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Biomedical subjects

H Jiang

Publications and source records attributed to H Jiang.

At least 19 recordsLinked to original sources
PubMed2000

Viral escape by selection of cytotoxic T cell-resistant variants in influenza A virus pneumonia.

Antigenic variation is a strategy exploited by influenza viruses to promote survival in the face of the host adaptive immune response and constitutes a major obstacle to efficient vaccine development. Thus, variation in the surface glycoproteins hemagglutinin and neuraminidase is reflected by changes in susceptibility to antibody neutralization. This has led to the current view that antibody-mediated selection of influenza A viruses constitutes the basis for annual influenza epidemics and periodic pandemics. However, infection with this virus elicits a vigorous protective CD8(+) cytotoxic T lymphocyte (CTL) response, suggesting that CD8(+) CTLs might exert selection pressure on the virus. Studies with influenza A virus-infected transgenic mice bearing a T cell receptor (TCR) specific for viral nucleoprotein reveal that virus reemergence and persistence occurs weeks after the acute infection has apparently been controlled. The persisting virus is no longer recognized by CTLs, indicating that amino acid changes in the major viral nucleoprotein CTL epitope can be rapidly accumulated in vivo. These mutations lead to a total or partial loss of recognition by polyclonal CTLs by affecting presentation of viral peptide by class I major histocompatibility complex (MHC) molecules, or by interfering with TCR recognition of the mutant peptide-MHC complex. These data illustrate the distinct features of pulmonary immunity in selection of CTL escape variants. The likelihood of emergence and the biological impact of CTL escape variants on the clinical outcome of influenza pneumonia in an immunocompetent host, which is relevant for the design of preventive vaccines against this and other respiratory viral infections, are discussed.

Animals↗
PubMed2000

Zinc and cadmium can promote rapid nuclear translocation of metal response element-binding transcription factor-1.

Metal response element-binding transcription factor-1 (MTF-1) is a six-zinc finger protein that plays an essential role in activating metallothionein expression in response to the heavy metals zinc and cadmium. Low affinity interactions between zinc and specific zinc fingers in MTF-1 reversibly regulate its binding to the metal response elements in the mouse metallothionein-I promoter. This study examined the subcellular distribution and DNA binding activity of MTF-1 in cells treated with zinc or cadmium. Immunoblot analysis of cytosolic and nuclear extracts demonstrated that in untreated cells, about 83% of MTF-1 is found in the cytosolic extracts and is not activated to bind to DNA. In sharp contrast, within 30 min of zinc treatment (100 microM), MTF-1 is detected only in nuclear extracts and is activated to bind to DNA. The activation to bind to DNA and nuclear translocation of MTF-1 occurs in the absence of increased MTF-1 content in the cell. Furthermore, immunocytochemical localization and immunoblotting assays demonstrated that zinc induces the nuclear translocation of MTF-1-FLAG, expressed from the cytomegalovirus promoter in transiently transfected dko7 (MTF-1 double knockout) cells. Immunoblot analysis of cytosolic and nuclear extracts from cadmium-treated cells demonstrated that concentrations of cadmium (10 microM) that actively induce metallothionein gene expression cause only a small increase in the amount of nuclear MTF-1. In contrast, an overtly toxic concentration of cadmium (50 microM) rapidly induced the complete nuclear translocation and activation of DNA binding activity of MTF-1. These studies are consistent with the hypothesis that MTF-1 serves as a zinc sensor that responds to changes in cytosolic free zinc concentrations. In addition, these data suggest that cadmium activation of metallothionein gene expression may be accompanied by only small changes in nuclear MTF-1.

Animals↗
PubMed2000

Phosphorylation of the RAP74 subunit of TFIIF correlates with Tat-activated transcription of the HIV-1 long terminal repeat.

Transcription from the HIV-1 long terminal repeat (LTR) is regulated by the viral transactivator Tat, which increases RNA polymerase II (RNAP II) processivity. Previous reports have demonstrated that phosphorylation of the RNAP II carboxy-terminal domain by TFIIH and P-TEFb is important for Tat transactivation. Our present results demonstrate that phosphorylation of the RAP74 subunit of TFIIF is also an important step in Tat transactivation. Interestingly, while the general transcription factor TFIIF is required for both basal and Tat-activated transcription, phosphorylation of the RAP74 subunit occurs in the presence of Tat and correlates with a high level of transcription activity. Using a biotinylated DNA template transcription assay, we provide evidence that RAP74 is phosphorylated by TAF(II)250 during Tat-activated transcription. Depletion of RAP74 from the HeLa nuclear extract inhibited HIV-1 LTR-driven basal transcription and Tat transactivation. The addition of TFIIF, reconstituted from recombinant RAP30 and RAP74, to the depleted HeLa nuclear extract resulted in restoration of Tat transactivation. Of importance, the exogenous RAP74 was rapidly phosphorylated in the presence of Tat. These results suggest that RAP74 phosphorylation is one important step, of several, in the Tat transactivation cascade.

Biopolymers↗
PubMed2000

Cloning and characterization of the promoter region of the rat epidermal growth factor receptor gene and its transcriptional regulation by nerve growth factor in PC12 cells.

Our previous studies have shown that treatment of PC12 cells with nerve growth factor (NGF) causes a profound down-regulation of the epidermal growth factor receptor (EGFR) mRNA and protein. Further, the NGF-induced down-regulation of the EGFR is under transcriptional control. To elucidate the molecular mechanism of this down-regulation we have cloned a 2.7-kilobase sequence from the promoter region of the rat EGFR from a rat P1 library. Six transcriptional start sites were identified by 5'-rapid amplification of cDNA ends and primer extension. Sequence analysis showed a 62% overall homology with the human EGFR promoter region. To investigate its transcription, 1.1 kilobases of the 5'-flanking sequence were fused to a luciferase reporter gene. This sequence exhibited functional promoter activity in transient transfection experiments with PC12, C6, and CV-1 cells. Treatment of PC12 cells with NGF inhibited promoter activity. By transfection of promoter deletion constructs, a silencer element was found between nucleotides -260 and -181, and TCC repeat sequences appeared to be at least partially responsible for the down-regulation of the EGFR by NGF. Supportive evidence for the relevance of this sequence was obtained from gel mobility shift assays and by transfection of TCC mutation constructs. Our results demonstrate that TCC repeat sequences are required for the down-regulation of rat EGFR by NGF in PC12 cells and may lead to the identification of the NGF-responsive transcription factors.

Animals↗
PubMed2000

Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction.

The roles of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) in chemoattractant-elicited responses were studied in mice lacking these key enzymes. PI3Kgamma was required for chemoattractant-induced production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5)P3] and has an important role in chemoattractant-induced superoxide production and chemotaxis in mouse neutrophils and in production of T cell-independent antigen-specific antibodies composed of the immunoglobulin lambda light chain (TI-IglambdaL). The study of the mice lacking PLC-beta2 and -beta3 revealed that the PLC pathways have an important role in chemoattractant-mediated production of superoxide and regulation of protein kinases, but not chemotaxis. The PLC pathways also appear to inhibit the chemotactic activity induced by certain chemoattractants and to suppress TI-IglambdaL production.

Animals↗
PubMed2000

Nuclear factor-kappaB activation by the photochemotherapeutic agent verteporfin.

The nuclear factor-kappa B (NF-kappaB) gene transactivator serves in the formation of immune, inflammatory, and stress responses. In quiescent cells, NF-kappaB principally resides within the cytoplasm in association with inhibitory kappa (IkappaB) proteins. The status of IkappaB and NF-kappaB proteins was evaluated for promyelocytic leukemia HL-60 cells treated at different intensities of photodynamic therapy (PDT). The action of the potent photosensitizer, benzoporphyrin derivative monoacid ring A (verteporfin), and visible light irradiation were assessed. At a verteporfin concentration that produced the death of a high proportion of cells after light irradiation, evidence of caspase-3 and caspase-9 processing and of poly(ADP-ribose) polymerase cleavage was present within whole cell lysates. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) effectively blocked these apoptosis-related changes. Recent studies indicate that IkappaB proteins may be caspase substrates during apoptosis. However, the level of IkappaBbeta was unchanged for HL-60 cells undergoing PDT-induced apoptosis. IkappaBalpha levels decreased during PDT-induced apoptosis, though ZVAD.fmk did not affect this change. At a less intensive level of photosensitization, cellular IkappaBalpha levels were transiently depressed after PDT. At these times, p50 and RelA NF-kappaB species were increased within nuclear extracts, as revealed by electrophoretic mobility supershift assays. HL-60 cells transiently transfected with a kappaB-luciferase reporter construct exhibited elevated luciferase activity after PDT or treatment with tumor necrosis factor-alpha, a well-characterized NF-kappaB activator. Productive NF-kappaB activation and associated gene transcription may influence the phenotype and behavior of cells exposed to less intensive PDT regimens. However, IkappaBalpha is not subject to caspase-mediated degradation as a component of PDT-induced apoptosis. (Blood. 2000;95:256-262)

Amino Acid Chloromethyl Ketones↗
PubMed2000

Blood pressure changes after intravenous administration of cell-free hemoglobin A and hemoglobin H in the rat.

Hemoglobin H (HbH) is a tetramer of four beta chains (present in erythrocytes of alpha thalassemia), whereas hemoglobin A is a tetramer of two alpha and two beta chains. Since HbH is known to bind four times more nitric oxide (a vasodilator) at its sulfhydryls compared to HbA, the present studies were conducted to see the effect of HbH and HbA on rat blood pressure. The acute administration (20-2000 nmol/kg) of both HbH and HbA produced a dose-dependent effect on blood pressure. The net change in mean arterial pressure was significantly higher with HbH compared to HbA. Partially nitrosylated (in which SH groups are occupied with NO) HbH retained the property of raising blood pressure to some extent while HbA lost this property. Completely nitrosylated (in which both heme and SH groups are occupied with NO) derivatives of both HbH and HbA reduced the blood pressure to the same extent. The preliminary studies with chronic administration of HbA and HbH resulted in nonsignificant increase in blood pressure. It is concluded that these findings may explain the earlier observations of increased risk of hypertension in individuals with alpha thalassemia.

Analysis of Variance↗
PubMed2000

Protein phosphatase type-1 regulatory subunits Reg1p and Reg2p act as signal transducers in the glucose-induced inactivation of maltose permease in Saccharomyces cerevisiae.

The REG1 gene encodes a regulatory subunit of the type-1 protein phosphatase (PP1) G1c7 in Saccharomyces cerevisiae, which directs the catalytic subunit to substrates involved in glucose repression. Loss of REG1 relieves glucose repression of many genes, including the MAL structural genes that encode the maltose fermentation enzymes. In this report, we explore the role of Reglp and its homolog Reg2p in glucose-induced inactivation of maltose permease. Glucose stimulates the proteolysis of maltose permease and very rapid loss of maltose transport activity - more rapid than can be explained by loss of the permease protein alone. In a reg1delta strain we observe a significantly reduced rate of glucose-induced proteolysis of maltose permease, and the rapid loss of maltose transport activity does not occur. Instead, surprisingly, the slow rate of proteolysis of maltose permease is accompanied by an increase in maltose transport activity. Loss of Reg2p modestly reduces the rates of both glucose-induced proteolysis of maltose permease and inactivation of maltose transport activity. Overexpression of Reg2p in a reg1delta strain suppresses the effect on maltose permease proteolysis and partially restores the inactivation of maltose transport activity, but does not affect the insensitivity of MAL gene expression to repression by glucose observed in this strain. Thus, protein phosphatase type-1 (Glc7p-Reglp and Glc7p-Reg2p) plays a role in transduction of the glucose signal during glucose-induced proteolysis of maltose permease, but only Glc7p-Reglp is involved in glucose-induced inactivation of maltose transport activity and glucose repression of MAL gene expression. Overexpression of REG1 partially restores proteolysis of maltose permease in a grr1delta strain, which lacks glucose signaling, but does not rescue rapid inactivation of maltose transport activity or sensitivity to glucose repression. A model for the role of Reglp and Reg2p in glucose signaling pathways is discussed. We also uncovered a previously unrecognized G2/M delay in the grr1delta but not the reg1delta strains, and this delay is suppressed by REG1 overexpression. The G1/S delay seen in grr1delta mutants is slightly suppressed as well, but REG1 overexpression does not suppress other grr1delta phenotypes such as insensitivity to glucose repression.

Blotting, Western↗
PubMed2000

Responses of cells to stationary and moving sound stimuli in the anterior ectosylvian cortex of cats.

The azimuthal, directional and angular speed sound selectivities of single units were examined in the posterior part of the anterior ectosylvian cortex. Broadband noise bursts and simulated moving sounds were delivered from 16 loudspeakers fixed on the horizontal plane in a quasi-anechoic sound-isolation chamber. The activity of 78 neurons was recorded and quantitatively analyzed. Most cells responded to at least the static sound. The relative strengths of their responses suggested that the cells could be classed as omnidirectional (37.2%), contralateral hemifield (29.5%), ipsilateral hemifield (2.5%) and azimuth (7.7%) selective. The remaining 23.1% could not be classified. All cells responded to a simulated moving sound displaced at five different speeds. A majority (88%) of them showed some directional preference in that they discharged at least twice as strongly for one direction as for the other for at least one speed. 14.7% displayed angular speed selectivity. Different patterns of neuronal discharges were evoked. For static sounds, most of the cells gave ON-type responses. A large proportion (60%) of the cells responded in a sustained manner to maintained stimulation. Among these, 68% also gave sustained discharges to moving sounds. The spatial tuning and the directional and angular speed selectivity of neurons in the posterior part of the AEC suggest that this area is involved in the processing of static and moving sounds.

Acoustic Stimulation↗
PubMed2000

In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection.

Ribonuclease-resistant RNA molecules that bind to infectious human cytomegalovirus (HCMV) were isolated in vitro from a pool of randomized sequences after 16 cycles of selection and amplification. The two ligands (L13 and L19) characterized exhibited high HCMV-binding affinity in vitro and effectively inhibited viral infection in tissue culture. Their antiviral activity was also specific as they only reacted with two different strains of HCMV but not with the related herpes simplex virus 1 and human cells. These two ligands appeared to function as antivirals by blocking viral entry. Ultraviolet (UV) crosslinking studies suggested that L13 and L19 bind to HCMV essential glycoproteins B and H, respectively. Thus, RNA ligands that bind to different surface antigens of HCMV can be simultaneously isolated by the selection procedure. Our study demonstrates the feasibility of using these RNA ligands as a research tool to identify viral proteins required for infectivity and as an antiviral agent to block viral infection.

Antiviral Agents↗
PubMed2000

Analysis of the mechanism by which glucose inhibits maltose induction of MAL gene expression in Saccharomyces.

Expression of the MAL genes required for maltose fermentation in Saccharomyces cerevisiae is induced by maltose and repressed by glucose. Maltose-inducible regulation requires maltose permease and the MAL-activator protein, a DNA-binding transcription factor encoded by MAL63 and its homologues at the other MAL loci. Previously, we showed that the Mig1 repressor mediates glucose repression of MAL gene expression. Glucose also blocks MAL-activator-mediated maltose induction through a Mig1p-independent mechanism that we refer to as glucose inhibition. Here we report the characterization of this process. Our results indicate that glucose inhibition is also Mig2p independent. Moreover, we show that neither overexpression of the MAL-activator nor elimination of inducer exclusion is sufficient to relieve glucose inhibition, suggesting that glucose acts to inhibit induction by affecting maltose sensing and/or signaling. The glucose inhibition pathway requires HXK2, REG1, and GSF1 and appears to overlap upstream with the glucose repression pathway. The likely target of glucose inhibition is Snf1 protein kinase. Evidence is presented indicating that, in addition to its role in the inactivation of Mig1p, Snf1p is required post-transcriptionally for the synthesis of maltose permease whose function is essential for maltose induction.

Base Sequence↗
PubMed2000

Amplitude correction in image deconvolution for determining crystal defects at atomic level

A method is proposed to improve the image deconvolution technique based on the weak-phase object approximation for determining the crystal defects at atomic level. Originally, both the amplitude and phase modulation caused by the contrast transfer function (CTF) was removed for all reflections in the diffractogram of the image and then the deconvoluted image was obtained by carrying out the inverse FT. In this paper after removing the CTF modulation the amplitudes of reflections are further corrected by constraining the integral amplitudes to be equal to the corresponding structure factor amplitudes of perfect crystals. [110] high-resolution electron microscope images were simulated for a structure model of Si crystal containing the 60 degrees dislocation with different thickness. A comparison between deconvoluted images with the amplitudes corrected by the present method and those uncorrected is given to show the effectiveness of the method. The amplitude-crystal thickness dependence for images simulated close to the Scherzer defocus condition has been analysed and the result serves as an empirical basis of the method. The validity and limitation of the method are discussed.

Journal Article↗
PubMed2000

A localization algorithm and error analysis for stereo x-ray image guidance.

Stereo x-ray radiography attracts increasing attention in major clinical applications. The purpose of this paper is to analyze the 3D localization error for breast biopsy procedures and provide guidelines for improving its accuracy. Our prototype is a CCD based digital stereo x-ray imaging system. The mathematical model consists of two x-ray sources and one stationary detector plane. A closed form least-squares solution is derived for 3D localization of feature points, particularly a biopsy needle tip, from a pair of 2D digital radiographs. Based on the least-squares formula and its first order approximation, the 3D localization error is analyzed in terms of object location, measurement error, separation between the two x-ray sources, and distance from the source to the detector. The stereo imaging and error estimation formulas are numerically simulated and experimentally validated. The data are in agreement with theoretical prediction. These results can be used for the purpose of system design and protocol optimization.

Algorithms↗
PubMed2000

Lens distortion in optically coupled digital x-ray imaging.

UNLABELLED: The objectives of this research are to analyze geometrical distortions introduced by relay lenses in optically coupled digital x-ray imaging systems and to introduce an algorithm to correct such distortions. METHODS: The radial and tangential errors introduced by a relay lens in digital x-ray imaging were experimentally measured, using a lens-coupled CCD (charge coupled device) prototype. An algorithm was introduced to correct these distortions. Based on an x-ray image of a standard calibration grid, the algorithm first identified the location of the optical axis, then corrected the radial and tangential distortions using polynomial transformation technique. RESULTS: Lens distortions were classified and both radial and tangential distortions introduced by lenses were corrected using polynomial transformation. For the specific lens-CCD prototype investigated, the mean positional error caused by the relay lens was reduced by the correction algorithm from about eight pixels (0.69 mm) to less than 1.8 pixels (0.15 mm). Our investigation also shows that the fourth order of polynomial for the correction algorithm provided the best correction result. CONCLUSIONS: Lens distortions should be considered in position-dependent, quantitative x-ray imaging and such distortions can be minimized in CCD x-ray imaging by appropriate algorithm, as demonstrated in this paper.

Algorithms↗
PubMed2000

Metabolic signals trigger glucose-induced inactivation of maltose permease in Saccharomyces.

Organisms such as Saccharomyces capable of utilizing several different sugars selectively ferment glucose when less desirable carbon sources are also available. This is achieved by several mechanisms. Glucose down-regulates the transcription of genes involved in utilization of these alternate carbon sources. Additionally, it causes posttranslational modifications of enzymes and transporters, leading to their inactivation and/or degradation. Two glucose sensing and signaling pathways stimulate glucose-induced inactivation of maltose permease. Pathway 1 uses Rgt2p as a sensor of extracellular glucose and causes degradation of maltose permease protein. Pathway 2 is dependent on glucose transport and stimulates degradation of permease protein and very rapid inactivation of maltose transport activity, more rapid than can be explained by loss of protein alone. In this report, we characterize signal generation through pathway 2 using the rapid inactivation of maltose transport activity as an assay of signaling activity. We find that pathway 2 is dependent on HXK2 and to a lesser extent HXK1. The correlation between pathway 2 signaling and glucose repression suggests that these pathways share common upstream components. We demonstrate that glucose transport via galactose permease is able to stimulate pathway 2. Moreover, rapid transport and fermentation of a number of fermentable sugars (including galactose and maltose, not just glucose) are sufficient to generate a pathway 2 signal. These results indicate that pathway 2 responds to a high rate of sugar fermentation and monitors an intracellular metabolic signal. Production of this signal is not specific to glucose, glucose catabolism, glucose transport by the Hxt transporters, or glucose phosphorylation by hexokinase 1 or 2. Similarities between this yeast glucose sensing pathway and glucose sensing mechanisms in mammalian cells are discussed.

Fermentation↗
PubMed2000

Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme.

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation of cis-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compared to the enantiomerically pure [>99% (+)-1R,2S] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.

Amino Acid Substitution↗