Biomedical subjects

W Huang

Publications and source records attributed to W Huang.

At least 343 records · Page 19Linked to original sources

PubMed1998

EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine.

The conformations of isolated rabbit fast myosin light chains (LCs) were modified using trifluoperazine (TFP), the hydrophobic calmodulin inhibitor. CD spectroscopy showed that TFP altered secondary structural content of the LCs, with half-maximal effects at TFP concentrations of approximately 14-50 microM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. & Ogawa, Y. (1988) J. Physiol. 403, 407-424]. EPR spectroscopy provided structural information from paramagnetic probes on C-terminal domain surfaces. In the absence of TFP, tauR (rotational correlation time) was 1.6 ns for both alkali light chains (ALCs) and 1.8 ns for light chain 2 (LC2). This was faster than expected for proteins of this size (approximately 10 ns). TFP progressively recruited the probes into populations with tauR sevenfold to 12-fold slower, with half-maximal effects at a TFP concentration of approximately 370-800 microM. The differences probably indicate that CD spectroscopy detects changes in protein conformation due to 'specific' TFP binding at the LC hydrophobic core, while less specific binding at higher TFP concentrations is required to effect conformational changes on the protein surfaces near the paramagnetic probes. TFP binding was generally not cooperative. Comparative sequence analysis between calmodulin, troponin C, and myosin LCs indicated considerable conservation between residues expected to bind TFP.

Amino Acid Sequence↗

PubMed1998

Mapping protein-ligand interactions using whole genome phage display libraries.

The function of many genes cannot be deduced from sequence similarity, and biochemical methods are usually required. Whole genome sequences can be thought of as not only a set of genes but also collections of functional domains. These domains can be studied by affinity methods whereby identification of the ligand can provide information on biochemical function. To take advantage of this method, one must express all functional domains in a form suitable for affinity studies. Phage display technology provides a means for accomplishing this. The pJuFo phage display system, based on the interaction between the leucine zippers Jun and Fos, has been modified and used to create a genomic phage display library from Escherichia coli MG1655. The system has been tested by using the library to map the dominant binding epitopes for an anti-RecA protein polyclonal antibody sera. This methodology provides a general biochemical approach to functional analysis of protein-ligand interactions on a genomewide basis.

Antibodies, Monoclonal↗

PubMed1998

Interaction of platelet-activating factor, spleen and atrial natriuretic peptide in plasma volume regulation during endotoxaemia in rats.

1. We studied endotoxin (lipopolysaccharide, LPS)-induced platelet-activating factor (PAF) production in various visceral organs, and the effect of PAF antagonists or splenectomy on LPS-induced changes. 2. PAF production in response to LPS was highest in the spleen, followed by ileum, heart, lung and kidneys. None was found in the liver. The splenic response was rapid, reaching 10 times the basal level at 30 min. The increased PAF content in each organ was unrelated to the enzyme activity of either macrophages or neutrophils. 3. LPS-induced hypotension and haemoconcentration were largely prevented by PAF antagonists and splenectomy. 4. Plasma volume fell, and plasma atrial natriuretic peptide (ANP) rose, after LPS administration. Splenectomy or pretreatment with PAF antagonists almost completely prevented these LPS-induced changes at 30 min, but only partially reversed them at 90 min. 5. These results suggest that during endotoxaemia: (a) the spleen is the site of the highest endogenous PAF production; (b) the initial release of ANP is dependent on the production of endogenous PAF, and a PAF-ANP interaction mediates the early plasma volume reduction; (c) plasma volume reduction as well as ANP release depend on the spleen; (d) PAF mediated the hypotensive response and its action in the spleen; and (e) sequestered neutrophils are probably not the main source of PAF in the spleen.

Animals↗

PubMed1998

Genetic relationship of populations in China.

Despite the fact that the continuity of morphology of fossil specimens of modern humans found in China has repeatedly challenged the Out-of-Africa hypothesis, Chinese populations are underrepresented in genetic studies. Genetic profiles of 28 populations sampled in China supported the distinction between southern and northern populations, while the latter are biphyletic. Linguistic boundaries are often transgressed across language families studied, reflecting substantial gene flow between populations. Nevertheless, genetic evidence does not support an independent origin of Homo sapiens in China. The phylogeny also suggested that it is more likely that ancestors of the populations currently residing in East Asia entered from Southeast Asia.

Animals↗

PubMed1998

Histological characterization and pharmacological control of chronic rejection in xenogeneic and allogeneic heart transplantation.

BACKGROUND: Chronic allograft rejection remains a major barrier to successful long-term allograft transplantation in humans. Chronic allograft rejection is characterized by the appearance of arterial lesions with concentric intimal thickening. This study investigates the development and control of chronic rejection in hamster cardiac xenografts transplanted into Lewis rats. METHODS: Chronic rejection in the xenograft model involves transplantation of hamster hearts into Lewis rats treated with leflunomide (Lef) continuously at 15 mg/kg/day. The allograft model involves transplantation of Lewis hearts into Fisher-334 rats treated with cyclosporine (CsA) at 2.5 mg/kg for 5 days. RESULTS: The average scores of arterial intimal thickening on day 45 after transplantation were 1.89+/-0.43 in the xenograft and 2.50+/-0.72 in the allograft. The basic pathology of both xenografts and allografts undergoing chronic rejection was arterial intimal thickening comprising smooth muscle cell proliferation, mononuclear cell infiltration, and fibrosis. The majority of cells infiltrating the arterial intima and myocardium were T cells and macrophages. Compared with the allograft, intimal edema, matrix deposition and fibrinoid necrosis were specifically presented in the xenografts and generally involved the larger arteries. The predominant isotype of antibody deposited was IgM in xenografts and IgG in allografts. When combined Lef and CsA therapy was initiated on day 45 after transplantation, the changes of chronic rejection were reversed in both xenografts and allografts by day 90. The scores of intimal thickening were significantly reduced to 0.97+/-0.45 and 1.48+/-0.56, respectively. CONCLUSIONS: We conclude that chronic rejection can be induced in xenografts under conditions of inadequate immunosuppression. Chronic rejection in xenografts involves arterial lesions that bear some histological similarities to, as well as differences from, those observed in chronically rejected allografts. Finally, combination therapy with CsA and Lef reduced the incidence and severity of the intimal lesions in both chronically rejecting xenografts and allografts.

Animals↗

PubMed1998

The N-degron protein degradation strategy for investigating the function of essential genes: requirement for replication protein A and proliferating cell nuclear antigen proteins for nucleotide excision repair in yeast extracts.

Nucleotide excision repair (NER) of DNA in the yeast Saccharomyces cerevisiae and in human cells has been shown to be a biochemically complex process involving multiple gene products. In yeast, the involvement of the DNA replication accessory proteins, replication protein A (RPA1) and proliferating cell nuclear antigen (PCNA) in NER has not been demonstrated genetically. In this study we have generated temperature-degradable rfa1 and pcna mutants and show that these mutants are defective in NER in vitro under conditions that promote degradation of the RFA1 and PCNA gene products. We also demonstrate a physical interaction between RPA1 protein and subunits of the RNA polymerase II basal transcription factor IIH (TFIIH).

DNA Repair↗

PubMed1998

Initial performance evaluation of an indirect-detection, active matrix flat-panel imager (AMFPI) prototype for megavoltage imaging.

PURPOSE: The development of the first prototype active matrix flat-panel imager (AMFPI) capable of radiographic and fluoroscopic megavoltage operation is reported. The signal and noise performance of individual pixels is empirically quantified. Results of an observer-dependent study of imaging performance, using a contrast-detail phantom, are detailed and radiographic patient images are shown. Finally, a theoretical investigation of the zero-frequency detective quantum efficiency (DQE) performance of such imagers, using a cascaded systems formalism, is presented. METHODS AND MATERIALS: The imager is based on a 508-microm pitch, 26 x 26 cm2 array which detects radiation indirectly via an overlying copper plate + phosphor screen converter. RESULTS: Due to its excellent optical coupling, the imager exhibits sensitivity superior to that of video-based systems. With an approximately 133 mg/cm2 Gd2O2S:Tb screen the system is x-ray quantum-noise-limited down to approximately 0.3 cGy, conservatively, and extensions of this behavior to even lower doses by means of reduced additive electronic noise is predicted. The observer-dependent study indicates performance superior to that of conventional radiotherapy film while the patient images demonstrate good image quality at 1 to 4 MU. The theoretical studies suggest that, with a 133 mg/cm2 Gd2O2S:Tb screen, the system would provide DQE performance equivalent to that of video-based systems and that almost a factor of two improvement in DQE is achievable through the incorporation of a 400 mg/cm2 screen. CONCLUSION: The reported prototype imager is the first megavoltage AMFPI having performance characteristics consistent with practical clinical operation. The superior contrast-detail sensitivity of the imager allows the capture of high-quality 6- and 15-MV images at minimal dose. Moreover, significant performance improvements, including extension of the operational range up to full portal doses, appear feasible. Such capabilities could be of considerable practical benefit in patient localization and verification.

Aged↗

PubMed1998

A dual thrombin receptor system for platelet activation.

Platelet-dependent arterial thrombosis triggers most heart attacks and strokes. Because the coagulation protease thrombin is the most potent activator of platelets, identification of the platelet receptors for thrombin is critical for understanding thrombosis and haemostasis. Protease-activated receptor-1 (PAR1) is important for activation of human platelets by thrombin, but plays no apparent role in mouse platelet activation. PAR3 is a thrombin receptor that is expressed in mouse megakaryocytes. Here we report that thrombin responses in platelets from PAR3-deficient mice were markedly delayed and diminished but not absent. We have also identified PAR4, a new thrombin-activated receptor. PAR4 messenger RNA was detected in mouse megakaryocytes and a PAR4-activating peptide caused secretion and aggregation of PAR3-deficient mouse platelets. Thus PAR3 is necessary for normal thrombin responses in mouse platelets, but a second PAR4-mediated mechanism for thrombin signalling exists. Studies with PAR-activating peptides suggest that PAR4 also functions in human platelets, which implies that an analogous dual-receptor system also operates in humans. The identification of a two-receptor system for platelet activation by thrombin has important implications for the development of antithrombotic therapies.

Amino Acid Sequence↗

PubMed1998

Synthesis and Characterization of a New p-n Diblock Light-Emitting Copolymer.

In this paper we present the synthesis and characterization of a new light-emitting copolymer constituted of regularly alternating segments of 3,3'-didecyl-2,2'-bithiophene and 2,6-bis(1,3,4-oxadiazolyl)toluene, which are p-dopable and n-dopable, respectively. The new polymer emits intense green light under the irradiation of UV light in its film states and shows strong solvatochromism both on absorption and on emission. The n-doping potential of the polymer is measured to be similar with that of 2-(4-biphenylyl)-5-(4-tert-butylphenyl)-1,3,4-oxadiazole (PBD) and other reported oxadiazole-containing polymers, the currently excellent electron-transporting materials, while its p-doping potential is much lower than those of other oxadiazole-containing light-emitting polymers and is comparable with that of poly(p-phenylenevinylene) (PPV) and polythiophenes (PT), the typical p-dope type materials. The presented synthesis reveals a possible approach to balance the rates of injection of electrons and holes from opposite contacts into an emissive layer in polymer light-emitting diodes by controlling the intrinsic properties of light-emitting materials.

Journal Article↗

PubMed1998

Further characterization of stimulus interaction of cat carotid chemoreceptors.

The hypothesis that the maximal response to pCO2 of carotid body chemoreceptors would be the same regardless of pO2, if the receptor molecule behaves like a hemoglobin molecule, was investigated using single or a few fiber carotid body chemoreceptors in cats in vivo which were anesthetized and artificially ventilated. In one series, graded levels of CO2 inhalation in steady-state at p(a)O2 = 354 +/- 19 Torr showed a linear response from 1 to 20.1 +/- 2.3 imp/s for p(a)CO2 increase from 32 to 178 +/- 18 Torr, and at p(a)O2 of 48 +/- 3.8 Torr, from 3.8 to 18.6 +/- 1.7 imp/s for p(a)CO2 increase from 21 to 109 +/- 11 Torr, levelling off thereafter. In another series of multi-fiber preparation, close intra-arterial injection of blood plus saline containing pCO2 of about 270 Torr gave peak responses of 44 +/- 9, 42 +/- 6 and 42 +/- 7 imp/s at p(a)O2 of 40 +/- 4, 82 +/- 6 and 388 +/- 18 Torr, respectively. Thus, the chemosensory responses to p(a)CO2 reached the same level of maximal activity regardless of p(a)O2. Taken together, the maximal responses in both steady-state and transient-state to p(a)CO2 appeared to be the same at hypoxic and hyperoxic p(a)O2. This stimulus-response relationship of the receptor molecule is analogous to O2-CO2 interaction with hemoglobin molecule with a Bohr effect, reaching a saturation point at a finite pO2.

Acidosis↗

PubMed1998

Valacyclovir: a substrate for the intestinal and renal peptide transporters PEPT1 and PEPT2.

Valacyclovir is a prodrug of the antiviral agent acyclovir and it does not contain a peptide bond in its structure. We studied the interaction of valacyclovir with the peptide transporters in the human intestinal cell line Caco-2 and the rat kidney proximal tubular cell line SKPT which differentially express peptide transporters PEPT1 and PEPT2. The results of the studies done with these cell lines were confirmed with the cloned peptide transporters human PEPT1 and rat PEPT2, expressed heterologously in HeLa cells. The activity of the peptide transporters was assessed by measuring the uptake of radiolabeled glycylsarcosine in the presence of a H+ gradient. Valacyclovir inhibited the uptake of glycylsarcosine with an inhibition constant (Ki) of 0.49 +/- 0.04 mM in Caco-2 cells and 0.17 +/- 0.01 mM in SKPT cells. In both cell types, the inhibition was competitive. Acyclovir, in contrast to valacyclovir, did not interact with the peptide transporters. Similar results were obtained with heterologously expressed human PEPT1 and rat PEPT2. Valacyclovir inhibited the hPEPT1-mediated glycylsarcosine transport competitively with a Ki value of 0.74 +/- 0.14 mM. The rPEPT2-mediated transport of glycylsarcosine was also inhibited by valacyclovir competitively and the Ki value for the process was 0.39 +/- 0.03 mM. Acyclovir did not interact with either of these cloned peptide transporters. We conclude that valacyclovir is a substrate for the peptide transporters PEPT1 and PEPT2 and that a peptide bond is not a prerequisite for recognition as a substrate by the peptide transporters.

Acyclovir↗

PubMed1998

Protection of the arterial internal elastic lamina by inhibition of the renin-angiotensin system in the rat.

Spontaneous rupture of the internal elastic lamina (IEL) occurs in some arteries of the rat during growth and aging. Inbred, normotensive, Brown Norway (BN) rats are particularly susceptible to rupture of the IEL, especially in the abdominal aorta (AA). Preliminary experiments showed that different angiotensin-converting enzyme (ACE) inhibitors protect against rupture of the IEL in the BN rat to a greater extent than hydralazine, suggesting a role of the renin-angiotensin system (RAS) in this phenomenon. To explore this possibility, we have treated male BN rats from 4.5 to 14 weeks of age with either enalapril or losartan (both at 1, 3, and 10 mg x kg(-1) x d(-1)) or with the calcium antagonists mibefradil (at 3, 10, 30, and 45 mg x kg(-1) x d(-1)) and amlodipine (at 30 mg x kg(-1) x d(-1)). Systolic blood pressure (SBP) was measured weekly, and at the end of treatment we (1) recorded body and heart weights, (2) measured various parameters of the RAS in plasma, (3) quantified interruptions in the IEL on "en face" preparations of AA, and (4) quantified elastin, collagen, and cell proteins in the media of the thoracic aorta. Results showed that enalapril and losartan similarly decrease SBP and rupture of the IEL in the AA, suggesting that enalapril inhibits the latter via a decrease in the production of angiotensin II (Ang II) and not via another effect on ACE. The decrease in IEL rupture and in SBP, as well as the modifications in the parameters of the RAS, were all dose dependent. Mibefradil had little effect on the RAS and, at the highest doses, decreased SBP to an extent similar to that for enalapril at 3 mg x kg(-1) x d(-1) but did not significantly inhibit IEL rupture. Amlodipine decreased SBP, increased plasma renin concentration, and was without effect on IEL rupture. All treatments at the highest doses had a hypotrophic effect on the aortic media but differed in their effects on the heart, with enalapril and losartan decreasing and mibefradil and amlodipine increasing heart weight, suggesting that the inhibition of IEL rupture may be related to a cardiac hypotrophic effect. All these results, taken together, suggest that Ang II plays a role in the rupture of the IEL that is, in part, independent of SBP.

Amlodipine↗

PubMed1998

Engineering analysis of biological variables: an example of blood pressure over 1 day.

Almost all variables in biology are nonstationarily stochastic. For these variables, the conventional tools leave us a feeling that some valuable information is thrown away and that a complex phenomenon is presented imprecisely. Here, we apply recent advances initially made in the study of ocean waves to study the blood pressure waves in the lung. We note first that, in a long wave train, the handling of the local mean is of predominant importance. It is shown that a signal can be described by a sum of a series of intrinsic mode functions, each of which has zero local mean at all times. The process of deriving this series is called the "empirical mode decomposition method." Conventionally, Fourier analysis represents the data by sine and cosine functions, but no instantaneous frequency can be defined. In the new way, the data are represented by intrinsic mode functions, to which Hilbert transform can be used. Titchmarsh [Titchmarsh, E. C. (1948) Introduction to the Theory of Fourier Integrals (Oxford Univ. Press, Oxford)] has shown that a signal and i times its Hilbert transform together define a complex variable. From that complex variable, the instantaneous frequency, instantaneous amplitude, Hilbert spectrum, and marginal Hilbert spectrum have been defined. In addition, the Gumbel extreme-value statistics are applied. We present all of these features of the blood pressure records here for the reader to see how they look. In the future, we have to learn how these features change with disease or interventions.

Animals↗

PubMed1998

[Fluorescence-based semi-automated gene scan with microsatellite markers by multiplex PCR techniques].

OBJECTIVE: To develop a high output and low cost multiplex PCR approach to facilitating large scale gene scan and gene typing with microsatellite markers. METHODS: 5-15 pairs of primers of microsatellite loci were added in one single tube with 5ul reaction volume, containing 10 mmol/L Tris-HCl (pH8.3), 50 mmol/L KCl,0. 1mg/ml gelatin, 3.0 mmol/L MgCl2, 200micromol/L dNTPs (each), 0.25U Taq DNA polymerase, Taq Start antibody and DNA templates. PCR thermocycles were carried out on Perkin Elmer Gene Amp PCR System 9600 with touch-down algorithm (the annealing temperature was decreased by 0.5 C in each cycle) to meet the different demands of different primers. RESULTS: Up to 10-15 loci were labeled by different fluorescents or by the same fluorescent, but their PCR amplification fragments did not overlap in size. Almost all target microsatellite loci were successfully co-amplified in a 5 microliter reaction volume,electrophoresized and analyzed in a single gel lane with Perkin Elmer ABI 373A DNA sequencer, and 9 microsatellite loci were well genotyped. CONCLUSION: This high output and low cost genotyping protocol is applicable to gene mapping, human evolution and forensic analysis.

Genotype↗

PubMed1998

Modular folding and evidence for phosphorylation-induced stabilization of an hsp90-dependent kinase.

The de novo folding of the individual domains of the src family kinase p56(lck) was examined within the context of full-length p56(lck) molecules produced in rabbit reticulocyte lysate containing active chaperone machinery. The catalytic domain required geldanamycin-inhibitable heat shock protein 90 (hsp90) function to achieve its active protease-resistant conformation, but the src homology 2 (SH2) domain acquired phosphopeptide-binding competence independently of hsp90 function. The SH2 domain of hsp90-bound p56(lck) was folded and functional. In addition to the facilitation by hsp90 of kinase biogenesis, a conditional role in maintenance folding could be demonstrated; although wild type p56(lck) molecules with a negative-regulatory C-terminal tyrosine matured to a nearly hsp90-independent state, p56(lck) molecules with a mutated C-terminal tyrosine continued to require hsp90-mediated maintenance. De novo folding could be distinguished from maintenance folding on the basis of proteolytic fingerprints and the effects of different temperatures on folding behavior. Results indicate that during p56(lck) biogenesis, the SH2 domain rapidly folds independently of hsp90 function, followed by the slower hsp90-dependent folding of the catalytic domain and suggest the final stabilization of p56(lck) structure by phosphorylation-mediated interdomain interactions.

Animals↗

PubMed1998

Crystal structure of beta-ketoacyl-acyl carrier protein synthase II from E.coli reveals the molecular architecture of condensing enzymes.

In the biosynthesis of fatty acids, the beta-ketoacyl-acyl carrier protein (ACP) synthases catalyze chain elongation by the addition of two-carbon units derived from malonyl-ACP to an acyl group bound to either ACP or CoA. The crystal structure of beta-ketoacyl synthase II from Escherichia coli has been determined with the multiple isomorphous replacement method and refined at 2.4 A resolution. The subunit consists of two mixed five-stranded beta-sheets surrounded by alpha-helices. The two sheets are packed against each other in such a way that the fold can be described as consisting of five layers, alpha-beta-alpha-beta-alpha. The enzyme is a homodimer, and the subunits are related by a crystallographic 2-fold axis. The two active sites are located near the dimer interface but are approximately 25 A apart. The proposed nucleophile in the reaction, Cys163, is located at the bottom of a mainly hydrophobic pocket which is also lined with several conserved polar residues. In spite of very low overall sequence homology, the structure of beta-ketoacyl synthase is similar to that of thiolase, an enzyme involved in the beta-oxidation pathway, indicating that both enzymes might have a common ancestor.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed1998

Transport mechanisms for vitamin C in the JAR human placental choriocarcinoma cell line.

We investigated the transport pathways available for the uptake of vitamin C in the human placental choriocarcinoma cell line, JAR. These cells were found to possess the capacity to accumulate the vitamin when presented either in the oxidized form (dehydroascorbic acid) or in the reduced form (ascorbate). Dithiothreitol and 5,5'-dithiobis(2-nitrobenzoic acid) were used to maintain vitamin C as ascorbate and dehydroascorbic acid, respectively. The uptake of these two forms of vitamin C in JAR cells was found to occur by different mechanisms. The uptake of the dehydroascorbic acid was Na(+)-independent and was mediated by facilitative glucose transporters as evidenced from the inhibition of the uptake process by glucose. On the other hand, the uptake of ascorbate was Na(+)-dependent and was not sensitive to inhibition by glucose. Substitution of Na+ with other monovalent cations abolished the uptake of ascorbate completely. The uptake process was, however, not influenced by anions. Kinetic analysis indicated the presence of a single saturable transport system for ascorbate with a Michaelis-Menten constant of 22 +/- 1 microM. The dependence of the uptake rare of ascorbate on Na+ concentration exhibited sigmoidal kinetics, suggesting interaction of more than one Na+ ion with the transporter. The Hill coefficient for the Na+ interaction was 2, indicating that the Na(+)-dependent ascorbate transport is electrogenic. The Na(+)-dependent stimulation of ascorbate uptake was primarily due to an increase in the affinity of the transporter for ascorbate in the presence of Na+. It is concluded that the JAR placental trophoblast cell line expresses two different transport systems for vitamin C: one for the reduced form of the vitamin ascorbate; and the other for the oxidized form of the vitamin dehydroascorbic acid.

Ascorbic Acid↗

PubMed1998

Acute effects of morphine on the expression of mRNAs for NMDA receptor subunits in the rat hippocampus, hypothalamus and spinal cord.

The acute effects of subcutaneously injected morphine on transcripts of the NMDA receptor subunits NR1, NR2A and NR2B in certain areas of the central nervous system of male rats were examined by Northern blot analysis. The result clearly indicated that a single dose (10 mg/kg) of the opioid alters the expression of the mRNA for receptor subunits in the hippocampus and hypothalamus 4 h after drug injection. No change in the mRNA levels was observed 30 min following injection, and after 24 h most of the levels were restored to control values. The observation suggests that morphine affects this type of glutamate receptor already in the acute phase of its administration.

Analgesics, Opioid↗