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Biomedical subjects

W Huang

Publications and source records attributed to W Huang.

At least 127 records · Page 7Linked to original sources
PubMed2001

Tissue remodeling of rat pulmonary artery in hypoxic breathing. I. Changes of morphology, zero-stress state, and gene expression.

The remodeling of the pulmonary arterial tissue in response to a step change of the oxygen concentration in the gas in which a rat lives was recorded as function of time and function of O2 concentration. Three steps of changing from 20.9% to 17.2%, 13.6%, and 10% O2 were imposed. Earlier work in our laboratory has shown that pulmonary arterial tissue remodeling is significant in the first 24 h after a step change of oxygen tension. Hence we made measurements in this period. Furthermore, data were obtained for tissue remodeling of circumferential and axial lengths of the pulmonary arteries. We recorded the activities of gene expressions in the lung tissues by microarray, determined the dose response curves of gene expression in the homogenized whole lungs with respect to four levels of O2 concentration, and obtained the time courses of gene expression in the lung parenchyma in 30 days after a step decrease of O2 concentration from 20.9% to 10%. We would like to suggest that the correlation of gene expression with physiological function parameters, i.e., time, O2 tension, blood pressure, opening angle, wall thicknesses, etc., is the way to narrow down the search for specific genes for specific physiological functions.

Animals↗
PubMed2001

Tissue remodeling of rat pulmonary artery in hypoxic breathing. II. Course of change of mechanical properties.

When cells and the matrix of a tissue remodel, the mechanical properties of the tissue do change. The mechanical properties are expressed by constitutive equations. In this article the remodeling of the constitutive equation of the pulmonary artery is studied. The remodeling was induced in a rat breathing a gas whose oxygen concentration was suddenly decreased as a step function of time and maintained constant (17.2%, 13.6%, or 10%) afterwards. Since the mathematical form of the constitutive equation has been identified in earlier papers, we need to determine only the elastic constants that change in the process of tissue remodeling. We consider arteries subjected to blood pressure and longitudinal stretch, and limit ourselves to two-dimensional problems involving only circumferential and longitudinal stress and strain. In the neighborhood of an in vivo state, the perturbations of stresses and strains are related by linear, anisotropic, tensor equations involving three elastic constants: the incremental Young's modulus in the circumferential direction Ythetaz, that in the longitudinal direction Yzz, and the cross modulus Ythetaz. Over a 24 h period, changes of Ythetatheta between 164 and 187 kN/m2, Yzz between 64 and 92 kN/m2, and Ythetaz between 61 and 88 kN/m2 are statistically insignificant.

Animals↗
PubMed2001

The surface-tension-driven flow of blood from a droplet into a capillary tube.

In tissue, medical, or dental engineering, when blood comes into contact with a new artificial material, the flow may be influenced by surface tension between the blood and the surface of the material. The effect of surface tension on the flow of blood is significant, especially in microscale. The leading edge of the flowing blood is the triple point where the blood, the material surface, and a stationary gas orfluid meet. The movement of the triple point, i.e., the advancing front of the flow, is driven by surface tension, resisted by viscous shear stress, and balanced by the inertial force (-mass x acceleration). In this article, the dynamics is illustrated in detail in the case of blood flowing into a capillary tube by contact. The capillar, tube draws the blood into it. It is shown theoretically that initially the flow of blood in the capillary has a large acceleration, followed by a relatively large deceleration over the next short period of time, then the acceleration becomes small and oscillatory. The velocity history appears impulsive at first, then slows down. The history of the length of blood column appears smooth after integration. Existing solutions of the Navier-Stokes equation permit the analysis of simpler cases. Further fluid mechanics development is needed to meet the practical needs of bioengineering. The importance of experimental study of surface tension and contact angle over a biological surface or a man-made material as a future direction of research is pointed out.

Biomechanical Phenomena↗
PubMed2001

Binding properties of a peptide derived from beta-lactamase inhibitory protein.

To overcome the antibiotic resistance mechanism mediated by beta-lactamases, small-molecule beta-lactamase inhibitors, such as clavulanic acid, have been used. This approach, however, has applied selective pressure for mutations that result in beta-lactamases no longer sensitive to beta-lactamase inhibitors. On the basis of the structure of beta-lactamase inhibitor protein (BLIP), novel peptide inhibitors of beta-lactamase have been constructed. BLIP is a 165-amino-acid protein that is a potent inhibitor of TEM-1 beta-lactamase (K(i) = 0.3 nM). The cocrystal structure of TEM-1 beta-lactamase and BLIP indicates that residues 46 to 51 of BLIP make critical interactions with the active site of TEM-1 beta-lactamase. A peptide containing this six-residue region of BLIP was found to retain sufficient binding energy to interact with TEM-1 beta-lactamase. Inhibition assays with the BLIP peptide reveal that, in addition to inhibiting TEM-1 beta-lactamase, the peptide also inhibits a class A beta-lactamase and a class C beta-lactamase that are not inhibited by BLIP. The crystal structures of class A and C beta-lactamases and two penicillin-binding proteins (PBPs) reveal that the enzymes have similar three-dimensional structures in the vicinity of the active site. This similarity suggests that the BLIP peptide inhibitor may have a broad range of activity that can be used to develop novel small-molecule inhibitors of various classes of beta-lactamases and PBPs.

Bacterial Proteins↗
PubMed2001

Efficient c-kit receptor-targeted gene transfer to primary human CD34-selected hematopoietic stem cells.

We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit(+) human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer.

Adenoviridae↗
PubMed2001

Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus.

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.

Alpharetrovirus↗
PubMed2001

Oxytocin antagonist disrupts hypotension-evoked renin secretion and other responses in conscious rats.

Previous experiments have indicated that arterial hypotension increases plasma oxytocin (OT) levels in rats and that OT infused intravenously causes an increase in plasma renin activity (PRA). The goal of the present study was to determine whether systemic administration of an OT receptor antagonist would attenuate the increase in PRA that is normally evoked by arterial hypotension in rats. In conscious male rats, intravenous injection of hydralazine or diazoxide produced sustained hypotension and evoked a significant increase in PRA, as expected. Intravenous infusion of an OT receptor antagonist did not alter the hypotension induced by hydralazine or diazoxide, but it did markedly blunt the induced increase in PRA. The OT receptor antagonist also blunted the hypotension-evoked increase in heart rate and plasma vasopressin levels, suggesting that the antagonist may have generally disrupted afferent signaling of hypotension. Thus hypotension-evoked OT secretion may contribute to cardiovascular homeostasis by enhancing baroreceptor signals that stimulate increases in renin secretion, vasopressin secretion, and heart rate during arterial hypotension in rats.

Animals↗
PubMed2001

Modification of gold surface by grafting of poly(ethylene glycol) for reduction in protein adsorption and platelet adhesion.

Gold surfaces were first treated in an alkanethiol solution to form self-assembled monolayers (SAMs). The thiolated Au surface was then subjected to Ar plasma pretreatment, followed by air exposure and UV-induced graft polymerization of poly(ethylene glycol) methacrylate (PEGMA) macromonomer. In comparison with the 3-mercaptopropionic acid-2-ethylhexyl ester (MPAEE) SAM, the (3-mercaptoproply)trimethoxysilane (MPTMS) SAM on Au exhibited higher stability under the conditions of Ar plasma pretreatment. The graft concentration of the PEGMA polymer on SAM-modified Au surface increased with increasing PEGMA macromonomer concentration and UV-graft polymerization time. The modified-Au surfaces were characterized by X-ray spectroscopy (XPS), atomic force microscopy (AFM), and water contact angle measurement. The Au surface with a high concentration of grafted PEGMA polymer could completely repel protein adsorption and platelet adhesion.

Adsorption↗
PubMed2001

Na+ - and Cl- -coupled active transport of nitric oxide synthase inhibitors via amino acid transport system B(0,+).

Nitric oxide synthase (NOS) inhibitors have therapeutic potential in the management of numerous conditions in which NO overproduction plays a critical role. Identification of transport systems in the intestine that can mediate the uptake of NOS inhibitors is important to assess the oral bioavailability and therapeutic efficacy of these potential drugs. Here, we have cloned the Na+ - and Cl- -coupled amino acid transport system B(0,+) (ATB(0,+)) from the mouse colon and investigated its ability to transport NOS inhibitors. When expressed in mammalian cells, ATB(0,+) can transport a variety of zwitterionic and cationic amino acids in a Na+ - and Cl- -coupled manner. Each of the NOS inhibitors tested compete with glycine for uptake through this transport system. Furthermore, using a tritiated analog of the NOS inhibitor N(G)-nitro-L-arginine, we showed that Na+ - and Cl- -coupled transport occurs via ATB(0,+). We then studied transport of a wide variety of NOS inhibitors in Xenopus laevis oocytes expressing the cloned ATB(0,+) and found that ATB(0,+) can transport a broad range of zwitterionic or cationic NOS inhibitors. These data represent the first identification of an ion gradient-driven transport system for NOS inhibitors in the intestinal tract.

Amino Acid Transport System ASC↗
PubMed2001

The novel gene locus for agenesis of permanent teeth (He-Zhao deficiency) maps to chromosome 10q11.2.

He-Zhao deficiency has been recently characterized with a distinct form of agenesis of permanent teeth that is different from other previously reported disorders of tooth agenesis. This inherited abnormality suggests that some gene(s) associated with the development of permanent teeth may mutate. In this study, we map the gene locus to chromosome 10q11.2. The DNA pooling method combined with two-point and multi-point linkage analysis has been successfully applied. The maximum LOD (Zmax) scores for two-point and multi-point analyses are 13.29 (on marker D10S196) at recombination fraction (theta) = 0 and 18.09 (between markers D10S1772 and D10S1766), respectively. Haplotype analysis confined the locus within an interval of 5.5 cM flanked by markers D10S604 and D10S568. This study has demonstrated a novel gene locus responsible for He-Zhao deficiency and provides a good likelihood for the discovery of one of the genes determining permanent tooth formation and development.

Alleles↗
PubMed2001

cDNA library screening using the SOS recruitment system.

The SOS recruitment system (SRS), a recently developed method for detecting protein-protein interactions, provides an attractive alternative to identify biologically important protein interactions. In SRS, the protein-protein interactions take place in the cytoplasm instead of the nucleus, as is the case in the conventional two-hybrid system. Although the SRS has overcome some of the disadvantages of the conventional two-hybrid system, it still has several problems and limitations. Here, we describe a new protocol for SRS library screening. A new combination of growth media to avoid the tedious step of replica plating greatly increases the number of independent colonies in a single library screening. Furthermore, we designed a pair of ras-specific primers and a one-step simple PCR to rule out the most abundant false positive, the mammalian ras cDNA, in SRS library screening.

Animals↗
PubMed2001

Discovery of transition state factor Xa inhibitors as potential anticoagulant agents.

Factor Xa is an attractive biological target in the discovery and development of either parenteral or orally active anticoagulant agents. Several strategies have been utilized at COR Therapeutics in the pursuit of tri-peptide based transition state mimetic factor Xa inhibitors with high aqueous solubility. Some of these inhibitors have displayed excellent in vitro potency in inhibiting factor Xa in the prothrombinase complex. More importantly, these compounds showed strong in vivo antithrombotic efficacy without significant bleeding complications in several animal thrombosis models. These results demonstrated that small molecule factor Xa inhibitors could be advantageous over Warfarin and LMWH. For the discovery and development of orally active anticoagulant agents, small organic molecules as reversible factor Xa inhibitors were explored. From a medicinal chemistry perspective, significant insight has been gained regarding the in vivo antithrombotic efficacy and pharmacokinetic behaviors of each class of factor Xa inhibitors. This review will focus on the design and discovery of transition state factor Xa inhibitors as potential parenteral anticoagulant agents. Several excellent comprehensive review articles on factor Xa inhibitors have appeared recently [1-4].

Animals↗
PubMed2001

Structure, function, and regulation of human cystine/glutamate transporter in retinal pigment epithelial cells.

PURPOSE: The purpose of this investigation was to provide evidence for the expression of the cystine/glutamate transporter (x(c)(-)) in the human retinal pigment epithelial cell line ARPE-19, clone the light chain of the transporter from an ARPE-19 cell cDNA library and study its function, and investigate the regulation of this transporter by nitric oxide (NO) in ARPE-19 cells. METHODS: Uptake of radiolabeled cystine and glutamate was measured in ARPE-19 cells. The functional identity of x(c)(-) in these cells was established by substrate specificity and Na(+)-independence of the uptake process. The human x(c)(-) light chain (human xCT) was cloned from an ARPE-19 cell cDNA library. The functional identity of the cloned human xCT was investigated by heterologous coexpression of the light chain with the heavy chain (human 4F2hc) in HeLa cells. ARPE-19 cells were treated with or without the NO donor 3-nitroso-N:-acetylpenicillamine (SNAP) and the expression of x(c)(-) was studied at the functional and molecular levels. RESULTS: ARPE-19 cells take up cystine as well as glutamate in the absence of Na(+). Substrate specificity studies indicate that although the uptake of cystine in the absence of Na(+) is mediated by multiple amino acid transport systems including x(c)(-), the uptake of glutamate in the absence of Na(+) occurs exclusively via x(c)(-). The human xCT cloned from ARPE-19 cells is a protein of 501 amino acids. These cells express the heavy chain 4F2hc as evidenced from RT-PCR analysis. Coexpression of human xCT with 4F2hc in HeLa cells leads to the induction of cystine and glutamate uptake with characteristics similar to that of x(c)(-). The activity of x(c)(-) in ARPE-19 cells is upregulated by SNAP, and the process is associated with an increase in the expression of xCT with no detectable change in the expression of 4F2hc. CONCLUSIONS: ARPE-19 cells express the cystine/glutamate transporter x(c)(-) (the light chain xCT and the heavy chain 4F2hc) as is evident from functional and molecular studies. NO upregulates this transport system and the process is associated with an increase in xCT mRNA but with no change in 4F2hc mRNA.

Amino Acid Sequence↗
PubMed2001

Variables in human liver microsome preparation: impact on the kinetics of l-alpha-acetylmethadol (LAAM) n-demethylation and dextromethorphan O-demethylation.

We examined three primary variables in the preparation of human liver microsomes. In three experiments, each using three livers, we manipulated 1) the force of the first centrifugation (9,000, 10,500, or 12,000g); 2) the presence of sucrose in the homogenization buffer; and 3) the number of homogenizing strokes (6, 8, or 10). Sedimentation plots for the marker enzymes succinate dehydrogenase, NADPH cytochrome P450 reductase (reductase), and glutathione S-transferase in the resulting premicrosomal, microsomal, and cytosolic fractions suggest that enhanced purity of microsomes can be obtained by reducing force of centrifugation, including sucrose, and increasing the number of homogenization strokes. Each microsomal fraction was also assayed for protein content, cytochrome P450, NADH cytochrome b(5) reductase, cytochrome b(5), absorbance at 420, p-nitrophenol hydroxylation, tolbutamide hydroxylation, dextromethorphan N- and O-demethylation, glucuronidation of morphine and 1-naphthol, and ester cleavage of p-nitrophenolacetate. These microsomal indicators were ranked and tested for statistical differences. The use of 9000g statistically increased optimal recovery (per gram of liver) and specific activity (per milligram of protein). The inclusion of sucrose improved activity specific to reductase activity. Ten homogenization strokes improved activity specific to reductase activity. Substrate-dependent activities of dextromethorphan O-demethylation to dextrorphan and the N-demethylation of l-alpha-acetylmethadol (LAAM) to norLAAM and dinorLAAM were compared in microsomes prepared with or without sucrose and microsomes prepared using 9,000 or 12,000g force, respectively. No significant differences were found in the concentration-dependent activities. Variation of the methods used to prepare human liver microsomes can significantly affect the recovery and specific activity of microsomal components; however, they do not appear to affect enzyme kinetics.

Adolescent↗
PubMed2001

Correlation of gene expression with physiological functions: Examples of pulmonary blood vessel rheology, hypoxic hypertension, and tissue remodeling.

Microarray gene chip technology is a powerful invention looking for applications. A general principle is proposed here to direct the power of the technology toward physiology, medicine, and pharmacology. Our principle is to match quantitative measures of gene expression with the trend of mathematical parameters that describe biological functions. Mathematical parameterization is the heart. The procedure is illustrated by lung physiology, including the hypoxic hypertension, rheological properties of the tissues, and the remodeling of the pulmonary arterial wall under hypertensive stress. We show first how to reduce the experimental results on these physiological functions into mathematical formulas, and how the parameters of these formulas describe the functional trends precisely. Then under the assumption that the microarray reveals gene activities quantitatively, we match the trends of the gene activity with the trends of the functional parameters. Genes whose trends do match are interpreted as relevant to the functions. Those that do not match are considered irrelevant to the functions. The more functions we consider, the fewer will be the number of genes that are relevant to all functions. Thus we learn about the generality and specificity of the influence of genes on physiology.

Animals↗
PubMed2001

[Comparison of denaturing high performance liquid chromatography with direct sequencing in the detection of single nucleotide polymorphism].

OBJECTIVE: Investigate the sensitivity and accuracy of the denaturing high performance liquid chromatography(DHPLC) technique for the detection of single nucleotide polymorphism(SNP). METHODS: Forty-one samples were detected by both DHPLC and direct sequencing. RESULTS: The comparison demonstrated that DHPLC detected all heterozygous sequences found by direct sequencing. No false-positive signals were seen in the cases of homozygous sequences. Furthermore, no false-negative results were ever obtained with heterozygous mutations or polymorphisms, or both. CONCLUSION: DHPLC is a potent method for SNP identification especially SNP typing in large scale screening.

Chromatography, High Pressure Liquid↗