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Biomedical subjects

Z Huang

Publications and source records attributed to Z Huang.

At least 19 recordsLinked to original sources
PubMed1995

Molecular basis for nonadditive mutational effects in Escherichia coli dihydrofolate reductase.

Recently, two sets of single, double, and quadruple residue changes within the hydrophobic substrate binding pocket of Escherichia coli dihydrofolate reductase (5,6,7,8-tetrahydrofolate+ oxidoreductase, EC 1.5.1.3) were shown to exhibit nonadditive mutational effects [Huang, Z., Wagner, C. R., & Benkovic, S. J. (1994) Biochemistry 33, 11576--11585]. In particular, the analysis of data for the L28Y, L54F, and L28Y-L54F mutations revealed nonadditive changes in the free energy associated with the substrate and cofactor binding, hydride transfer, and product release steps. Construction of a related set of mutant proteins including L28F and L28F-L54F permits a comparison of similar energy changes and provides a means for assessing differences in the interactions of Phe28 and Tyr28 with both the ligands and the side chains at residue 54. We find a single functional group change, from Phe C4-H to Tyr C4-OH, can influence the additivity of mutational effects and serve as a probe to monitor the appearance of differing enzyme conformations along the reaction pathway through changes in the interaction energy (delta GI). The comparison of additivity/nonadditivity in free energy changes for three interrelated double mutational cycles (WT --> L28F-L54F, WT --> L28Y-L54F, and L28F --> l28Y-L54F) demonstrates that the side chains of positions 28 and 54 interact cooperatively to facilitate hydride transfer by preferentially influencing the enzyme--substrate ground-state complexes. The delta GI data for individual steps also provide evidence for multiple conformations of the enzyme operating during the catalytic cycle. The fact that there are no published examples of the synergistic enhancement of favorable mutational effects is consistent with the expectation that the binding/active site surface of wild-type dihydrofolate reductase has been optimized.

Base Sequence↗
PubMed1995

Hypertension in mice lacking the gene for endothelial nitric oxide synthase.

Nitric oxide (NO), a potent vasodilator produced by endothelial cells, is thought to be the endothelium-dependent relaxing factor (EDRF) which mediates vascular relaxation in response to acetylcholine, bradykinin and substance P in many vascular beds. NO has been implicated in the regulation of blood pressure and regional blood flow, and also affects vascular smooth-muscle proliferation and inhibits platelet aggregation and leukocyte adhesion. Abnormalities in endothelial production of NO occur in atherosclerosis, diabetes and hypertension. Pharmacological blockade of NO production with arginine analogues such as L-nitroarginine (L-NA) or L-N-arginine methyl ester affects multiple isoforms of nitric oxide synthase (NOS), and so cannot distinguish their physiological roles. To study the role of endothelial NOS (eNOS) in vascular function, we disrupted the gene encoding eNOS in mice. Endothelium-derived relaxing factor activity, as assayed by acetylcholine-induced relaxation, is absent, and the eNOS mutant mice are hypertensive. Thus eNOS mediates basal vasodilation. Responses to NOS blockade in the mutant mice suggest that non-endothelial isoforms of NOS may be involved in maintaining blood pressure.

Amino Acid Oxidoreductases↗
PubMed1995

Induction of nitric oxide synthase activity in rodent brain following middle cerebral artery occlusion.

The conversion of L-[3H]arginine to L-[3H]citrulline in the absence of calcium can be used to assay selectively the activity of inducible nitric oxide synthase (NOS) in rat spleen homogenates 6 h after lipopolysaccharide administration. Using similar assay conditions, changes in inducible NOS activity were measured within ischemic brain tissue between 2 h and 7 days following permanent middle cerebral artery (MCA) occlusion in Sprague-Dawley rats and SV-129 mice. Total (constitutive and inducible) NOS activity was measured in the presence of 0.5 mM CaCl2. Whereas total NOS activity in rat decreased dramatically to 16% and 6% of baseline 6 and 12 h after MCA occlusion, inducible NOS activity remained undetectable before 2 days after occlusion, became maximal at 3 days, and decreased to less than 10% of maximal iNOS activity at 7 days. In the mouse, total NOS activity decreased after MCA occlusion but inducible NOS activity was undetectable from 2 h to 4 days after occlusion. Sustained NO production by inducible NOS activity does not contribute to ischemic injury within 24 h after MCA occlusion, but may contribute to infarct maturation 2-4 days after ischemia in some but not all species.

Animals↗
PubMed1995

Interaction of retinoblastoma gene product with transcription factors ATFa and ATF2.

Two highly related transcription factors, activating transcription factors a and 2 (ATFa and ATF2) are able to activate expression in CHO cells of a reporter gene driven by the human transforming growth factor beta 2 gene promoter. This action is dependent on the intact motif CGTCA which is found as part of the cAMP responsive element in a number of promoters and to which both factors can bind in vitro. The retinoblastoma gene product also weakly stimulates expression of this reporter gene but, in combination with the factors, it exhibits a differential action: additive or greater stimulatory effects with ATF2 but strong inhibition of the actions of ATFa. Thus, although both of these two related factors are able to exert the same apparent effect on this promoter, coexpression of the retinoblastoma gene product reveals at least one significant difference in their actions.

Activating Transcription Factor 1↗
PubMed1995

Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505.

Cytosolic PLA2 (cPLA2) has been implicated in the release of the arachidonic acid utilized in the inflammatory cascade. Phosphorylation of cPLA2 on Ser-505 by MAP kinase in response to agonist treatment, is thought to be one of the mechanisms required for activation of the enzyme in the cell. In order to obtain enough material for enzymological studies as well as to investigate the role of phosphorylation in the activation of cPLA2, the human enzyme was overexpressed in insect cells using a recombinant baculovirus. We report here on the characterization of the phosphorylation state of cPLA2 overexpressed in Sf9 cells. The level of overexpressed cPLA2 was shown to peak between 48 and 60 h post-infection, by this time the phosphorylated enzyme could easily be detected because of its reduced mobility on polyacrylamide gels. The reduced mobility or gel-shift has been shown to be due to phosphorylation of Ser-505. To determine whether this was also the case for insect cell overexpressed cPLA2, Ser-505 was replaced by Ala, and this mutant (cPLA2S505A) was expressed in Sf9 cells. Analysis of the overexpressed cPLA2S505A showed that it migrated only as the lower unshifted cPLA2 band confirming that the baculovirus overexpressed cPLA2 is extensively phosphorylated on Ser-505. Furthermore, treatment of infected Sf9 cells expressing the wild-type cPLA2 with phorbol 12-tetradecanoate 13-acetate (TPA) shifted all of the overexpressed cPLA2 to the phosphorylated Ser-505 form. When infected Sf9 cells were labelled with [32P], in addition to labelling of Ser-505 other sites were also labelled. Both cPLA2 and cPLA2S505A were purified from infected Sf9 cells and the specific activity for each of the enzymes was measured in a phosphatidylcholine vesicle fluorescence assay using 1-(10-pyrenedecanyl)arachidonyl-sn-glycero-3-phosphocholine as substrate. Under these conditions the specific activity of cPLA2 was, 2 mumol/min per mg, whereas cPLA2S505A was 7-fold less active. These findings suggest that Sf9 cells have a mechanism for phosphorylating cPLA2 similar to that found in mammalian cells which probably proceeds through a MAP kinase. Thus, insect cell overexpressed cPLA2 is a very good source for the Ser-505 phosphorylated enzyme.

Animals↗
PubMed1995

The cytoplasmic tail of the G-protein-coupled receptor for parathyroid hormone and parathyroid hormone-related protein contains positive and negative signals for endocytosis.

The present studies were done to evaluate the role of the cytoplasmic tail of the G-protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) in the endocytosis of agonist-occupied receptors. PTH/PTHrP receptor mutants progressively truncated from the C terminus were expressed in COS-7 cells, and their ability to internalize 125I-PTHrP(1-34)amide was determined. Most of the C-terminal tail (91 of 127 residues) could be deleted without affecting internalization. However, further truncation removing residues 475-494 resulted in a 50-60% decrease in ligand internalization. A mutant with an internal deletion of these 20 amino acids showed a similar reduction in internalization, confirming the presence of a positive endocytic signal. No additional positive signals were found in the membrane-proximal region of the tail. However, alanine mutagenesis of the membrane-proximal residues 459-461 (EVQ-->AAA) resulted in a mutant PTH/PTHrP receptor displaying a 40% increase in ligand endocytosis, indicating that EVQ functions as a negative signal. Treatment of COS-7 cells with hypertonic sucrose (to disrupt clathrin lattices) markedly suppressed (by > 80%) PTH/PTHrP receptor internalization. These results demonstrate the presence of both positive and negative endocytic signals in the membrane-proximal cytoplasmic tail of the PTH/PTHrP receptor and suggest that these signals regulate the ability of the receptor to accumulate in clathrin-coated pits.

Amino Acid Sequence↗
PubMed1995

The change of carnitine content in seminal plasma after reversible injection occlusion of vas deferens.

The change in carnitine content in seminal plasma after reversible injection occlusion of vas deferens (RIOVD) was observed. RIOVD is a safe, effective and simple method of male fertility control. Carnitine was determined by microenzymatic method. The incidence of sperm disappearance increased with the duration of RIOVD and reached 90% at the end of 12 months after operation. Before RIOVD, the mean value of carnitine in seminal plasma was 336.9 +/- 78.1 nmol/ml (X +/- SD, n = 58); after RIOVD, the mean value of seminal plasma carnitine was 112.7 +/- 50.7 nmol/ml (n = 172) in the group with sperm disappearance, and 172.5 +/- 71.7 nmol/ml (n = 51) in the group without sperm disappearance. There was a significant difference in carnitine content between pre-operation and postoperation (p < 0.01). After RIOVD, the carnitine concentration in seminal plasma of the group with sperm disappearance was lower than that of the group without sperm disappearance (p < 0.01). The results suggest that carnitine content in seminal plasma following RIOVD may be a reference index for judging the success or failure of the operation.

Carnitine↗
PubMed1995

Molecular cloning of the coding sequence of an interleukin-2 receptor alpha subunit cDNA in murine brain.

Interleukin-2 (IL-2) has various trophic and neuromodulatory actions in the mammalian central nervous system (CNS). The interleukin-2 receptor alpha (IL-2R alpha) is an accessory subunit of the IL-2 receptor heterotrimer complex which is essential for 'high' affinity IL-2 binding. Although an IL-2R alpha (or IL-2R alpha-like) epitope has been localized in brain by immunohistocytochemistry, it was unknown whether the IL-2R alpha subunit expressed in brain was derived from the same or a different gene than the lymphocyte IL-2R alpha. Therefore, in the present study, the cDNA comprising the full length coding region was cloned and sequenced from saline-perfused forebrain. The brain IL-2R alpha cDNA was found to be 100% homologous with the corresponding lymphocyte IL-2R alpha cDNA sequence. IL-2R alpha mRNA was expressed at very low levels in saline-perfused forebrain of non-challenged BALB/c mice as well as in saline-perfused forebrain from severe combined immunodeficiency (SCID) mice. The present data, demonstrating IL-2R alpha gene expression in both well-perfused normal and SCID mouse forebrain from which no CD3 gamma gene expression was detected by PCR, provides evidence that the IL-2R alpha clones isolated are from resident brain cells and not from blood lymphocytes (e.g. T lymphocytes). Thus, these findings demonstrate that the protein coding sequence of the mouse brain IL-2R alpha is derived from the same gene coding sequence as the lymphocyte IL-2R alpha, and indicate that previously reported differences in the size of their respective mRNA transcripts appear to be due to differences in untranslated regions.

Animals↗
PubMed1995

Poly(binaphthyl-20-crown-6) as receptor based molecular selective potentiometric electrodes for catecholamines and other 1,2-dihydroxybenzene derivatives.

A novel type of poly(crown ether) electrode that is capable of selectively determining some 1,2-dihydroxybenzenes has been developed. A lipophilic macrocyclic crown ether, binaphthyl-20-crown-6, is electrochemically deposited on a platinum disc electrode. The film obtained is used as a sensory element for a potentiometric electrode for the determination of some neurotransmitters, namely, catecholamines. The new electrode is also capable of discriminating the steric shapes of 1,2-dihydroxybenzene moieties. The response of the new electrode is based on the principle of 'host-guest' chemistry. The potentiometric response is dependent on the pH of the solution and the nature of the buffer medium. The new sensor electrode has a useful analytical range of 1.5 x 10(-8) M-2 x 10(-5) M with a linear dynamic range between about 1 x 10(-7) M-5 x 10(-4) M with a 'super-Nernstian' slope of 110-130 mV/decade. The detection limit in phosphate buffer (0.1 M, pH 9.4) is ca. 3 x 10(-8) M for catecholamine. The sensor electrode is virtually insensitive towards interference from most inorganic ions and circumvents the interference from ascorbic acid, which is often found using amperometric methods in biological samples. A partial response mechanism of the present electrode is discussed, supported by results of electron dispersive analysis by x-rays (EDAX).

Animals↗
PubMed1995

Cerebrovascular responses under controlled and monitored physiological conditions in the anesthetized mouse.

Control of physiological parameters such as respiration, blood pressure, and arterial blood gases has been difficult in the mouse due to the lack of technology required to monitor these parameters in small animals. Here we report that anesthetized and artificially ventilated mice can be maintained under physiological control for several hours with apparently normal cerebrovascular reactivity to hypercapnia and mechanical vibrissal stimulation. SV-129 mice were anesthetized with urethane (750 mg/kg i.p.) and alpha-chloralose (50 mg/kg i.p.), intubated, paralyzed, and artificially ventilated. Respiratory control was maintained within physiological range by reducing the inspiratory phase of the respiratory cycle to < 0.1 s and by adjusting end-tidal CO2 to give a PCO2 of 35 +/- 3 mm Hg. In these mice, mean arterial pressure (95 +/- 9 mm Hg), heart rate (545 +/- 78 beats/min), and arterial pH (7.27 +/- 0.10) could be maintained for several hours. Body temperature was kept at 36.5-37.5 degrees C. We observed stable regional CBF (rCBF) measurements (as determined by laser-Doppler flowmetry) when systemic arterial blood pressure was varied between 40 and 130 mm Hg. Hypercapnia led to a 38 +/- 15% (5% CO2) and 77 +/- 34% (10% CO2) increase in rCBF. Mechanical stimulation of contralateral vibrissae for 1 min increased rCBF by 14 +/- 4%. Changes in rCBF compare favorably with those observed previously in another rodent species, the Sprague-Dawley rat. After placement of a closed cranial window, cerebrovascular reactivity to hypercapnia and whisker stimulation was intact and well maintained during 2-h superfusion with artificial CSF.

Anesthesia↗
PubMed1995

Gastric inhibitory polypeptide release from a tumor-derived cell line.

A cell line derived from intestinal tumors of transgenic mice (STC-1) was subcloned to produce a stable line with approximately 30% immunoreactive gastric inhibitory polypeptide (irGIP)-containing cells (STC 6-14). High-performance liquid chromatography (HPLC) of STC 6-14 extracts indicated that the tumor cell-derived irGIP had the same retention time as synthetic porcine GIP-(1-42) (pGIP). Approximately 30% of the cells also contained immunoreactive somatostatin (irSS), which eluted as a single peak on HPLC, corresponding with SS-(1-14). On average, each well of extracted cells (5.0 x 10(5) cultured 4 days) contained 33.3 +/- 1.4 ng irGIP and 18.4 +/- 1.5 ng irSS. Basal release of irGIP in the presence of 5 mM glucose was 733 +/- 58 pg.ml cells-1.2h-1 (2.20 +/- 0.17% of total cell content; TCC) and doubled at 20 mM glucose (4.20 +/- 0.42% TCC). The response to glucose was augmented by addition of a SS neutralizing antibody (SOMA-10) and suppressed by 10 nM SS. Basal release of irSS in 5 mM glucose was 377 +/- 35 pg.ml cells-1.2h-1 (2.05 +/- 0.19% TCC) and was increased by glucose (> or = 15 mM) and the addition of pGIP (> or = 1 nM). The STC 6-14 cell line represents a model to study the synthesis, storage, and release of GIP and SS in a controlled environment.

Animals↗
PubMed1995

Mutational analysis of the cytoplasmic tail of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein: effects on receptor expression and signaling.

The present studies were undertaken to examine the role of the cytoplasmic tail of the G protein-coupled receptor for PTH and PTH-related protein (PTHrP) on receptor signaling and expression. The wild type (WT) receptor (585 amino acids) and five truncated receptors whose cytoplasmic tails terminated at residues 507, 494, 474, 466, and 458 were expressed in COS-7 cells. Based on [125I]PTHrP binding, mutants T507, T494, and T466 displayed progressively decreased levels of expression, compared with WT. The tailless mutant T458 was not expressed in a functional form, whereas T474 was expressed at a level similar to WT. Comparable results were obtained when expression levels of WT and mutated PTH/PTHrP receptors were evaluated by Western blotting. Binding affinities were similar for all mutated receptors (IC50 = 1-2 nM). Immunocytochemistry showed that WT and mutated receptors were diffusely distributed, presumably at the cell surface, except for the tailless mutant T458, which displayed striking perinuclear localization. T458 did not display an adenylyl cyclase response to PTH, while the other mutants were similar to WT both with respect to their maximal adenylyl cyclase responses to PTH and to their EC50 values. Cai2+ signaling properties of these mutants were assessed as PTH-stimulated 45Ca efflux from Xenopus oocytes that had been injected with in vitro transcribed PTH/PTHrP receptor cRNAs. The WT and mutated receptors (except for T458) responded to PTH with significant (6- to 27-fold) increases in 45Ca efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1995

[The protective effect of IL-1 beta on stress-induced gastric mucosal damage in rat].

The effect of recombinant IL-1 beta on stress-induced gastric mucosal lessions was studied in rats. Pretreatment with IL-1 beta prevented formation of gastric mucosal damage in a dose-dependent manner. IL-1 receptor antagonist peptide (IRAP) could totally reverse the protective effect of IL-1 beta. IL-1 fragment peptide (163-171) had no effect on gastric ulcer formation in the experimental model, whereas sulfhydryl blocker N-ehtylmaleimide partially blocked the protective effect of IL-1 beta. The concentration of protein and noneprotein sulfhydryls in the gastric mucosa was significantly decreased 3 h after stress, and this decrease was partially prevented by IL-1 beta. IL-1 beta also decreased the concentration of molondialdehyde (MDA) in the gastric mucosa after 3 h stress. The results suggest that IL-1 beta could effectively lessen the degree of stress-induced gastric mucosal damage, due possibly to the production of endogenous sulfhydryl compounds in the gastric mucosa.

Animals↗
PubMed1995

[Basaloid squamous carcinoma of the oesophagus: a distinctive clinico-pathological entity].

5 cases of basaloid-squamous carcinoma (BSC) of oesophagus were reported. Their pathological features were: 1. The main component of the tumors were basaloid carcinoma cells. 2. Concomitant squamous cell differentiation. 3. Comedo-like necrosis in the basaloid carcinoma component of the tumor. 4. Hyaline degeneration within the stroma of the basaloid carcinoma nests (PAS+). The immunohistochemistry of keratin 10.11, CEA and EMA in the basaloid carcinoma component of BSC were negative or weak positive, while actin and S-100 were positive in some parts of the tumor sections. This suggested that the carcinoma component was poorly differentiated and somewhat tended to differentiate toward myoepithelia or other directions. We therefore consider that the origin of BSC may be the primitive totipotential cell. BSC occurred more frequently in elderly males. The biological behavior of BSC was highly malignant. Regional lymph nodes or distant organ metastasis were usually found at the first operation. The mean survival period after operation was very short, BSC was therefore considered to be a specific clinicopathological entity.

Actins↗
PubMed1995

Percutaneous endovascular embolization of intracerebral arteriovenous malformations. Experience in 72 cases.

Endovascular therapeutic embolization of arteriovenous malformations (AVMs) of the brain was performed in 72 patients between October 1986 and March 1993. From October 1986 to May 1991, 38 patients in this series were treated with isobuty1-2-cyanoacrylate (IBCA) glue. After June 1991, in the remaining 34 patients the embolic materials used included surgical silk (5-0), polyvinyl alcohol (PVA), ethanol, and estrogen. After treatment, 29 patients (40.3%) had complete angiographic obliteration of AVM, 15 (21%) transient neurologic postembolization deficit, and 3 (4%) permanent deficit without death. Embolic agents and procedures are discussed as to their curative effects, safety, normal perfusion pressure breakthrough (NPPB) problem, with silk mixture fluid being considered preferable.

Adolescent↗
PubMed1995

[Effect of electroacupuncture on the discharges of pain-sensitive neurons in the hypothalamic dorsomedial nucleus of rats].

The discharges of pain-sensitive neurons of the hypothalamic dorsomedial nucleus (DMH) in Sprague-Dawley rats were recorded with glass microelectrodes, After electroacupuncture acupoints "Zusanli" and "Sanyinjiao", the rate of spontaneous discharges and the rate and duration of the pain-evoked discharges of pain-excitatory units of DMH were profoundly decreased, and the spontaneous firing rate of pain-inhibitory units was increased while their inhibitory response to nociceptive stimulation was released by the electroacupuncture. The results mentioned above suggest that DMH participates in the activity of acupuncture analgesia.

Acupuncture Analgesia↗
PubMed1995

[A study on the relative factors for secondary parathyroidosis and renal osteodystrophy in long-term hemodialysis patients].

The authors classified sixty chronic renal failure (CRF) patients receiving hemodialysis (HD) treatment into four groups for clinical study. Forty patients received HD for more than five years and the remaining twenty patients received HD for less than two years. These two groups were further divided into two subgroups according to whether they took Rocaltrol or not. The levels of parathyroid hormone (PTH), calcitonin (CT), alkaline phosphatase (AKP) and bone X-ray were studied in each patient. The results showed: the levels of PTH and CT were obviously increased in all the patients. The levels of PTH and CT were higher in the patients having received HD for more than five years than those for less than two years. They were also higher in patients who had not taken Rocatrol than those who had. Ninety-five percent of the patients having received HD for more than five years had renal osteodystrophy (ROD) without receiving Rocaltrol treatment, while only sixty-five percent of the patients developed ROD with Rocatrol treatment. The longer the HD time, the higher the ROD incidence. The cause, prevention and treatment of ROD were discussed.

Adult↗