Biomedical subjects

Z Wang

Publications and source records attributed to Z Wang.

At least 19 recordsLinked to original sources

PubMed1992

Characterization of the Ca(2+)-triggered conformational transition in troponin C.

Troponin C is the Ca(2+)-binding subunit of troponin in vertebrate striated muscle. Binding of Ca2+ to troponin C is thought to induce a conformational change that triggers subsequent events in the initiation of muscle contraction. A molecular modeling study has proposed that, when Ca2+ binds to the N-terminal triggering sites, helices B and C separate from the helices D and A, thereby exposing a crucial interaction site for troponin I, the inhibitory subunit of troponin [Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644]. In the present study the question of whether this separation actually occurs is addressed directly. A mutant rabbit skeletal troponin C containing a pair of cysteines at position 12 in helix A and position 49 in the polypeptide segment linking helices B and C was created by site-directed mutagenesis. Pyrene excimer fluorescence and resonance energy transfer studies on the labeled mutant troponin C reveal a Ca(2+)-induced increase in distance between the two cysteines. Under certain assumptions, the distance increase could be estimated from the extent of energy transfer to be approximately 13 A, in good agreement with the distance increase predicted by molecular modeling. Our results provide further experimental support for the model proposed by Herzberg et al. (above).

Animals↗

PubMed1992

Fatty-acid-binding protein in locust flight muscle. Developmental changes of expression, concentration and intracellular distribution.

Fatty-acid-binding protein (FABP) from the flight muscle of the locust, Schistocerca gregaria, is similar to mammalian heart FABP in its primary structure and biochemical characteristics. We have studied developmental changes using enzyme-linked immunosorbent assays, RNA hybridization and electron microscopy of immunogold-labeled sections. Locust muscle FABP is the most abundant soluble muscle protein in fully developed adult locusts, comprising 18% of the total cytosolic protein. At the beginning of the adult stage, however, no FABP is detectable. Its concentration rises during the following 10 days, after which it reaches its maximal value. FABP mRNA is present shortly after adult ecdysis; its concentration increases for 10 days, before it diminishes and reaches a constant, low level, probably needed to maintain the established FABP level. The protein is abundant in cytosol and nuclei, but virtually absent in mitochondria.

Animals↗

PubMed1992

Functional analysis of the human platelet-derived growth factor A-chain promoter region.

The platelet-derived growth factor (PDGF) A-chain gene is a developmentally regulated gene that is expressed in high levels in a limited number of normal and transformed cell lines and in cells stimulated by cytokines, including PDGF itself. We have now analyzed potential regulatory elements in 3.6 kilobase pairs (kb) of the 5'-flanking sequences of the human PDGF A-chain gene using reporter gene constructs and transient transfection analyses. The region between base pairs (bp) -618 and +392 (relative to the transcription initiation site) is sufficient for optimal promoter activity. A highly G + C region containing three contiguous Sp1 binding sites between bp -150 and -33 contributes over 80% of promotor activity. DNase I footprinting analyses indicates that Sp1 binds to and protects over 57 bp of this G + C region. A functional serum response element is located within bp -477 and -468 and positively regulates induction of PDGF A by PDGF. A negative regulatory (silencer) element is located from -1.9 to -0.9 kb. The results suggest that the major constitutive expression of the PDGF A-chain gene requires a highly G + C-rich region containing three Sp1 binding sites and that induction of the PDGF A-chain gene by PDGF is mediated by a SRE located at bp -477 to -468.

Base Sequence↗

PubMed1992

The inverted hexagonal phase is more sensitive to hydroperoxidation than the multilamellar phase in phosphatidylcholine and phosphatidylethanolamine aqueous dispersions.

The effect of phase behaviour (hexagonal II phase and lamellar phase) on the peroxidation of membrane phospholipids has been investigated in dilinoleoyl phosphatidylcholine (DLPC)/dilinoleoyl phosphatidylethanolamine (DLPE) aqueous dispersions. Peroxidation was initiated with a water-soluble radical inducer 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPN). The phospholipid morphology was monitored by 31P-nuclear magnetic resonance (NMR). Phospholipid hydroperoxides (PCOOH and PEOOH) were determined by chemiluminescence high-performance liquid chromatography (CL-HPLC). In pH-induced phase transition systems, DLPE in the bilayer state was much less oxidized than in the hexagonal II state. In composition-induced phase transition systems, the formation of total hydroperoxides and the consumption of alpha-tocopherol in the hexagonal II phase were greater than in the bilayer phase. These data suggest that the hexagonal II phase is more sensitive to hydroperoxidation than the bilayer phase in phospholipid aqueous dispersions.

Chromatography, High Pressure Liquid↗

PubMed1992

Differential susceptibility of cultured human melanoma cell lines to enhancement by retinoic acid of intercellular adhesion molecule 1 expression.

The potential role of intercellular adhesion molecule 1 (ICAM-1) in the biology of human melanoma cells has stimulated interest in the characterization of its modulation. The present study has shown that the differentiating agent retinoic acid (RA) up-regulates ICAM-1 expression by melanoma cells in a dose- and time-dependent fashion. The enhancement of ICAM-1 cell surface expression is paralleled by an increase in ICAM-1 mRNA. Therefore, ICAM-1 represents an additional gene which may be transcriptionally regulated by RA. The five melanoma cell lines tested displayed a differential susceptibility to the modulation of ICAM-1 expression by RA, since the cell line MeWo did not change in its ICAM-1 expression following incubation with RA. Nevertheless, RA-insensitive as well as RA-sensitive melanoma cell lines displayed a higher increase in ICAM-1 expression following incubation with RA and cytokines than following incubation with each of them. Analysis of the distribution in the melanoma cell lines of retinoic acid receptors (RARs) showed a relationship between susceptibility to a RA-mediated increase of ICAM-1 expression and RAR beta expression, suggesting that the latter receptor may play a role in the phenomenon. RAR alpha and RAR gamma were present in RA-sensitive and -insensitive melanoma cell lines, suggesting that they play a role in the enhancement by RA of cytokine-mediated up-regulation of ICAM-1 expression. The melanoma cell lines we have described may represent a useful system for investigating the role of RAR in the regulation of gene expression and the mechanism(s) which underlie this effect.

Carrier Proteins↗

PubMed1992

Modulation of transcription of the platelet-derived growth factor A-chain gene by a promoter region sensitive to S1 nuclease.

Homodimers of the platelet-derived growth factor (PDGF) A-chain are strong mitogens for cells of mesenchymal origin. Differences in the levels of expression of the PDGF A-chain gene have been reported in both normal and transformed cell lines, suggesting that transcription of the PDGF A-chain gene is highly regulated. We have now identified two S1-hypersensitive sites which flank a 13-base pair oligo(dG).oligo(dC) sequence located 70-82 base pairs upstream of the transcription initiation site. Three lines of evidence suggest that these S1-sensitive sites contribute to optimum promoter activity. Nuclear protein(s) binding to these sites were detected in gel mobility shift assays. Deletion of the S1-sensitive sites results in a 2-3-fold decrease in the transcriptional activity and eliminated sensitivity to S1 nuclease. Deletions in the oligo(dG).oligo(dC) motif also eliminated sensitivity to S1 and resulted in a 2.5-fold decrease of the promoter activity in the stable transfection assays. The results suggest that the highly G+C-rich region in the PDGF A-chain gene promoter locally induces the formation of non-B-form DNA under torsional stress which appears to be important in the transcriptional regulation of the PDGF A-chain gene in vivo.

Base Sequence↗

PubMed1992

1H NMR and NOE studies of the purple acid phosphatases from porcine uterus and bovine spleen.

The diiron active sites of the purple acid phosphatases from porcine uterus (also called uteroferrin, Uf) and bovine spleen (BSPAP) and their complexes with tungstate are compared by 1H NMR and NOE techniques. The paramagnetically shifted features of the 1H NMR spectrum of reduced BSPAP are similar to those of reduced Uf, while the spectra of the tungstate complexes are almost identical. These observations suggest that the two active sites are quite similar, in agreement with the greater than 90% sequence homology found in the two enzymes. Nuclear Overhauser effect (NOE) experiments on the His N-H resonances show that the Fe(III)-His residue is N epsilon-coordinated, while the Fe(II)-His is H delta-coordinated in both enzymes. On the basis of the above NMR and NOE results, our previously proposed model for the dinuclear iron active site of Uf [Scarrow, R. C., Pyrz, J. W., & Que, L., Jr. (1990) J. Am. Chem. Soc. 112, 657-665] is corroborated, refined, and found to represent the diiron center of BSPAP as well.

Acid Phosphatase↗

PubMed1992

Inositol trisphosphate-induced hyperpolarization in rat dorsal root ganglion neurons.

Inositol 1,4,5-trisphosphate (1,4,5-InsP3) was perfused into rat dorsal root ganglion (DRG) neurons by whole-cell patch-clamp electrodes, while measuring the membrane potential. This operation evoked a transient (2-3 min) membrane hyperpolarization of about -15 mV (from -42 mV) followed by a depolarization. The membrane hyperpolarization was abolished when 30 mM EGTA was perfused together with 1,4,5-InsP3 or when 0.2 mM quinine was added to the bath solution. The hyperpolarizing response was enhanced when a low-Ca2+ EGTA-free intracellular solution was used. Two InsP2 isomers induced a different response. Our results suggest that the hyperpolarization is due to 1,4,5-InsP3-induced Ca2+ release which may trigger Ca-sensitive K+ channels to open. Present results show that cultured DRG neurons are able to respond to 1,4,5-InsP3 perfusion in the whole-cell configuration.

Animals↗

PubMed1992

Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae.

Excision repair of DNA is an important cellular response to DNA damage caused by a broad spectrum of physical and chemical agents. We have established a cell-free system in which damage-specific DNA repair synthesis can be demonstrated in vitro with nuclear extracts from the yeast Saccharomyces cerevisiae. Repair synthesis of UV-irradiated plasmid DNA was observed in a radiation dose-dependent manner and was unaffected by mutations in the RAD1, RAD2, RAD3, RAD4, RAD10, or APN1 genes. DNA damaged with cis-platin was not recognized as a substrate for repair synthesis. Further examination of the repair synthesis observed with UV-irradiated DNA revealed that it is dependent on the presence of endonuclease III-sensitive lesions in DNA, but not pyrimidine dimers. These observations suggest that the repair synthesis observed in yeast nuclear extracts reflects base excision repair of DNA. Our data indicate that the patch size of this repair synthesis is at least seven nucleotides. This system is expected to facilitate the identification of specific gene products which participate in base excision repair in yeast.

Cell Nucleus↗

PubMed1992

Examination of the functional roles of 5 highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides.

The cytochrome b subunit of the bc1 complexes contains two cytochrome components (bL and bH) and is the locus of both a quinol-oxidizing site (Qo or Qz) and a quinone-reducing site (Qi or Qc). Sequence alignments of this subunit from over 20 eukaryotic and prokaryotic species have revealed a remarkable degree of conservation, including approximately 20 totally conserved residues. In this paper, site-directed mutagenesis has been used to examine the structural or functional roles of 5 of these highly conserved residues, Gly48, Gln58, Ser102, Phe104, and Pro202, all predicted to be within transmembrane alpha-helical segments. The mutants were made in the bc1 complex of Rhodobacter sphaeroides, a photosynthetic bacterium. The ability to use spectroscopic, electrochemical, and flash-induced kinetic methods allows the mutants to be analyzed for influences both on cytochrome spectra and thermodynamic properties and on the kinetics of specific electron transfer reactions. The results show that none of the 5 residues is absolutely essential. Substitution of aspartate or valine for Gly48 results in the loss of photosynthetic growth. The G48V mutant assembles a bc1 complex, but with modified cytochromes bH and bL, and a dysfunctional quinone reductase (Qc) site; an alanine is tolerated at this position. Possibly, a small residue is important here for heme packing. Gln58 and Ser102 are the only highly conserved polar residues predicted to be within the transmembrane spans, apart from the histidines which are heme axial ligands. Neither Gln58 nor Ser102 is essential for assembly or function of the bc1 complex, although substitution of other amino acids in these positions does cause subtle, but measurable changes. Phe104 lies midway between the axial ligands to cytochromes bL and bH and can be modeled to project in the space separating the two hemes. Replacement of this highly conserved aromatic residue by isoleucine has no measurable influence on the rate of electron transfer through the cytochrome b chain containing the two hemes. Finally, Pro202 is a totally conserved proline which is in the middle of transmembrane helix D, in between the 2 histidines which provide ligands to the hemes. No major inhibition of electron transfer resulted from replacing this proline by a leucine, although subtle changes in spectra of the b cytochromes and their electrochemical properties were noted.

Amino Acid Sequence↗

PubMed1992

Polypoid lesions of the gallbladder: diagnosis and indications for surgery.

One hundred and eighty-two patients with an ultrasonographic and/or pathological diagnosis of polypoid lesions of the gallbladder (PLG) were reviewed to determine the reliability of ultrasonography in the diagnosis of PLG and the indications for operation in this disease. Of the 182 patients operated on, PLG were demonstrated by the gross appearance of the resected gallbladder in 172. Histologically benign lesions were present in 159 gallbladders and malignant lesions in 13. Cholesterol polyps accounted for most benign PLG. The sensitivity of ultrasonography in detecting PLG was 90.1 per cent, significantly higher than that of oral cholecystography, computed tomography or endoscopic retrograde cholangiopancreatography (P less than 0.01). The specificity of ultrasonography in the diagnosis of PLG was 93.9 per cent. Therefore, ultrasonography is a highly sensitive method for investigating PLG, and the preoperative diagnosis of PLG in this unit has now become entirely dependent on this technique. The size and number of PLG, the presence of gallstones and the patient's age all correlate with the nature of PLG, and these features are helpful in differentiating malignant from benign lesions before operation. Surgical treatment is indicated when PLG exceed 1.0 cm in diameter, when PLG are single in number, when PLG are associated with gallstones, when patients with PLG are over the age of 50 years, or when clinical symptoms of PLG are apparent.

Adolescent↗

PubMed1992

Movement of fluorescence pattern after photobleaching: an accelerated procedure for DNA electrophoretic mobility analysis.

A new approach which is compatible with many of the existing procedures for the analysis of DNA species in gel electrophoresis is being demonstrated. It takes advantage of fluorescence photobleaching in order to create a sharp boundary between the stained and the (partially) photobleached DNA. By arbitrarily creating a stained DNA band of narrower width, the sensitivity to detect (averaged) DNA band movements has been increased. This feature permits measurements of time-dependent electrophoretic mobility over very short time periods. The approach can be used to shorten the running time of gel electrophoresis experiment and to increase the resolution because of the sharper boundary and narrower band width. With faster running time, diffusion of both DNA and dye in the gel also becomes less serious. Movement of fluorescence pattern after photobleaching also permits measurements of localized motions when the gel pores are small in comparison with DNA sizes. Experiments demonstrating some aspects of the proposed technique, as well as the anticipated limitations, are presented and discussed.

Autoanalysis↗

PubMed1992

Sensitivity of in vivo MRS of the N-delta proton in proximal histidine of deoxymyoglobin.

The sensitivity of in vivo MRS of the N-delta proton of the proximal histidine of deoxymyoglobin in human skeletal muscles is discussed. Longitudinal relaxation time T1 of this deoxymyoglobin signal was measured in cuffed human forearms at 1.5 T and found to be 9.9 ms. Deoxymyoglobin spectra can be obtained from a forearm in seconds. The detection sensitivity of deoxymyoglobin in fully ischemic skeletal muscles and that of 31P MRS of PCr in normal resting muscles are compared.

Adult↗

PubMed1992

BMY-14802, a sigma ligand and potential antipsychotic drug, reverses amphetamine-induced changes in neostriatal single-unit activity in freely moving rats.

The effects of BMY-14802 (5, 10, or 20 mg/kg), a sigma-receptor ligand showing preclinical evidence of antipsychotic efficacy, were tested on single-unit activity in the neostriatum of freely moving rats with or without pretreatment with 1.0 mg/kg D-amphetamine. Relative to resting baseline, amphetamine activated the large majority of neurons that changed firing rate in close temporal association with movement. All doses of BMY-14802 reversed this neuronal response, but the effect was most pronounced at 20 mg/kg. This dose, however, was equally likely to reverse or to induce a haloperidol-like potentiation of those neurons inhibited by amphetamine. In contrast, 10 mg/kg BMY-14802 consistently reversed amphetamine-induced neuronal inhibitions. All doses of BMY-14802 attenuated the locomotor effects of amphetamine, but only the higher doses also blocked other aspects of the amphetamine behavioral response. By itself, BMY-14802 dose dependently inhibited motor-related neurons, but elicited less behavioral activation than amphetamine. BMY-14802 (20 mg/kg) also induced hindlimb ataxia and occasional backwards locomotion. Haloperidol (1.0 mg/kg) reliably suppressed both behavior and neuronal activity when injected 30 min after BMY-14802, whether or not amphetamine pretreatment was given. Thus, BMY-14802 shares with other neuroleptics the capacity to reverse amphetamine-induced excitations of neostriatal motor-related neurons, whereas other effects of BMY-14802 reveal some haloperidol-like actions at 20 mg/kg that do not occur at lower doses.

Amphetamine↗

PubMed1992

Investigation of stroke in sickle cell disease by 1H nuclear magnetic resonance spectroscopy.

Localized proton nuclear magnetic resonance spectroscopy (MRS), obtained with stimulated echo and spin echo sequences, MR imaging (MRI) and MR angiography (MRA) were used to study the brain in 13 children and adolescents with sickle cell disease. Regions of interest (ROI) studied by MRS included regions appearing normal on MRI as well as regions showing complications of sickle cell disease, including focal deep white matter areas of high signal intensity (deep white matter ischemia, DWMI) seen on long TR images, focal atrophic brain areas, and infarcts. The findings in these studies are summarized as follows: Normal-appearing regions on MRI have normal MRS. In ROI including small areas of DWMI, lactate elevation was not detected, but the levels of N-acetyl-aspartate (NAA) appeared slightly elevated. In areas of DWMI 1-2 cm in size, reduced blood flow could be seen on MRA and lactate elevation could be detected with MRS. When blood flow to a DWMI region was normal, NAA was reduced and there was little lactate elevation, as cell death had already occurred. ROI consisting of atrophic tissue had reduced NAA levels but total creatine levels were not changed. Sometimes lipids, presumably from broken cell membrane, could be detected. In regions of past massive stroke, all metabolites were absent except for small amounts of lactate or lipids.

Adolescent↗

PubMed1992

Muscarinic receptor subtypes in equine tracheal smooth muscle.

Selective muscarinic receptor antagonists were used to identify muscarinic receptor subtypes in equine trachealis strips. The M1 receptor antagonist pirenzepine (10(-7) mol/L to 3 x 10(-5) mol/L) and the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 10(-9) mol/L to 3 x 10(-7) mol/L3) dose dependently inhibited the contractile responses to electrical field stimulation (EFS) and exogenous acetylcholine (ACh). Schild plots yielded a pA2 value for pirenzepine vs ACh of 6.75 +/- 0.09, which is consistent with the affinity for M2 or M3 receptors, and a pA2 value for 4-DAMP vs ACh of 8.47 +/- 0.09, which is in agreement with the affinity for M3 receptors. The M2 receptor antagonist gallamine (10(-5) mol/L and 10(-4) mol/L) did not affect the response of trachealis to exogenous ACh and low-frequency EFS (0.1-2 Hz) but decreased the responses to high-frequency EFS (4-16 Hz). These results suggest that the muscarinic receptors mediating contractions induced by ACh in equine tracheal smooth muscle are of the M3 subtype. The lack of an increase in the response to EFS following gallamine suggests that functional prejunctional inhibitory M2 receptors are not present on the cholinergic nerves innervating equine tracheal smooth muscle.

Acetylcholine↗

PubMed1992

A simple electronic device for time-lapse recording of neural and other cell movements using a home video cassette recorder.

This article describes a simple electronic unit to obtain time-lapse recordings with the use of a common remote-controlled home video cassette recorder, for example a VHS recorder. The electronic unit is a timer to be connected to the remote-control unit. The video cassette recorder itself remains unchanged. Replay of the recorded images speeds up the original process by a factor of 2-100 x or more. This technique has been applied in video micrographic studies of (1) the development of dorsal root ganglion (DRG) cells in culture, including growth cone and Schwann cell movements, and (2) tumor cell killing by natural killer (NK) cells.

Animals↗

PubMed1992

Alpha 2 adrenergic receptor subtypes in depression: a candidate gene study.

Alpha 2 adrenergic receptors play an important role in regulating the neuronal release of norepinephrine through presynaptic feedback inhibition in the locus ceruleus. Therefore, alpha 2 adrenergic autoreceptors may underlie some aspects of the pathogenesis and symptomatic expression of depressive illness. We studied two brain-expressed alpha 2 adrenergic receptor genes as genetic markers in linkage analyses in 17 multiplex pedigrees of unipolar depression. Neither of the genes was supportive of linkage to depression. Lod scores of less than -2 were found in both familial pure depressive disease pedigrees and in depression spectrum disease pedigrees. Therefore, we conclude that depression in our pedigrees is not related to mutations in the two alpha 2 adrenergic receptor genes tested.

Adult↗