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Biomedical subjects

Z Huang

Publications and source records attributed to Z Huang.

At least 433 records · Page 24Linked to original sources
PubMed1994

Proposed three-dimensional structure for the cellular prion protein.

Prion diseases are a group of neurodegenerative disorders in humans and animals that seem to result from a conformational change in the prion protein (PrP). Utilizing data obtained by circular dichroism and infrared spectroscopy, computational studies predicted the three-dimensional structure of the cellular form of PrP (PrPc). A heuristic approach consisting of the prediction of secondary structures and of an evaluation of the packing of secondary elements was used to search for plausible tertiary structures. After a series of experimental and theoretical constraints were applied, four structural models of four-helix bundles emerged. A group of amino acids within the four predicted helices were identified as important for tertiary interactions between helices. These amino acids could be essential for maintaining a stable tertiary structure of PrPc. Among four plausible structural models for PrPc, the X-bundle model seemed to correlate best with 5 of 11 known point mutations that segregate with the inherited prion diseases. These 5 mutations cluster around a central hydrophobic core in the X-bundle structure. Furthermore, these mutations occur at or near those amino acids which are predicted to be important for helix-helix interactions. The three-dimensional structure of PrPc proposed here may not only provide a basis for rationalizing mutations of the PrP gene in the inherited prion diseases but also guide design of genetically engineered PrP molecules for further experimental studies.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of a partial cDNA of the interleukin-2 receptor-beta in normal mouse brain: in situ localization in the hippocampus and expression by neuroblastoma cells.

While interleukin-2 (IL-2) has been shown to produce a variety of effects in the CNS and has recently been implicated as an endogenous brain neurokine, little is known about the molecular biology of IL-2 receptors in normal brain. The present investigation provides the first evidence that mRNA for IL-2 receptor-beta (IL-2R beta), an essential subunit for signal transduction by peripheral immune cells, is expressed in normal murine forebrain. Using polymerase chain reaction (PCR) cloning, a partial cDNA (349 bp) corresponding to the extracellular domain was cloned and found to have the identical sequence as the lymphocyte IL-2R beta. IL-2R beta mRNA expression was confirmed by a ribonuclease protection assay, and using in situ hybridization histochemistry, IL-2R beta mRNA was localized in the hippocampus where an intense signal was present over the neuron-rich granule cells of the dentate gyrus and Ammon's horn. Moreover, cDNA clones obtained from two murine neuroblastoma cell lines exhibited the same sequence as IL-2R beta cDNA from normal brain. IL-2R beta gene expression was also detected in the frontal cortex and striatum using PCR. Further in situ hybridization studies will be important to extend this initial observation to determine the brain regional localization and cell-specific anatomy of IL-2R beta mRNA in the CNS.

Animals↗
PubMed1994

Spectroscopic characterization of conformational differences between PrPC and PrPSc: an alpha-helix to beta-sheet transition.

Although no chemical modifications have been found to distinguish the cellular prion protein PrPC from its infectious analogue PrPSc, spectroscopic methods such as Fourier transform infrared (FTIR) spectroscopy reveal a major conformational difference. PrPC is rich in alpha-helix but is devoid of beta-sheet, whereas PrPSc is high in beta-sheet. N-terminal truncation of PrPSc by limited proteolysis does not destroy infectivity but it increases the beta-sheet content and shifts the FTIR absorption to lower frequencies, typical of the cross beta-pleated sheets of amyloids. Thus the formation of PrPSc from PrPC involves a conformational transition in which one or more alpha-helical regions of the protein is converted to beta-sheet. This transition is mimicked by synthetic peptides, allowing predictions of domains of PrP involved in prion diseases.

Amino Acid Sequence↗
PubMed1994

A continuous fluorescence-based assay for the human high-molecular-weight cytosolic phospholipase A2.

A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase A2 (cPLA2) is described. Recombinant cPLA2 efficiently hydrolyzes fatty acid esters of 7-hydroxycoumarin, producing the free fatty acid and the highly fluorescent 7-hydroxycoumarin. All of the observed 7-hydroxycoumarinyl ester hydrolase activity (7-HCEase) in a preparation of the purified recombinant cPLA2 was due to this enzyme since: (1) all of the ester hydrolase activity comigrated on nondenaturing polyacrylamide gel with a protein characterized as the cPLA2 by Western analysis; (2) the immunoreactive protein also possessed both phospholipase A2 and lysophospholipase activities; and (3) arachidonyl trifluoromethyl ketone, a potent inhibitor of the phospholipase A2 activity of cPLA2, also inhibited the 7-HCEase activity. A study of the 7-HCEase activity demonstrated that when 7-hydroxycoumarinyl gamma-linolenate was dispersed in a phospholipid matrix it was hydrolyzed by cPLA2 at a rate comparable to that of an arachidonyl-containing phospholipid substrate and with an identical reaction progress curve. In the presence of phospholipid vesicles, the cPLA2-catalyzed hydrolysis of hydrophobic 7-hydroxycoumarinyl esters was stimulated by submicromolar concentration of free calcium and showed a preference for polyunsaturated substrates. The cPLA2-catalyzed hydrolysis of the water-soluble substrate 7-hydroxycoumarinyl 6-heptenoate was catalyzed by cPLA2 in the absence of calcium and other lipids.

Calcium↗
PubMed1994

Effect of retinal impulse blockage on cytochrome oxidase-poor interpuffs in the macaque striate cortex: quantitative EM analysis of neurons.

One of the hallmarks of the primate striate cortex is the presence of cytochrome oxidase-rich puffs in its supragranular layers. Neurons in puffs have been classified as type A, B, and C in ascending order of cytochrome oxidase content, with type C cells being the most vulnerable to retinal impulse blockade. The present study aimed at analysing cytochrome oxidase-poor interpuffs with reference to their metabolic cell types and the effect of intraretinal tetrodotoxin treatment. The same three metabolic types were found in interpuffs, except that type B and C neurons were smaller and less cytochrome oxidase-reactive in interpuffs than in puffs. Type A neurons had small perikarya, low levels of cytochrome oxidase, and received exclusively symmetric axosomatic synapses. The largest neurons were pyramidal, type B cells with moderate cytochrome oxidase activity and were also contacted exclusively by symmetric axosomatic synapses. Type C cells medium-sized with a rich supply of large, darkly reactive mitochondria and possessed all the characteristics of GABAergic neurons. They were the only cell type that received both symmetric and asymmetric axosomatic synapses. Two weeks of monocular tetrodotoxin blockade in adult monkeys caused all three major cell types in deprived interpuffs to suffer a significant downward shift in the size and cytochrome oxidase reactivity of their mitochondria, but the effects were more severe in type B and C neurons. In nondeprived interpuffs, all three cell types gained both in size and absolute number of mitochondria, and type A cells also had an elevated level of cytochrome oxidase, indicating that they might be functioning at a competitive advantage over cells in deprived columns. However, type B and C neurons showed a net loss of darkly reactive mitochondria, indicating that these cells became less active. Thus, mature interpuff neurons remained vulnerable to retinal impulse blockade and the metabolic capacity of these cells remains tightly regulated by neuronal activity.

Animals↗
PubMed1994

Molecular cloning of NPY-Y1 receptor cDNA from rat splenic lymphocytes: evidence of low levels of mRNA expression and [125I]NPY binding sites.

The present study addressed the question of whether the effects of neuropeptide Y (NPY) on parameters of cellular immune activity are mediated by the direct action of this neuropeptide on lymphocyte NPY receptors. A partial cDNA corresponding to bp 3-585 of the NPY-Y1 receptor coding sequence was cloned from rat splenic lymphocytes and found to have 100% nucleotide sequence homology with that segment of the NPY-Y1 receptor in brain. Basal levels of NPY-Y1 mRNA expression and [125I]NPY binding sites of rat splenic lymphocytes were markedly lower than in frontal cortex. These data provide the first direct evidence that cells of the immune system possess NPY receptors, and suggest that further study will be necessary to determine their physiological significance.

Animals↗
PubMed1994

Translational diffusion of bovine prothrombin fragment 1 weakly bound to supported planar membranes: measurement by total internal reflection with fluorescence pattern photobleaching recovery.

Previous work has shown that bovine prothrombin fragment 1 binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca(2+)-specific manner. The apparent equilibrium dissociation constant is 1-15 microM, and the average membrane residency time is approximately 0.25 s-1. In the present work, fluorescence pattern photobleaching recovery with evanescent interference patterns (TIR-FPPR) has been used to measure the translational diffusion coefficients of the weakly bound fragment 1. The results show that the translational diffusion coefficients on fluid-like PS/PC planar membranes are on the order of 10(-9) cm2/s and are reduced when the fragment 1 surface density is increased. Control measurements were carried out for fragment 1 on solid-like PS/PC planar membranes. The dissociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also used to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diffusion coefficient was approximately 10(-8) cm2/s, which is consistent with that measured by conventional fluorescence pattern photobleaching recovery. This work represents the first measurement of a translational diffusion coefficient for a protein weakly bound to a membrane surface.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens.

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.

Amino Acid Sequence↗
PubMed1994

L-arginine infusion promotes nitric oxide-dependent vasodilation, increases regional cerebral blood flow, and reduces infarction volume in the rat.

BACKGROUND AND PURPOSE: We previously reported that L-arginine infusion increased pial vessel diameter by nitric oxide-dependent mechanisms, improved regional cerebral blood flow (rCBF) distal to middle cerebral artery (MCA) occlusion, and reduced infarction volume in spontaneously hypertensive rats when administered intraperitoneally before and after MCA occlusion. In this report we extend our findings (1) by examining the time course of L-arginine on rCBF and pial vessel diameter under basal conditions and on rCBF after MCA occlusion and (2) by reproducing the protective effect of L-arginine on infarct volume when given intravenously immediately after the onset of MCA occlusion in both normotensive and hypertensive models of focal cerebral ischemia. METHODS: Changes in pial vessel diameter (closed cranial window) and rCBF (laser-Doppler flowmetry) were measured over time after L-arginine infusion into anesthetized Sprague-Dawley rats. rCBF was also measured distal to MCA occlusion in a brain region showing rCBF reductions in the range of 80% of baseline. The effects of infusing L-arginine (300 mg/kg for 10 minutes beginning 5 minutes after occlusion) were assessed on infarction volume in Sprague-Dawley rats after proximal MCA occlusion and in spontaneously hypertensive rats after common carotid artery plus distal MCA occlusion. RESULTS: L-Arginine (300 mg/kg IV) elevated rCBF by 20% when measured in the dorsolateral cortex of Sprague-Dawley rats and caused L-nitroarginine-methyl ester-inhibitable increases in pial vessel diameter. L-Arginine (> or = 30 mg/kg IV) increased blood flow distal to MCA occlusion by 50%. These effects were sustained throughout the observation period (70 to 105 minutes). Changes in mean arterial blood pressure were not observed. L-Arginine (300 mg/kg IV) reduced infarction volume by 35% and 28% in Sprague-Dawley and spontaneously hypertensive rats, respectively, when examined 24 hours after vessel occlusion. CONCLUSIONS: These studies extend our previous findings by demonstrating that exogenous L-arginine induces sustained rCBF increases in normal brain as well as in a marginally perfused brain region distal to MCA occlusion. Our data in Sprague-Dawley rats support the conclusion that L-arginine-induced increases in rCBF can decrease infarction volume. We conclude that nitric oxide-mediated mechanisms increase rCBF and decrease infarction volume after MCA occlusion in both normotensive and hypertensive animals.

Amino Acid Oxidoreductases↗
PubMed1994

Cystic fibrosis transmembrane conductance regulator mutations that disrupt nucleotide binding.

Increasing evidence suggests heterogeneity in the molecular pathogenesis of cystic fibrosis (CF). Mutations such as deletion of phenylalanine at position 508 (delta F508) within the cystic fibrosis transmembrane conductance regulator (CFTR), for example, appear to cause disease by abrogating normal biosynthetic processing, a mechanism which results in retention and degradation of the mutant protein within the endoplasmic reticulum. Other mutations, such as the relatively common glycine-->aspartic acid replacement at CFTR position 551 (G551D) appear to be normally processed, and therefore must cause disease through some other mechanism. Because delta F508 and G551D both occur within a predicted nucleotide binding domain (NBD) of the CFTR, we tested the influence of these mutations on nucleotide binding by the protein. We found that G551D and the corresponding mutation in the CFTR second nucleotide binding domain, G1349D, led to decreased nucleotide binding by CFTR NBDs, while the delta F508 mutation did not alter nucleotide binding. These results implicate defective ATP binding as contributing to the pathogenic mechanism of a relatively common mutation leading to CF, and suggest that structural integrity of a highly conserved region present in over 30 prokaryotic and eukaryotic nucleotide binding domains may be critical for normal nucleotide binding.

Adenosine Triphosphate↗
PubMed1994

Hyperinsulinemia is associated with altered insulin receptor mRNA splicing in muscle of the spontaneously obese diabetic rhesus monkey.

The human insulin receptor has two isoforms derived from alternative splicing of exon 11 of the insulin receptor gene. The type B (containing exon 11, or exon 11+) isoform binds insulin with twofold lower affinity than the type A (lacking exon 11, or exon 11-) isoform. In efforts to resolve the controversy over whether altered splicing is involved in the development of insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM), the spontaneously obese diabetic rhesus monkey, a unique model that is extraordinarily similar to human NIDDM, was used. Cross-sectional studies of insulin receptor mRNA splicing variants in vastus lateralis muscle were performed on 19 rhesus monkeys. When monkeys were divided into four groups based upon the known stages of progression to NIDDM: normal (normoglycemic/normoinsulinemic), prediabetic (normoglycemic/hyperinsulinemic), early NIDDM (hyperglycemic/hyperinsulinemic), and late NIDDM (hyperglycemic/hypoinsulinemic), both hyperinsulinemic groups had significantly higher percentages of the exon 11- mRNA splicing variant compared to the normal (74.8 +/- 1.7 vs 59.0 +/- 2.3%; P < 0.005) and late NIDDM groups (74.8 +/- 1.7 vs 64.2 +/- 3.9%; P < 0.05). Our findings provide the first direct evidence linking hyperinsulinemia to alterations in insulin receptor mRNA splicing, and suggest that alterations of insulin receptor mRNA splicing in muscle is an early molecular marker that may play an important role in NIDDM.

Adipose Tissue↗