacemaker.

Adult↗

PubMed1995

New myrsinol-related polyfunctional pentacyclic diterpene esters from roots of Euphorbia prolifera.

Five Euphorbia substances, SPr1-SPr5, were isolated from the roots of Euphorbia prolifera. They were found to have similar structures but were inactive in a mouse ear inflammation assay. By nmr analysis and after single-crystal X-ray crystallography the structure of SPr5 was established as a hexaester (tetraacetate-benzoate-propionate) of a hitherto unknown polyfunctional pentacyclic diterpene parent alcohol, structurally related to myrsinol. As judged from its nmr spectra, SPr4 is an analogue of SPr5, carrying an isobutyrate substituent in place of a benzoate ester functionality. SPr1-SPr3 were partially characterized by their mass spectra as esters of diterpene parent alcohols possibly related to the myrsinol structure. SPr1-SPr5 may represent one of the product lines branching off the proposed main route of biogenesis of the oligocyclic diterpenoid skin irritants and tumor promoters occurring in many, but not all, of the species in the plant families Thymelaeaceae and Euphorbiaceae.

Animals↗

PubMed1995

Antioxidants and immune response in aged persons: overview of present evidence.

The oxidant-antioxidant balance is an important determinant of immune cell function, including maintaining the integrity and functionality of membrane lipids, cellular proteins, and nucleic acids and controlling signal transduction and gene expression in immune cells. Optimal amounts of antioxidants are needed for maintenance of the immune response across all age groups. This need might be more critical, however, in aged persons. Age-associated dysregulation of immune response, particularly of T cell-mediated function, is well documented. The well-known age-related increase in free radical formation and lipid peroxidation contributes, at least in part, to this phenomenon. We summarize animal and human studies undertaken by ourselves as well as other investigators on the effects of antioxidants, vitamin E, beta-carotene, and glutathione on the immune response of aged persons. The underlying mechanisms for the antioxidant nutrients' effects as well as their health implications for aged persons are discussed.

Adjuvants, Immunologic↗

PubMed1995

Temporal patterns of uncoupling between oxidative metabolism and regional cerebral blood flow demonstrated by functional magnetic resonance imaging.

RATIONALE AND OBJECTIVES: Brain activation can be detected by functional magnetic resonance imaging. Termination of stimulation can cause drop of postactivation signal below preactivation baseline; the nature of this "undershoot" remains controversial. The authors investigated postactivation signal after stimulation of the visual cortex with photic stimuli of different duration. METHODS: Activation of visual cortex in 11 healthy subjects was studied. The authors underwent short and long stimulation. The relation between activation and postactivation signal was investigated after both durations of stimulation. RESULTS: Average postactivation signal after short stimulation was -1.71 +/- 2.66% and after long stimulation 0.82 +/- 1.59% (P < or = 0.01). Significant "undershoot" was detected after short stimulation in the majority of cases, although this was typically absent after long stimulation. CONCLUSIONS: The authors propose that "undershoot" is caused by decreased oxygen content in venous blood compared with the preactivation level and may reflect increased extraction of oxygen as a result of glycolytic metabolism. The absence of "undershoot" after longer stimulation suggests a gradual shift from uncoupling between regional cerebral blood flow and oxygen consumption toward a steady state.

Adult↗

PubMed1995

Pharmacokinetics of dextromethorphan and metabolites in humans: influence of the CYP2D6 phenotype and quinidine inhibition.

Dextromethorphan is primarily metabolized to dextrorphan by cytochrome P450 2D6 (CYP2D6), a genetically polymorphic enzyme in humans. Dextrorphan is an active metabolite that produces phencyclidine-like behavioral effects in animals and exhibits anticonvulsant and neuroprotective properties in a variety of experimental models. In these studies, we examined the effects of CYP2D6 phenotype and quinidine inhibition on the pharmacokinetics of dextromethorphan and its metabolites in humans. After a single oral dose of dextromethorphan HBr (30 mg), the major metabolites in the plasma of extensive metabolizers (N = 5) were conjugated dextrorphan and conjugated 3-hydroxymorphinan. Free dextrorphan concentrations were about 100-fold less than the conjugated dextrorphan, and dextromethorphan was not detectable. Pretreatment of these subjects with 100 mg of quinidine, a selective inhibitor of CYP2D6, significantly suppressed the formation of dextrorphan and elevated the concentrations of dextromethorphan (t1/2, 16.4 hours). In poor metabolizers (N = 4) given the same dose, dextromethorphan was the major component in the plasma with a t1/2 of 29.5 hours. Present at concentrations 5- to 10-fold less were conjugated dextrorphan and the other two metabolites. Urinary recovery studies indicated that the inhibition by quinidine was reversible and that the elimination of dextromethorphan primarily depends on CYP2D6 activity rather than renal elimination. These data demonstrated that the CYP2D6 phenotype and the concurrent administration of quinidine significantly affect the disposition of dextromethorphan and the formation of the active metabolite dextrorphan and are important factors to be considered in studies of the pharmacologic and behavioral effects of dextromethorphan.

Adult↗

PubMed1995

Immunohistochemical analyses of pancreatic macrophages and CD4 and CD8 T cell subsets prior to and following diabetes in the NOD mouse.

CD4 and CD8 T cells and macrophages have been implicated as cellular mediators of beta cell destruction in insulin-dependent diabetes mellitus (IDDM). The ratios of the two T cell subsets were, therefore, quantified in nonobese diabetic (NOD) mouse islets prior to IDDM, at the onset of the disease, and following onset by immunohistochemistry. The number of periislet-, intraislet-, and exocrine-located macrophages were also determined during these stages. At all time points studied (day 90, day 250, at diabetes onset, 4-6 weeks after diabetes), CD4 cells were 2.5 times higher than CD8 cells except at day 250, on which the CD4:CD8 ratio was 3.8. At days 90 and 250, islets were heavily infiltrated with both the T cell subsets associated with lower numbers of macrophages. At onset of the disease and after insulin treatment, although the CD4:CD8 ratios were similar, the absolute numbers of the two subsets were reduced-considerably. At these stages a majority of the islets was atrophied, some were still surrounded by T cells and macrophages and were enriched with glucagon cells. CD4 and CD8 cells were also observed in the exocrine region at the two stages. Macrophages located in the periislet areas were significantly higher in number than in the intraislet positions in all study groups (p = 0.001). They also showed a gradual decline from day 90 to clinical diabetes. Periislet-located macrophages were significantly higher at day 90 than after onset and in control Swiss mice (p < 0.05). The numbers of macrophages in the exocrine areas were similar in all groups of NOD mice. In control Swiss mice they were significantly lower in the periislet, intraislet, and exocrine regions.

Animals↗