Biomedical subjects

Z Huang

Publications and source records attributed to Z Huang.

At least 55 records · Page 3Linked to original sources

PubMed2001

The stability of tetramine, morphine and meperidine in formalin solution.

The stability of tetramine, morphine and meperidine in formalin solution is an important factor for drug analysis in forensic investigation. In this paper, the tissues (liver, kidney, lung and heart) from poisoned rabbits were immersed in 50 ml 10% formalin solutions for 4 months before examination. We compared the levels of tetramine, morphine, meperidine and the main metabolite normeperidine, measured by GC/NPD or GC-MS, in frozen rabbit tissues, formalin-fixed rabbit tissues, and formalin solution. There was a decrease in the levels of tetramine, morphine, meperidine in formalin-preserved tissues compared with the levels of these drugs in the frozen tissues. It is suggested that the formalin-fixed tissues and formalin solution should be analyzed at the same time to assure the accurate results.

Animals↗

PubMed2001

Purification and characterization of the human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells.

The human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells was found to be heavily phosphorylated on both serines of the conserved SPS motif by mass spectrometric analysis. The purified protein exists as a tetramer at a concentration approximately 1 mg/ml from light scattering measurement and has a Km of 2 microM in hydrolyzing cAMP. In comparison, a partially purified PDE4A330-723 expressed in Escherichia coli has an apparent Km of 10 microM. The EC50 values for the Mg2+- or Co2+-mediated cAMP hydrolysis between the two enzymes differed by less than twofold. In addition, both enzymes exhibit similar sensitivities toward inhibition by a diverse set of inhibitors. Together with the fact that its adjacent peptide was covalently labeled by an electrophilic cAMP analogue, these results support that the SPS motif is not part of but is positioned near the active site. An efficient purification protocol that provides a highly purified PDE4A catalytic domain suitable for crystallization study is described.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2001

Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies.

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.

Aspartic Acid↗

PubMed2001

Matching conditions in atomistic-continuum modeling of materials.

A new class of matching conditions between the atomistic and continuum regions is presented for the multiscale modeling of crystals. They ensure the accurate passage of large scale information between the atomistic and continuum regions and at the same time minimize the reflection of phonons at the interface. These matching conditions can be made adaptive if we choose appropriate weight functions. Applications to dislocation dynamics and friction between two-dimensional atomically flat crystal surfaces are described.

Journal Article↗

PubMed2001

Dissecting the cofactor-dependent and independent bindings of PDE4 inhibitors.

Type 4 phosphodiesterases (PDE4s) are metallohydrolases that catalyze the hydrolysis of cAMP to AMP. At the bottom of its active site lie two divalent metal ions in a binuclear motif which are involved in both cAMP binding and catalysis [(2000) Science 288, 1822-1825; (2000) Biochemistry 39, 6449-6458]. Using a SPA-based equilibrium [(3)H]rolipram binding assay, we have determined that Mg(2+), Mn(2+), and Co(2+) all mediated a high-affinity (K(d) between 3 and 8 nM) and near stoichiometric (R)-rolipram binding to PDE4. In their absence, (R)-rolipram binds stoichiometrically to the metal ion-free apoenzyme with a K(d) of approximately 150 nM. The divalent cation dose responses in mediating the high-affinity rolipram/PDE4 interaction mirror their efficacy in catalysis, suggesting that both metal ions of the holoenzyme are involved in mediating the high-affinity (R)-rolipram/PDE4 interaction. The specific rolipram binding to the apo- and holoenzyme is differentially displaced by cAMP, AMP, and other inhibitors, providing a robust tool to dissect the components of metal ion-dependent and independent PDE4/ligand interactions. cAMP binds to the holoenzyme with a K(s) of 1.9 microM and nonproductively to the apoenzyme with a K(d) of 179 microM. In comparison, AMP binds to the holo- and apoenzyme with K(d) values of 7 and 11 mM, respectively. The diminished Mg(2+)-dependent component of AMP binding to PDE4 suggests that most of the Mg(2+)/phosphate interaction in the cAMP/PDE4 complex is disrupted upon the hydrolysis of the cyclic phosphoester bond, leading to the rapid release of AMP.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2001

Beta1-class integrins regulate the development of laminae and folia in the cerebral and cerebellar cortex.

Mice that lack all beta1-class integrins in neurons and glia die prematurely after birth with severe brain malformations. Cortical hemispheres and cerebellar folia fuse, and cortical laminae are perturbed. These defects result from disorganization of the cortical marginal zone, where beta1-class integrins regulate glial endfeet anchorage, meningeal basement membrane remodeling, and formation of the Cajal-Retzius cell layer. Surprisingly, beta1-class integrins are not essential for neuron-glia interactions and neuronal migration during corticogenesis. The phenotype of the beta1-deficient mice resembles pathological changes observed in human cortical dysplasias, suggesting that defective integrin-mediated signal transduction contributes to the development of some of these diseases.

Animals↗

PubMed2001

Dopamine and sensory tissue development in Drosophila melanogaster.

Dopamine is an important signaling molecule in the nervous system; it also plays a vital role in the development of diverse non-neuronal tissues in the fruit fly Drosophila melanogaster. The current study demonstrates that males depleted of dopamine as third instar larvae (via inhibition of the biosynthetic enzyme tyrosine hydroxylase) demonstrated abnormalities in courtship behavior as adults. These defects were suggestive of abnormalities in sensory perception and/or processing. Electroretinograms (ERGs) of eyes from adults depleted of dopamine for 1 day as third instar larvae revealed diminished or absent on- and off-transients. These sensory defects were rescued by the addition of L-DOPA in conjunction with tyrosine hydroxylase inhibition during the larval stage. Depletion of dopamine in the first or second larval instar was lethal, but this was not due to a general inhibition of proliferative cells. To establish that dopamine was synthesized in tissues destined to become part of the adult sensory apparatus, transgenic lines were generated containing 1 or 4 kb of 5' upstream sequences from the Drosophila tyrosine hydroxylase gene (DTH) fused to the E. coli beta-galactosidase reporter. The DTH promoters directed expression of the reporter gene in discrete and consistent patterns within the imaginal discs, in addition to the expected expression in gonadal, brain, and cuticular tissues. The beta-galactosidase expression colocalized with tyrosine hydroxylase protein. These results are consistent with a developmental requirement for dopamine in the normal physiology of adult sensory tissues.

Animals↗

PubMed2001

Myelin antigen-specific CD8+ T cells are encephalitogenic and produce severe disease in C57BL/6 mice.

Encephalitogenic T cells that mediate experimental autoimmune encephalomyelitis (EAE) are commonly assumed to be exclusively CD4+, but formal proof is still lacking. In this study, we report that synthetic peptides 35-55 from myelin oligodendrocyte glycoprotein (pMOG(35-55)) consistently activate a high proportion of CD8+ alphabetaTCR+ T cells that are encephalitogenic in C57BL/6 (B6) mice. The encephalitogenic potential of CD8+ MOG-specific T cells was established by adoptive transfer of CD8-enriched MOG-specific T cells. These cells induced a much more severe and permanent disease than disease actively induced by immunization with pMOG(35-55). CNS lesions in pMOG(35-55) CD8+ T cell-induced EAE were progressive and more destructive. The CD8+ T cells were strongly pathogenic in syngeneic B6 and RAG-1(-/-) mice, but not in isogeneic beta2-microglobulin-deficient mice. MOG-specific CD8+ T cells could be repeatedly reisolated for up to 287 days from recipient B6 or RAG-1(-/-) mice in which disease was induced adoptively with <1 x 10(6) T cells sensitized to pMOG(35-55). It is postulated that MOG induces a relapsing and/or progressive pattern of EAE by eliciting a T cell response dominated by CD8+ autoreactive T cells. Such cells appear to have an enhanced tissue-damaging effect and persist in the animal for long periods.

Adoptive Transfer↗

PubMed2001

Solid-phase microextraction using fused-silica fibers coated with sol-gel-derived hydroxy-crown ether.

A novel solid-phase microextraction (SPME) fiber containing hydroxydibenzo-14-crown-4 (OH-DB14C4)/hydroxy-terminated silicone oil (OH-TSO) was first prepared by a sol-gel method and investigated for the determination of phenols. The possible mechanism is discussed and confirmed by IR spectra. The coating has stable performance in high temperature (to 350 degrees C) and solvents (organic and inorganic) due to the chemical binding between the coating and the fiber surface. The addition of crown ether enhances the polarity of the coating compared with that of the sol-gel OH-terminated silicone oil fiber and, accordingly, provides higher extraction efficiency for polar phenolic compounds. On the other hand, OH-terminated silicone oil in the coating can not only increase the length of network but also help to spread the stationary phase on the silica surface uniformly. The fluorescence microscopy experiment suggests the benefit the more uniform surface of the sol-gel-derived OHDB14C4/OH-TSO fiber in comparison with sol-gelderived OH-DB14C4 fiber. Some parameters of the SPME fiber for the determination of phenols were investigated. Limits of detection of the phenols are below 1.0 ng/mL, and the precisions are from 2.9 to 4.6% (n = 6). Linear ranges were found to be 0.1-10 microg/mL The sensitivity of the method is enhanced at a low-pH level (pH approximately 1) and with the addition of salt. The method was applied to the analysis of wastewater sample from a paper mill.

Journal Article↗

PubMed2001

Exponential gain and saturation of a self-amplified spontaneous emission free-electron laser.

Self-amplified spontaneous emission in a free-electron laser has been proposed for the generation of very high brightness coherent x-rays. This process involves passing a high-energy, high-charge, short-pulse, low-energy-spread, and low-emittance electron beam through the periodic magnetic field of a long series of high-quality undulator magnets. The radiation produced grows exponentially in intensity until it reaches a saturation point. We report on the demonstration of self-amplified spontaneous emission gain, exponential growth, and saturation at visible (530 nanometers) and ultraviolet (385 nanometers) wavelengths. Good agreement between theory and simulation indicates that scaling to much shorter wavelengths may be possible. These results confirm the physics behind the self-amplified spontaneous emission process and forward the development of an operational x-ray free-electron laser.

Journal Article↗

PubMed2001

2-[N1-2-pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate: a potent antitumor agent.

2-[N'-2-Pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate has been prepared by reaction of chlorambucil with sulfadiazine derivative. Schiffs base has been used as the protective group of the aromatic amine in the synthesis. It can be completely removed by the irradiation of 365 nm UV light at room temperature. The title compound exhibits a high antitumor activity with a therapeutic index (TI) of 47.55 which is twice that of chlorambucil's (TI: 22.84).

Animals↗

PubMed2001

Acidic fibroblast growth factor (FGF-1) signaling inhibits peroxynitrite-induced cell death during pancreatic tumorigenesis.

Previous immunohistochemical studies have demonstrated enhanced appearance of FGF-1 and nitrotyrosine, a footprint of reactive nitrogen species peroxynitrite (ONOO(-)), in human pancreatic adenocarcinoma. We have examined the consequences of constitutive exposure to FGF-1 in nontumorigenic rat ductal epithelial cells (ARIP). ARIP cells were transduced with either a secreted chimera of FGF-1, ARIP(FGF-1), or a control plasmid, 65 RIP(betag). These cells were evaluated for alteration in growth and morphology, responses to ONOO(-) (protein tyrosine nitration/phosphorylation), and in vivo tumor formation. ARIP(FGF-1) cells, in contrast to 65 RIP(betag), demonstrated a transformed morphology, a 2-fold increased growth rate, and enhanced protein tyrosine phosphorylation. Treatment with 150 microM ONOO(-) resulted in 86 and 7% (p <.01) death of ARIP(betag) and ARIP(FGF-1), respectively. Exposure of 65 RIP(betag) cells to ONOO(-) enhanced tyrosine phosphorylation and tyrosine nitration of several polypeptides. Cell signaling by FGF-1 enhanced both phosphorylation and nitration of tyrosine residues in target proteins modified by ONOO(-). ARIP(betag) cells failed to exhibit tumor formation in nude mice, but at d 7 in vivo cells were TUNEL and nitrotyrosine positive and FGF-1 negative. ARIP(FGF-1) cells readily formed tumor nodules, exhibiting features of pancreatic adenocarcinoma and demonstrating FGF-1-positive, nitrotyrosine-positive, and TUNEL-negative epithelium. These results suggest an interdependent role between FGF-1 and ONOO(-) during the development and progression of pancreatic adenocarcinoma.

Animals↗

PubMed2001

Regulation of type II phosphatidylinositol phosphate kinase by tyrosine phosphorylation in bovine rod outer segments.

Type II phosphatidylinositol phosphate kinase (PIPKII) is an enzyme responsible for the synthesis of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)) from phosphatidylinositol-5-phosphate (PI-5-P). In this study, we demonstrate the presence of PIPKII alpha in bovine photoreceptor rod outer segments (ROS) and the involvement of tyrosine phosphorylation in the regulation of its activity. PIPKII activity in bovine ROS was verified by the preferential conversion of synthetic dipalmitoyl PI-5-P to PI-4,5-P(2), lack of effect of phosphatidic acid, inhibition by heparin, immunoreaction with an anti-PIPKII alpha antibody on Western blots, and immunocytochemical localization in bovine and rat ROS by anti-PIPKII alpha. Immunoprecipitates of bovine ROS with the anti-PIPKII alpha antibody possessed PIPK enzymatic activity and preferentially used PI-5-P as substrate for PI-4,5-P(2) biosynthesis. The activity of PIPKII was greatly increased under conditions favoring tyrosine phosphorylation in ROS, and PIPKII activity was immunoprecipitated with anti-phosphotyrosine (anti-PY) antibodies from tyrosine phosphorylated ROS. Preincubation of ROS with tyrosine kinase inhibitors almost abolished the kinase activity in the anti-PY immunoprecipitates. Immunoblot analysis showed that PIPKII alpha was present in anti-PY immunoprecipitates from phosphorylated ROS but not from nonphosphorylated controls. We conclude that PIPKII alpha is present in ROS and that its activity is regulated by tyrosine phosphorylation.

Animals↗

PubMed2001

Sustained activation of the extracellular signal-regulated kinase pathway is required for extracellular calcium stimulation of human osteoblast proliferation.

Elevated levels of [Ca(2+)](o) in bone milieu as a result of the resorptive action of osteoclasts are implicated in promoting proliferation and migration of osteoblasts during bone remodeling. However, mitogenic effects of [Ca(2+)](o) have only been shown in some, but not all, clonal osteoblast-like cells, and the molecular mechanisms underlying [Ca(2+)](o)-induced mitogenic signaling are largely unknown. In this study we demonstrated for the first time that [Ca(2+)](o) stimulated proliferation of primary human osteoblasts and selectively activated extracellular signal-regulated kinases (ERKs). Neither p38 mitogen-activated protein (MAP) kinase nor stress-activated protein kinase was activated by [Ca(2+)](o). Treatment of human osteoblasts with a MAP kinase kinase inhibitor, PD98059, impaired both basal and [Ca(2+)](o)-stimulated phosphorylation of ERKs and also reduced both basal and [Ca(2+)](o)-stimulated proliferation. [Ca(2+)](o) treatment resulted in two distinctive phases of ERK activation: an acute phase and a sustained phase. An inhibition time course revealed that it was the sustained phase, not the acute phase, that was critical for [Ca(2+)](o)-stimulated osteoblast proliferation. Our results demonstrate that mitogenic responsiveness to [Ca(2+)](o) is present in primary human osteoblasts and is mediated via prolonged activation of the MAP kinase kinase/ERK signal pathway.

3T3 Cells↗

PubMed2001

Cloning the full-length IL-2/15 receptor-beta cDNA sequence from mouse brain: evidence of enrichment in hippocampal formation neurons.

Numerous studies have implicated interleukin-2 (IL-2) in various brain processes, and more recently, several studies have also attributed neurobiological actions to interleukin-15 (IL-15). On lymphocytes, receptors for IL-2 and IL-15 share a common subunit, the IL-2/15 receptor-beta (IL-2/15Rbeta) that is essential for intracellular signaling. Although a short segment of IL-2/15Rbeta has been cloned (0.35 kb) from normal brain cells, attempts to isolate the full-length cDNA have been unsuccessful, suggesting the possibility that the genes expressed by brain cells and lymphocytes may differ. Using conventional and anchored PCR cloning strategies, we isolated the full-length cDNA of IL-2/15Rbeta (2038 bp) from well-perfused, normal mouse forebrain. The coding sequence and the adjacent 5' and 3' UTR sequences from brain and lymphocyte were found to be fully homologous. Although evidence of expression of IL-2/15Rbeta can be found in many brain regions using PCR, clear evidence of gene expression by in situ hybridization was detectable only in the hippocampal formation, habenula and piriform cortex. This same pattern of mRNA expression in situ was also observed for the common gamma subunit shared by IL-2 and IL-15. In the hippocampus, IL-2/15Rbeta expression was localized to neurons by high resolution in situ hybridization and evidence of IL-2 receptor protein expression was also detected by radioligand receptor binding using hippocampal homogenates. Comparison of undifferentiated and differentiated, immortalized H19-7 hippocampal neurons showed that IL-2/15Rbeta was constitutively expressed across disparate stages of hippocampal neuronal differentiation. These data indicate that IL-2/15Rbeta may serve to modulate neuronal processes in the hippocampus and associated limbic brain regions.

Animals↗

PubMed2001

Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice.

Measles remains a significant problem in both the developed and developing world, and new measles vaccination strategies need to be developed. This paper examines the strategy of utilizing transgenic plants expressing a measles antigen for the development of an oral sub-unit measles vaccine. A 1.8 kb fragment encompassing the coding region of the measles virus hemagglutinin (H) protein was cloned into a plant expression cassette. Three different expression constructs were tested: pBinH (H gene alone), pBinH/KDEL (addition of a C-terminal endoplasmic reticulum-retention sequence SEKDEL) and pBinSP/H/KDEL (further addition of an authentic N-terminal plant signal peptide). The highest levels of recombinant H protein production were observed in plants transformed with pBinH/KDEL. Mice inoculated intraperitoneally with transgenic plant derived recombinant H protein produced serum anti-H protein antibodies that neutralized the measles virus (MV) in vitro. Mice gavaged with transgenic tobacco leaf extracts also developed serum H protein-specific antibodies with neutralizing activity against MV in vitro. These results indicate that the plant-derived measles H protein is immunogenic when administered orally and that, with further development, oral vaccination utilizing transgenic plants may become a viable approach to measles vaccine development.

Administration, Oral↗

PubMed2001

Effect of a cyclic heptapeptide based on the human CD4 domain 1 CC' loop region on murine experimental allergic encephalomyelitis: inhibition of both primary and secondary responses.

The 802-2 peptide, designed from the conserved D1-CC' loop region of human CD4, can disrupt CD4(+) T cell activation in both human and murine systems. Here, 802-2 was investigated for efficacy in acute murine experimental allergic encephalomyelitis (EAE) models, and was found to significantly reduce the severity of disease when administered either before or after the onset of symptoms. 802-2 treatment during PLP139-151 induction of EAE rendered the mice more resistant to subsequent rechallenge with antigen, and was also efficacious when initially administered during a secondary EAE response. T cells from 802-2-treated mice proliferated poorly to in vitro restimulation with PLP139-151 and exhibited decreased frequencies of IL-2, IL-4, and IFN-gamma producing cells, but were still able to respond to third-party antigens. These combined results suggest the potential therapeutic value of 802-2 for inhibition of CD4(+) T cell neuroimmunological responses.

Animals↗

PubMed2001

A method for UV-bonding in the fabrication of glass electrophoretic microchips.

This paper presents an approach for the development of methodologies amenable to simple and inexpensive microchip fabrication, potentially applicable to dissimilar materials bonding and chip integration. The method involves a UV-curable glue that can be used for glass microchip fabrication bonding at room temperature. This involves nothing more than fabrication of glue "guide channels" into the microchip architecture that upon exposure to the appropriate UV light source, bonds the etched plate and cover plate together. The microchip performance was verified by capillary zone electrophoresis (CZE) of small fluorescent molecules with no microchannel surface modification carried out, as well as with a DNA fragment separation following surface modification. The performance of these UV-bonded electrophoretic microchips indicates that this method may provide an alternative to high temperature bonding.

Adhesives↗