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Biomedical subjects

J Zheng

Publications and source records attributed to J Zheng.

At least 325 records · Page 18Linked to original sources
PubMed1997

Application of a 3D volume 19F MR imaging protocol for mapping oxygen tension (pO2) in perfluorocarbons at low field.

A limited flip angle gradient-echo 3D volume acquisition imaging protocol for mapping partial pressure of oxygen (pO2) in perfluorocarbon compounds (PFCs) at low field (0.14 T) is presented. The PO2 measurement method is based on the paramagnetic effect of dissolved molecular oxygen (O2) which reduces the PFC 19F T1. Specific objectives related to imaging of PFCs through use of the protocol include improved image signal-to-noise characteristics and elimination of 19F chemical shift artifacts. A parametric Wiener deconvolution filtering algorithm is used for suppression of 19F chemical shift artifacts. Application of the protocol is illustrated in a series of calculated PO2 maps of a gas equilibrated, multi-chamber phantom containing perfluorotributylamine (FC-43). The utility of the protocol is demonstrated in vivo through images of a commercially available perfluorocarbon based blood substitute emulsion containing FC-43 sequestered in the liver and spleen of a rat.

Algorithms↗
PubMed1997

Anti-lipid peroxidization effect of huangdan on chronic renal failure in rats.

The activity of superoxide dismutase (SOD) in renal cortex and erythrocytes, the level of lipid peroxidant (LPO) in plasma, renal function and morphology of leftover kidney were examined in rats. The results showed that Huangdan capsule could slow down the increase of BUN and Scr, promote the SOD activity in renal cortex and erythrocytes, reduce the level of LPO in plasma and delay the renal pathological changes in 5/6 nephrectomized rats. The mechanism of Huangdan capsule's antiperoxidant effect, its effect on the renal pathological changes and relationship between the two effects were also discussed.

Animals↗
PubMed1997

Enhancement of cell growth on a porous membrane co-immobilized with cell-growth and cell adhesion factors.

Insulin was co-immobilized with cell adhesion factors such as fibronectin or polylysine on a porous poly(ethylene terephthalate) membrane. The surface of the poly(ethylene terephthalate) membrane was partially hydrolysed under alkaline conditions and the proteins were immobilized on the membrane surface using a water-soluble carbodiimide. The adhesion of STO mouse fibroblast cells onto the immobilized membrane was accelerated by the immobilization of fibronectin and polylysine. The cell growth was enhanced by immobilization of insulin. Co-immobilization of insulin with the adhesion factors markedly accelerated cell growth. No significant differences were found between the effects of fibronectin and polylysine. The absence of insulin release was confirmed by the fact that growth was no accelerated under essentially the same culture conditions without direct contact with the protein-immobilized membrane.

Animals↗
PubMed1997

Isolation of an enriched fraction of nerve terminals from fish brain using a discontinuous Percoll gradient.

A discontinuous gradient of Percoll has been used to prepare a subcellular fraction (fraction III) from fish brain which is greatly enriched in synaptic nerve endings (synaptosomes). Synaptosomes display well-preserved morphology, good functional performance as assessed by ability to release preloaded neurotransmitter following depolarization, and the fraction as a whole has an enzyme profile consistent with the presence of nerve endings. The procedure is rapid, highly reliable and provides a mean yield of 4.5 mg synaptosomes per g wet weight of brain.

Acetylcholinesterase↗
PubMed1997

Demonstration of membrane estrogen binding proteins in rat brain by ligand blotting using a 17beta-estradiol-[125I]bovine serum albumin conjugate.

This paper describes a ligand blotting procedure to visualize membrane estrogen receptors/binding proteins immobilized on nitrocellulose membranes. Using 17beta-estradiol covalently linked with [125I]bovine serum albumin (BSA) at the C-6 position (17beta-E-6-[125I]BSA) as a ligand, three major binding proteins with molecular masses of approximately 23, 28, and 32 kDa were identified from crude synaptosomal fractions (P2) of female rat brains. The binding of 17beta-E-6-[125I]BSA to these proteins is selective for 17beta-estradiol because BSA had no effect, and 17alpha-E-6-BSA was at least two orders of magnitude less potent than 17beta-E-6-BSA in displacing the binding. In addition, [125I]BSA and 17alpha-E-6-[125I]BSA at similar concentrations did not bind to these proteins. Competition and saturation assays indicate that the binding affinity of 17beta-E-6-[125I]BSA for these proteins was in the range of 1-10 nM. These proteins are not contaminants from cytosolic or serum estrogen binding proteins since no corresponding protein bands were found in cytosolic fractions. Three additional protein bands with molecular masses of approximately 18, 40, and 130kDa were also detected, although inconsistently. The 23 and 40 kDa proteins seem to be concentrated in mitochondrial fractions (mP2), whereas the 28 and 32 kDa proteins are enriched in microsomal fractions (P3). Application of digitonin-solubilized P2 fractions to 17beta-estradiol-coupled affinity columns resulted in significant purification of the 23 kDa protein as shown by ligand blotting. This protein was later identified as oligomycin-sensitivity conferring protein (OSCP), as reported previously. These data indicate that specific estrogen binding proteins different from classical nuclear estrogen receptor (66 kDa) are present in the cellular membranes of the female rat brain. The ligand blotting technique described here would also be applicable for the identification of other membrane steroid binding proteins/receptors using similar radiolabelled steroid-BSA conjugates.

Animals↗
PubMed1997

Molecular heterogeneity in prostate cancer: can TP53 mutation unravel tumorigenesis?

While prostate cancer is the most common malignant visceral neoplasm of men, its etiology remains largely unknown and its clinical course unpredictable. Molecular genetics of prostate cancer has become a fruitful area of investigation and might provide clues to understanding these phenomena. Mutation of the TP53 tumor suppressor gene (encoding the p53 protein) has been commonly reported as a critical event in human carcinogenesis, but recent findings in prostate cancer research call into question the correlation between TP53 mutation and prognosis for patients with this tumor. Whole-mount analysis has begun to address the histologic significance of the focal evolution of TP53 mutation in a pre-existing cancer and to reveal its role throughout the process of tumor progression. This model might also apply to other tumors.

Disease Progression↗
PubMed1997

Evidence of quinone metabolites of naphthalene covalently bound to sulfur nucleophiles of proteins of murine Clara cells after exposure to naphthalene.

Naphthalene-induced Clara cell toxicity in the mouse is associated with the covalent binding of electrophilic metabolites to cellular proteins. Epoxide and quinone metabolites of naphthalene are proposed to be the reactive metabolites responsible for covalent binding to proteins. To identify the nature of reactive metabolites bound to proteins (cysteine residues), we alkaline-permethylated proteins obtained from mouse Clara cells incubated with 0.5 mM naphthalene in vitro. Alkaline permethylation of protein adducts produced (methylthio)naphthalene derivatives detected by GC-MS. 3,4-Dimethoxy(methylthio)naphthalene was observed to be a predominant (methylthio)naphthalene derivative formed in the alkaline-permethylated protein sample obtained from Clara cells after exposure to naphthalene. This indicates that 1,2-naphthoquinone is a major metabolite covalently bound to cysteine residues of the cellular proteins. We have developed an immunoblotting approach to detect 1,2-naphthoquinone covalently bound to cysteine residues of proteins [Zheng, J., and Hammock, B. D. (1996) Chem. Res. Toxicol. 9, 904-909]. To identify 1,2-naphthoquinone covalently bound to sulfur nucleophiles of proteins, homogenates obtained from naphthalene-exposed Clara cells were separated by SDS-PAGE followed by Western blotting and immunostaining with the antibodies. Two protein bands with 24 and 25 kDa were detected by the antibodies, further supporting the view that 1,2-naphthoquinone is a reactive metabolite of naphthalene which binds to Clara cell proteins in vitro.

Animals↗
PubMed1997

Role of the S3-S4 linker in Shaker potassium channel activation.

Structural models of voltage-gated channels suggest that flexibility of the S3-S4 linker region may be important in allowing the S4 region to undergo large conformational changes in its putative voltage-sensing function. We report here the initial characterization of 18 mutations in the S3-S4 linker of the Shaker channel, including deletions, insertions, charge change, substitution of prolines, and chimeras replacing the 25-residue Shaker linker with 7- or 9-residue sequences from Shab, Shaw, or Shal. As measured in Xenopus oocytes with a two-microelectrode voltage clamp, each mutant construct yielded robust currents. Changes in the voltage dependence of activation were small, with activation voltage shifts of 13 mV or less. Substitution of linkers from the slowly activating Shab and Shaw channels resulted in a three-to fourfold slowing of activation and deactivation. It is concluded that the S3-S4 linker is unlikely to participate in a large conformational change during channel activation. The linker, which in some channel subfamilies has highly conserved sequences, may however be a determinant of activation kinetics in potassium channels, as previously has been suggested in the case of calcium channels.

Amino Acid Sequence↗
PubMed1997

Selectivity changes during activation of mutant Shaker potassium channels.

Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH2-terminal-truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179-182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in approximately 40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of approximately 1.6 e0. The fully open T442S chimeric channel has the conductance sequence Rb+ > NH4+ > K+. The opposite conductance sequence, K+ > NH4+ > Rb+, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb+.

Amino Acid Sequence↗
PubMed1997

Analysis of high-affinity binding determinants in the receptor binding epitope of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitope required for receptor binding of the growth factor. In previous studies, we identified Glu96 as an essential residue in this epitope using site-directed mutagenesis. Here we examined the role of solvent accessible neighboring residues of Glu96 of bFGF on receptor binding affinity. Wild-type bFGF and its muteins were cloned and expressed in Escherichia coli and evaluated for FGF receptor binding affinity. Replacement of Asn104 of bFGF by alanine reduced receptor binding affinity over 400-fold compared with wild-type bFGF. We next explored the effect of neighboring residues of Asn104 on receptor binding affinity-Muteins in which Arg97, Leu98, Glu99, Asn101, Asn102, Thr105 and Pro141 were individually replaced by alanine exhibited receptor binding similar to wild-type bFGF. By contrast, substitution of Tyr103 or Leu140 by alanine reduced receptor binding affinity about 400- and 150-fold, respectively, in accord with a previous report. We conclude that at least six solvent-accessible residues in bFGF are crucial for high-affinity receptor binding, as evidenced by at least a 10-fold diminution in the affinity of the corresponding alanine muteins. The polar residues Glu96 and Asn104 appear to form an area important for facilitating the initial contact between ligand and receptor, whereas Tyr24, Tyr103, Leu140 and Met142 form a hydrophobic patch that may stabilize the complex. The detailed structure of this functional epitope can be employed in the discovery and design of bFGF antagonists using computational methods.

Asparagine↗
PubMed1997

Expression of basic fibroblast growth factor, endothelial mitogenic activity, and angiotensin II type-1 receptors in the ovine placenta during the third trimester of pregnancy.

To evaluate expression of basic fibroblast growth factor (FGF), endothelial mitogenic activity, and angiotensin II type-1 receptors (AT1r), as well as the role of angiotensin II (ANG II) in regulating basic FGF production/secretion, placentae were obtained from ewes on Days 110, 120, 130, and 142 of pregnancy and were separated into fetal cotyledonary (COT) and intercotyledonary (ICOT), as well as maternal caruncular (CAR) and intercaruncular (ICAR) components. Using immunohistochemistry, basic FGF and AT1r were found for the most part to be colocalized in all placental components, primarily in epithelium, stroma, endothelium, and vascular smooth muscle. Changes in basic FGF levels in placental explant-conditioned media were observed in fetal, but not maternal, components. In COT, basic FGF levels increased 2.4-fold (r2 = 0.48, p < 0.04) from Day 110 to 130 and then declined at term. In ICOT, basic FGF levels increased 6.4-fold (r2 = 0.33, p < 0.05) from Day 110 to 142. The rank order of averaged basic FGF levels was COT > CAR > ICOT = ICAR (p < 0.05). Endothelial mitogenic activity of conditioned media was observed in COT from Day 130 pregnant ewes (232.5 +/- 38.7% of control; p < 0.05) but not in other components, and it was neutralized by a basic FGF antibody. ANG II did not alter basic FGF levels in any placental component. Thus, throughout the third trimester, 1) basic FGF and AT1r are present in placentae, 2) both basic FGF levels and endothelial mitogenic activity in COT increase, 3) basic FGF levels are associated with endothelial mitogenic activity of COT, and 4) ANG II has no effect on production/secretion of basic FGF by the placenta.

Angiotensin II↗
PubMed1997

A mutation in the hamster alpha1B-adrenergic receptor that differentiates two steps in the pathway of receptor internalization.

An NP(X)nY motif is highly conserved among G protein-coupled receptors and is similar to an NPXY motif involved in receptor-mediated endocytosis for several non-G protein-coupled receptors. We investigated the role of this motif in alpha1B-adrenergic receptor function and regulation. Y348A alpha1B-adrenergic receptors in which this sequence was mutated from NPIIY to NPIIA were prepared by site-directed mutagenesis and transfected into Chinese hamster ovary cells. Binding of the antagonist prazosin to Y348A receptors was similar to that of wild-type receptors, but affinity of the Y348A receptors for the agonist epinephrine was increased by approximately 10-fold. Despite this increase in agonist binding affinity, the Y348A mutation completely uncoupled the receptors from stimulation of phosphoinositide hydrolysis and mobilization of intracellular Ca2+. Exposure of cells expressing Y348A receptors to the agonist epinephrine resulted in receptor "sequestration," defined as a loss of cell surface receptors accessible to radioligand in binding assays with intact cells on ice, similar to that for the wild-type receptor. In contrast, Y348A receptors did not undergo "endocytosis" into the light vesicle fraction in sucrose density gradient centrifugation assays, as did the wild-type receptor. These results (i) indicate an important role for Tyr348 in coupling the alpha1B-adrenergic receptor to G protein and subsequent effector activation, (ii) provide further evidence that alpha1B-adrenergic receptor internalization can be separated into a sequestration step and an endocytosis step, (iii) indicate that effector activation and second messenger formation are not required for the sequestration of these receptors but may be involved in endocytosis, and (iv) provide a useful new tool for further investigation of the nature of the subcellular compartments and the molecular modifications involved in the multiple steps involved in internalization of G protein-coupled receptors.

Animals↗
PubMed1997

Neuropeptide Y inhibits chromaffin cell nicotinic receptor-stimulated tyrosine hydroxylase activity through a receptor-linked G protein-mediated process.

Acetylcholine stimulation of bovine chromaffin cells results in increased norepinephrine and epinephrine secretion accompanied by a corresponding increase in synthesis. The addition of neuropeptide Y (NPY) to the culture medium prevents the increase in catecholamine synthesis but not secretion. Treatment of chromaffin cells with nicotine produces a concentration-dependent increase in tyrosine hydroxylase activity (IC50 = 1.2 microM) that is reduced if NPY is present during stimulation. Tyrosine hydroxylase activity decreases in a concentration-dependent fashion if increasing amounts of NPY are included in the culture medium, IC50 = 0.2 nM. Treatment with pertussis toxin completely prevents the effect of NPY. The rank order of potency for inhibition of tyrosine hydroxylase activity is NPY > or = [Leu31,Pro34]NPY > or = peptide YY > NPY2-36 > NPY13-36 > NPY18-36 > or = NPY26-36 >> NPY1-30, suggesting a NPY-Y1 receptor subtype. Examination of the effect of NPY on nicotine stimulation of chromaffin cell protein phosphorylation showed that NPY produces a concentration-dependent decrease in a 60-kDa protein, IC50 = 6.4 nM. The effect of NPY is pertussis toxin-sensitive. The rank order of potency is [Leu31,Pro34]NPY > or = NPY >> NPY18-36. Immunoprecipitation confirmed the identity of the 60-kDa protein as tyrosine hydroxylase.

Animals↗
PubMed1997

Endothelial vasodilator production by uterine and systemic arteries. II. Pregnancy effects on NO synthase expression.

Pregnancy is characterized by elevations in uterine but not omental artery nitric oxide synthase (NOS)-specific activity. We hypothesized that increases in NO production during pregnancy are associated with elevations in protein expression of the constitutive isoform, endothelial cell NOS (ecNOS), in uterine but not systemic arteries. Arterial NOS-specific activity and guanosine 3',5'-cyclic monophosphate (cGMP) production were tested in pregnant sheep in the presence or absence [+5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] of Ca2+. With the use of Western analysis, ecNOS and neuronal NOS (nNOS) constitutive isoform expressions were evaluated in intact and denuded [vascular smooth muscle (VSM)] uterine and systemic (omental and renal) arteries as well as in isolated endothelium-derived proteins from nonpregnant and pregnant sheep. Uterine and omental artery NOS activity and cGMP production were inhibited 75-85% by Ca2+ removal. ecNOS was localized only in uterine and systemic artery endothelium (not VSM) by immunohistochemistry and Western analysis; nNOS was not detected. Compared with nonpregnant ewes, pregnancy increased expression of ecNOS in uterine [2.1- to 4.2-fold (P < 0.0001)] and omental [1.3- to 2.2-fold (P = 0.032)] but not renal (P = 0.1367) artery endothelium. Increases in uterine were greater than in omental artery endothelium. Levels of plasma and urinary cGMP were elevated (P < 0.01) proportionally (1.8- to 2.0-fold) in pregnant versus nonpregnant ewes. During pregnancy, expression of uterine artery endothelium-derived (not VSM) ecNOS constitutive isoform is increased, whereas expression in systemic vessels shows little or no change.

Animals↗
PubMed1997

Pregnancy induces an increase in angiotensin II type-1 receptor expression in uterine but not systemic artery endothelium.

During pregnancy, the uterine artery demonstrates refractoriness to vasoconstriction by infused angiotensin II (AII). AII increases prostacyclin (PGI2) production by uterine artery endothelium from pregnant ewes, and this response is mediated via the AT1 receptor (AT1-R). This response is also unique to pregnancy because AII does not stimulate PGI2 production by uterine artery endothelium from nonpregnant ewes. We therefore hypothesize that the increase in uterine artery PGI2 production in response to AII in pregnancy is associated in part with a concomitant increase in AT1-R expression in uterine artery endothelium. Endothelium-derived protein was directly removed from the lumenal surface of freshly isolated uterine and systemic (omental) arteries from nonpregnant and pregnant ewes. AT1-R expression was then measured in both the endothelium-derived fraction and endothelium-denuded vascular smooth muscle (VSM) fraction by Western analysis. AT1-R was detected as 54- and 65-kDa proteins in all samples, as well as adrenal cortex control. AT1-R expression increased more than 8-fold in uterine artery endothelium of pregnant ewes over that in nonpregnant ewes at each of four gestational ages (P < 0.05 at 110, 120, 130, 142 days, n = 4 each vs. n = 6 nonpregnant). No significant differences were seen, however, from 110 to 142 days of gestation. In contrast, whereas the level of AT1-R staining in omental artery endothelium in nonpregnant ewes was higher than in uterine artery, AT1-R increased less in pregnant ewes (2-fold) and only reached significance over nonpregnant values at 110 and 120 days, or when data was combined irrespective of gestational age (P < 0.05). Although AT1-R was also detected in uterine and omental artery VSM, little or no change in expression was observed in pregnancy. Results were confirmed by immunohistochemical staining of arterial cross sections, and the increase in AT1-R expression in uterine artery endothelium was confirmed by RT/PCR amplification of AT1-R messenger RNA from collagenase dispersed cells (n = 4 pregnant vs. n = 4 nonpregnant, mean 20-fold increase, P < 0.028). We conclude that increased uterine artery endothelial PGI2 responsiveness to AII during pregnancy is indeed associated with a correspondingly marked and localized increase in expression of the endothelial AT1-R receptor. We believe our findings allow a more detailed understanding of the molecular mechanisms that underlie increased uterine blood flow that is so central to the normal development of the growing fetus, and on dysfunction may lead to conditions such as preeclampsia.

Angiotensin II↗
PubMed1997

[Interleukin-6 behaves as an autocrine growth stimulator for a human lung giant cell carcinoma cell line].

OBJECTIVE: To study the effect of interleukin-6 (IL-6) on the growth of human pulmonary giant cell carcinoma cell line PG in vitro. METHODS: IL-6 mRNA level was detected by means of reverse transcriptase polymerase chain reaction (RT-PCR) and IL-6 receptor (IL-6R) mRNA was analyzed using Northern blotting hybridization. The production of IL-6 was detected by bioactive assay. The biological role of IL-6 produced by PG cells was assessed with anti-IL-6 neutralizing antibodies. RESULTS: PG cells expressed IL-6R mRNA. Recombinant human IL-6 promoted the proliferation of PG cells. PG cells also expressed IL-6 mRNA and produced bioactive IL-6. Treatment of PG cells with anti-IL-6 antibodies resulted in reduced growth of PG cells. CONCLUSION: The results indicate that IL-6 behaves as an autocrine growth stimulator for PG cells in vitro.

Antibodies↗
PubMed1997

[Regulatory effects of cAMP analogs on growth and differentiation of metastatic human lung cancer cells].

OBJECTIVE: To study the effects of two cAMP analogs with different site-selectivity on growth and differentiation of metastatic human lung cancer cells. METHODS: The methods used include cell culture, in vitro invasion assay, soft agar colony formation assay, immunocytochemistry and electron microscopy. A metastatic human lung cancer cell line gwas treated with dibutyryl cAMP (db-cAMP, non-site-selective) or 8-chloro-cAMP (8-Cl-cAMP, site-selective for type II PKA). RESULTS: Treatment of PG cells with 1 mmol/L of db-cAMP for 7 days resulted in 48% growth inhibition, while treatment with 20 mumol/L of 8-Cl-cAMP gave 70% growth inhibition. The growth inhibitory effect of db-cAMP was shown to be reversible, while that of 8-Cl-cAMP was not. The ability of PG cells to penetrate matrigel-coated membrane and to form colonies in soft agar was also significantly inhibited by treatment with these two drugs. Microscopic observation showed that cells formed elongated cytoplasmic processes and increased expression of neuron-specific enolase as well as chromogranin after treatment. CONCLUSION: The objective to inhibit malignancy could be reached by activation of specific PKA with site-selective cAMP analogs.

8-Bromo Cyclic Adenosine Monophosphate↗