Biomedical subjects

H Zhong

Publications and source records attributed to H Zhong.

At least 37 records · Page 2Linked to original sources

PubMed2002

A lamellar model for the X-ray rocking curves of sagittally bent Laue crystals.

The use of sagittally bent asymmetric Laue crystals in horizontally focusing monochromators for high-energy synchrotron X-rays necessitates simulation of the X-ray reflectivity by such crystals. Based on the theory of the lattice distortion in the diffraction plane of sagittally bent Laue crystals, a lamellar model was developed to predict their rocking curves. The model was experimentally verified by rocking-curve measurements from various reflections on silicon crystals of four representative orientations, sagittally bent to various radii, using X-rays of 67 keV energy.

Journal Article↗

PubMed2002

Rapamycin induces Smad activity in prostate cancer cell lines.

Rapamycin inhibits the FK506-binding protein 12 (FKBP12)/mammalian target of rapamycin (mTOR) complex and causes cell cycle arrest in G1. The precise mechanism of growth inhibition by rapamycin is only partly understood. Rapamycin led to growth inhibition in the human prostate cancer cell lines LNCaP and PC3 cells after 72 h, ID50: 93 and 50 nM, respectively. Filter cDNA array analysis showed down-regulation by more than 0.75x by rapamycin in PC3 cells and LNCaP cells of the following genes: follistatin, eukaryotic initiation factor-4E (eIF4E), glucose-6-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH)-A, ATP synthase, heat shock protein (HSP)-1. Upregulation by more than 1.5x was found for: bone morphogenetic protein (BMP)-4, FKBP12, carcinoma embryonic antigen (CEA) precursor, eukaryotic initiation factor (eIF)-3 p36 subunit, latent transforming growth factor (TGF) beta binding protein (LTBP)1. Rapamycin induced BMP4 and reduced follistatin expression in PC3 cells. This resulted in a dose-dependent nuclear expression of Smad4 and activated the SBE4 Smad-reporter, indicating activation of TGFbeta/BMP signaling. Combining rapamycin with PI3K inhibition (LY294002) increased growth inhibition. These findings illustrate that Smad signaling plays a role in the anticancer effects of rapamycin and show that combination with PI3K inhibition improves growth inhibition.

Antibiotics, Antineoplastic↗

PubMed2002

Gene therapy of prostate cancer: current and future directions.

Prostate cancer (PCA) is the second most common cause of death from malignancy in American men. Developing new approaches for gene therapy for PCA is critical as there is no effective treatment for patients in the advanced stages of this disease. Current PCA gene therapy research strategies include cytoreductive approaches (immunotherapy and cytolytic/pro-apoptotic) and corrective approaches (replacing deleted or mutated genes). The prostate is ideal for gene therapy. It is an accessory organ, offers unique antigens (prostate-specific antigen, prostate-specific membrane antigen, human glandular kallikrein 2 etc.) and is stereotactically accessible for in situ treatments. Viral and non-viral means are being used to transfer the genetic material into tumor cells. The number of clinical trials utilizing gene therapy methods for PCA is increasing. We review the multiple issues involved in developing effective gene therapy strategies for human PCA and early clinical results.

Forecasting↗

PubMed2001

BRCA1-induced large-scale chromatin unfolding and allele-specific effects of cancer-predisposing mutations.

The breast cancer susceptibility gene BRCA1 encodes a protein that has been implicated in multiple nuclear functions, including transcription and DNA repair. The multifunctional nature of BRCA1 has raised the possibility that the polypeptide may regulate various nuclear processes via a common underlying mechanism such as chromatin remodeling. However, to date, no direct evidence exists in mammalian cells for BRCA1-mediated changes in either local or large-scale chromatin structure. Here we show that targeting BRCA1 to an amplified, lac operator-containing chromosome region in the mammalian genome results in large-scale chromatin decondensation. This unfolding activity is independently conferred by three subdomains within the transactivation domain of BRCA1, namely activation domain 1, and the two BRCA1 COOH terminus (BRCT) repeats. In addition, we demonstrate a similar chromatin unfolding activity associated with the transactivation domains of E2F1 and tumor suppressor p53. However, unlike E2F1 and p53, BRCT-mediated chromatin unfolding is not accompanied by histone hyperacetylation. Cancer-predisposing mutations of BRCA1 display an allele-specific effect on chromatin unfolding: 5' mutations that result in gross truncation of the protein abolish the chromatin unfolding activity, whereas those in the 3' region of the gene markedly enhance this activity. A novel cofactor of BRCA1 (COBRA1) is recruited to the chromosome site by the first BRCT repeat of BRCA1, and is itself sufficient to induce chromatin unfolding. BRCA1 mutations that enhance chromatin unfolding also increase its affinity for, and recruitment of, COBRA1. These results indicate that reorganization of higher levels of chromatin structure is an important regulated step in BRCA1-mediated nuclear functions.

Alleles↗

PubMed2001

Central role of the CNGA4 channel subunit in Ca2+-calmodulin-dependent odor adaptation.

Heteromultimeric cyclic nucleotide-gated (CNG) channels play a central role in the transduction of odorant signals and subsequent adaptation. The contributions of individual subunits to native channel function in olfactory receptor neurons remain unclear. Here, we show that the targeted deletion of the mouse CNGA4 gene, which encodes a modulatory CNG subunit, results in a defect in odorant-dependent adaptation. Channels in excised membrane patches from the CNGA4 null mouse exhibited slower Ca2+-calmodulin-mediated channel desensitization. Thus, the CNGA4 subunit accelerates the Ca2+-mediated negative feedback in olfactory signaling and allows rapid adaptation in this sensory system.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Altering the DNA-binding specificity of the yeast Matalpha 2 homeodomain protein.

Homeodomain proteins are a highly conserved class of DNA-binding proteins that are found in virtually every eukaryotic organism. The conserved mechanism that these proteins use to bind DNA suggests that there may be at least a partial DNA recognition code for this class of proteins. To test this idea, we have investigated the sequence-specific requirements for DNA binding and repression by the yeast alpha2 homeodomain protein in association with its cofactors, Mcm1 and Mata1. We have determined the contribution for each residue in the alpha2 homeodomain that contacts the DNA in the co-crystal structures of the protein. We have also engineered mutants in the alpha2 homeodomain to alter the DNA-binding specificity of the protein. Although we were unable to change the specificity of alpha2 by making substitutions at residues 47, 54, and 55, we were able to alter the DNA-binding specificity by making substitutions at residue 50 in the homeodomain. Since other homeodomain proteins show similar changes in specificity with substitutions at residue 50, this suggests that there is at least a partial DNA recognition code at this position.

Amino Acid Substitution↗

PubMed2001

Hypoxia-inducible factor 1alpha and 1beta proteins share common signaling pathways in human prostate cancer cells.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor consisting alpha and beta subunits. It is critically involved in cancer cell hypoxia adaptation, glycolysis, and angiogenesis. HIF-1beta is associated with HIF-1 functions as a dimerization partner of HIF-1alpha, and is on the other hand associated with carcinogenesis via dioxin signaling. Regulation of HIF-1beta protein expression was investigated in human prostate cancer (PCA) cells. HIF-1beta protein was expressed constitutively under nonhypoxic conditions in all human PCA cells tested, and was up-regulated by hypoxia, CoCl2, EGF, serum, or PMA in moderate levels. Compared to that of HIF-1alpha, the constitutive, serum-, EGF-, and PMA-increased HIF-1beta protein expression were also inhibited by selective PI3K or FRAP/TOR inhibitors but in higher doses. Hypoxia partially reversed the dose dependent inhibition of HIF-1beta. These results suggest that HIF-1alpha and beta share common signaling pathways for nuclear protein accumulation.

Blood Proteins↗

PubMed2001

Allosteric modulation of Ca2+ channels by G proteins, voltage-dependent facilitation, protein kinase C, and Ca(v)beta subunits.

N-type and P/Q-type Ca(2+) channels are inhibited by neurotransmitters acting through G protein-coupled receptors in a membrane-delimited pathway involving Gbetagamma subunits. Inhibition is caused by a shift from an easily activated "willing" (W) state to a more-difficult-to-activate "reluctant" (R) state. This inhibition can be reversed by strong depolarization, resulting in prepulse facilitation, or by protein kinase C (PKC) phosphorylation. Comparison of regulation of N-type Ca(2+) channels containing Cav2.2a alpha(1) subunits and P/Q-type Ca(2+) channels containing Ca(v)2.1 alpha(1) subunits revealed substantial differences. In the absence of G protein modulation, Ca(v)2.1 channels containing Ca(v)beta subunits were tonically in the W state, whereas Ca(v)2.1 channels without beta subunits and Ca(v)2.2a channels with beta subunits were tonically in the R state. Both Ca(v)2.1 and Ca(v)2.2a channels could be shifted back toward the W state by strong depolarization or PKC phosphorylation. Our results show that the R state and its modulation by prepulse facilitation, PKC phosphorylation, and Ca(v)beta subunits are intrinsic properties of the Ca(2+) channel itself in the absence of G protein modulation. A common allosteric model of G protein modulation of Ca(2+)-channel activity incorporating an intrinsic equilibrium between the W and R states of the alpha(1) subunits and modulation of that equilibrium by G proteins, Ca(v)beta subunits, membrane depolarization, and phosphorylation by PKC accommodates our findings. Such regulation will modulate transmission at synapses that use N-type and P/Q-type Ca(2+) channels to initiate neurotransmitter release.

Allosteric Regulation↗

PubMed2001

Control of gating mode by a single amino acid residue in transmembrane segment IS3 of the N-type Ca2+ channel.

N-type Ca(2+) channels can be inhibited by neurotransmitter-induced release of G protein betagamma subunits. Two isoforms of Ca(v)2.2 alpha1 subunits of N-type calcium channels from rat brain (Ca(v)2.2a and Ca(v)2.2b; initially termed rbB-I and rbB-II) have different functional properties. Unmodulated Ca(v)2.2b channels are in an easily activated "willing" (W) state with fast activation kinetics and no prepulse facilitation. Activating G proteins shifts Ca(v)2.2b channels to a difficult to activate "reluctant" (R) state with slow activation kinetics; they can be returned to the W state by strong depolarization resulting in prepulse facilitation. This contrasts with Ca(v)2.2a channels, which are tonically in the R state and exhibit strong prepulse facilitation. Activating or inhibiting G proteins has no effect. Thus, the R state of Ca(v)2.2a and its reversal by prepulse facilitation are intrinsic to the channel and independent of G protein modulation. Mutating G177 in segment IS3 of Ca(v)2.2b to E as in Ca(v)2.2a converts Ca(v)2.2b tonically to the R state, insensitive to further G protein modulation. The converse substitution in Ca(v)2.2a, E177G, converts it to the W state and restores G protein modulation. We propose that negatively charged E177 in IS3 interacts with a positive charge in the IS4 voltage sensor when the channel is closed and produces the R state of Ca(v)2.2a by a voltage sensor-trapping mechanism. G protein betagamma subunits may produce reluctant channels by a similar molecular mechanism.

Amino Acid Substitution↗

PubMed2001

Signaling pathways activated by alpha1-adrenergic receptor subtypes in PC12 cells.

PC12 cells were used to compare signaling pathways activated by alpha1-adrenergic receptor (AR) subtypes. PC12 cells were transfected with human alpha1A, alpha1B, or alpha1D-ARs, and subclones stably expressing receptor densities in physiological ranges isolated and characterized. Norepinephrine (NE) activated a large number of signaling pathways in transfected cells, including inositol phosphate formation, intracellular calcium, all three arms of the mitogen activated protein kinase pathways, and a number of tyrosine kinases. Activation of mitogen activated protein kinase pathways and tyrosine kinases was not blocked by chelation of intracellular calcium with BAPTA or inhibition of protein kinase C. NE also activated luciferase reporter constructs for seven different transcription factors (AP1, SRE, CRE, NFkappaB, NFAT, Stat, GAS) following transfection into alpha1A-AR expressing PC12 cells. However, similar increases in inositol phosphate formation and intracellular Ca2+ caused by purinergic P2Y2 receptor activation did not activate any of these reporters. Comparison of alpha1-AR subtypes showed that the alpha1A activated all seven reporters, the alpha1B showed smaller effects, while the alpha1D was ineffective. NE caused differentiation of alpha1A, but not alpha1B or alpha1D, -AR expressing PC12 cells similar to that caused by NGF. This NE-induced differentiation was reduced or blocked by all inhibitors tested. We conclude that alpha1-ARs activate many signaling pathways and transcriptional responses in PC12 cells, which are not linearly related to second messenger production, and which may differ for different alpha1-AR subtypes.

Animals↗

PubMed2001

The nucleoporin Nup98 associates with the intranuclear filamentous protein network of TPR.

The Nup98 gene codes for several alternatively spliced protein precursors. Two in vitro translated and autoproteolytically cleaved precursors yielded heterodimers of Nup98-6kDa peptide and Nup98-Nup96. TPR (translocated promoter region) is a protein that forms filamentous structures extending from nuclear pore complexes (NPCs) to intranuclear sites. We found that in vitro translated TPR bound to in vitro translated Nup98 and, via Nup98, to Nup96. Double-immunofluorescence microscopy with antibodies to TPR and Nup98 showed colocalization. In confocal sections the nucleolus itself was only weakly stained but there was intensive perinucleolar staining. Striking spike-like structures emanated from this perinucleolar ring and attenuated into thinner structures as they extended to the nuclear periphery. This characteristic staining pattern of the TPR network was considerably enhanced when a myc-tagged pyruvate kinase-6kDa fusion protein was overexpressed in HeLa cells. Double-immunoelectron microscopy of these cells using anti-myc and anti-TPR antibodies and secondary gold-coupled antibodies yielded row-like arrangements of gold particles. Taken together, the immunolocalization data support previous electron microscopical data, suggesting that TPR forms filaments that extend from the NPC to the nucleolus. We discuss the possible implications of the association of Nup98 with this intranuclear TPR network for an intranuclear phase of transport.

Cell Nucleus↗

PubMed2001

Levels of hypoxia-inducible factor-1 alpha during breast carcinogenesis.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates gene expression in critical pathways involved in tumor growth and metastases. In this report, we investigated whether the level of HIF-1 alpha is increased during carcinogenesis in breast tissue and is associated with other tumor biomarkers. METHODS: Paraffin-embedded clinical specimens from five pathologic stages of breast tumorigenesis and from normal breast tissue were used. HIF-1 alpha protein and the biomarkers vascular endothelial growth factor (VEGF), HER-2/neu, p53, Ki-67, and estrogen receptor (ER) were identified immunohistochemically, and microvessel density (a measure of angiogenesis) was determined. Associations among levels of HIF-1 alpha and these biomarkers were tested statistically. All statistical tests are two-sided. RESULTS: The frequency of HIF-1 alpha-positive cells in a specimen increased with the specimen's pathologic stage (P<.001, chi(2) test for trend) as follows: normal breast tissue (0 specimens with > or = 1% HIF-1 alpha-positive cells in 10 specimens tested), ductal hyperplastic lesions (0 in 10), well-differentiated ductal carcinomas in situ (DCIS) (11 in 20), well-differentiated invasive breast cancers (12 in 20), poorly differentiated DCIS (17 in 20), and poorly differentiated invasive carcinomas (20 in 20). Increased levels of HIF-1 alpha were statistically significantly associated with high proliferation and increased expression of VEGF and ER proteins. In DCIS lesions, increased levels of HIF-1 alpha were statistically significantly associated with increased microvessel density. HIF-1alpha showed a borderline association with HER-2/neu but no association with p53. CONCLUSIONS: The level of HIF-1 alpha increases as the pathologic stage increases and is higher in poorly differentiated lesions than in the corresponding type of well-differentiated lesions. Increased levels of HIF-1 alpha are associated with increased proliferation and increased expression of ER and VEGF. Thus, increased levels of HIF-1 alpha are potentially associated with more aggressive tumors.

Biomarkers, Tumor↗

PubMed2001

SB203580 promotes EGF-stimulated early morphological differentiation in PC12 cell through activating ERK pathway.

MAP kinases have important role in PC12 cell differentiation, since the activities of both extracellular regulated protein kinase (ERK) and p38 have been indicated as necessary signal for PC12 cell differentiation. Epidermal growth factor (EGF) and NGF both activate ERK and p38 in PC12 cells, but only NGF trigger differentiation. It has been proposed that the duration of ERK activation determines the switch from proliferation to differentiation, since EGF causes more transient activation of ERK than NGF in PC12 cells. Here we report that treatment of PC12 cells with EGF in the presence of SB203580, a widely used p38 inhibitor, caused differentiation. The pro-differentiation effect of SB203580 in EGF-treated PC12 cells was found to be independent of its function of p38 inhibition but was through an effect on the ERK pathway that has been recently reported (Kalmes et al. [1999] FEBS Lett. 444: 71-74; Hall-Jackson et al. [1999] Onc. 18: 2047-2054). We found that SB203580 by itself did not affect the activity of ERK1/2 but significantly extended EGF-induced ERK activation in PC12 cells, which resulted in early morphological differentiation. Our data indicated that although both ERK and p38 are required for PC12 cell differentiation, activation of p38 is not required when ERK is superactivated. Our data provided further evidence for the threshold theory that differentiation is determined by the duration of ERK activation.

Adenoviridae↗

PubMed2001

A Chinese homozygote of familial hypercholesterolemia: identification of a novel C263R mutation in the LDL receptor gene.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene; it is characterized by a high concentration of LDL, which frequently gives rise to tendon xanthomas and premature coronary artery disease (CAD). Individuals with heterozygous FH in China often exhibit a milder phenotype than those in other countries. The diagnosis of heterozygous FH relies on the clinical phenotype and this does not always permit unequivocal diagnosis of the disease. In the course of investigation of FH in a Chinese population sample, we found a family whose proband showed a markedly raised concentration of LDL cholesterol in plasma, and the presence of skin and tendon xanthomata. We used single-strand conformation polymorphism (SSCP) analysis to screen all the 18 exons and the exon-intron boundaries of the LDLR gene. One novel homozygous mutation, replacing T by C at nucleotide 850 in exon 6 was identified. This change substituted cysteine for arginine at codon 263 (C263R) of the LDLR. By means of mutant allele-specific amplification, we unequivocally diagnosed six heterozygotes with this novel mutation in the proband's family. forms of heterozygous FH are characterized by high serum LDL cholesterol levels, which are usually associated with premature coronary heart disease (CHD) and tendon xanthomas in early middle age, while homozygous FH individuals frequently suffer fatal CHD by their third decade (Goldstein et al. 1995). To date, more than 200 different mutations of the LDLR gene have been characterized worldwide (Hobbs et al. 1990; Hobbs et al. 1992; Varret et al. 1997). Chinese individuals with heterozygous FH are often recognized by virtue of their being parents of offspring with mutations in both alleles of the LDLR gene (Mak et al. 1998). Unlike their heterozygous parents, the Chinese homozygous FH patients are as severely affected, as are those elsewhere (Sun et al. 1994). In this article, we present an investigation of a Chinese family whose proband, with severe hypercholesterolemia, carried a novel mutation of the LDLR gene in a homozygous form, while the other six patients in the family, who carried this mutation in a heterozygous form, have a milder phenotype of FH. The main objective of our analysis was to delineate the full spectrum of mutations that underline FH in the Chinese population; our work represents a first step towards the implementation of nationwide DNA testing for this disease.

Base Sequence↗

PubMed2001

Efficacy of ground-applied ultra-low-volume malathion on honey bee survival and productivity in open and forest areas.

A study was conducted to determine the efficacy of ground ultra-low-volume malathion sprays on honey bee (Apis mellifera L.) apiaries in open and forested areas downwind from the spray route. Impact on colonies 7.6, 15.2, 47.7, and 91.4 m downwind from sprays was assessed by recording individual bee mortality 12 and 36 h after treatment. In addition, hives were weighed before as well as during the study and cluster counts were conducted at each hive to determine colony strength before and after treatment. Spray drift was monitored by the use of caged mosquito (Culex quinquefasciatus Say) mortality and deposition on filter paper. During the study, significant bee mortality in the open area occurred on 2 occasions at 7.6 m (16.8 +/- 4.3 bees, 11.8 +/- 7.0 bees) and at 15.2 m (6.5 +/- 1.7 bees, 5.3 +/- 1.5 bees). Significant mortality in the forested area was observed only once and consisted of 2 bees at 7.6 m. In each case where bee mortality occurred, spray deposits on filter papers had exceeded 400 ng/cm2. Although mortality of caged mosquitoes indicated that malathion drifted through the study areas, little correlation was apparent between mortality and spray deposition on filter paper.

Animals↗

PubMed2001

Regulator of G protein signaling proteins: novel multifunctional drug targets.

G protein-coupled receptors (GPCRs) play a major role in signal transduction and are targets of many therapeutic drugs. The regulator of G protein signaling (RGS) proteins form a recently identified protein family, and they strongly modulate the activity of G proteins. Their best known function is to inhibit G protein signaling by accelerating GTP hydrolysis [GTPase activating protein (GAP)] thus turning off G protein signals. RGS proteins also possess non-GAP functions, through both their RGS domains and various non-RGS domains and motifs (e.g., GGL, DEP, DH/PH, PDZ domains and a cysteine string motif). They are a highly diverse protein family, have unique tissue distributions, are strongly regulated by signal transduction events, and will likely play diverse functional roles in living cells. Thus they represent intriguing, novel pharmacological/therapeutic targets. Drugs targeting RGS proteins can be divided into five groups: 1) potentiators of endogenous agonist function, 2) potentiators/desensitization blockers of exogenous GPCR agonists, 3) specificity enhancers of exogenous agonists, 4) antagonists of effector signaling by an RGS protein, and 5) RGS agonists. In addition, a novel subsite distinction within the RGS domain has been proposed with significant functional implications and defined herein as "A-site" and "B-site". Therefore, RGS proteins should provide exciting new opportunities for drug development.

Amino Acid Motifs↗

PubMed2001

Adenosine-mediated mast cell degranulation in adenosine deaminase-deficient mice.

Adenosine is a signaling nucleoside that has been suggested to play a role in asthma in part through its ability to influence mediator release from mast cells. Adenosine levels are elevated in the lungs of asthmatics, further implicating this molecule in the regulation of lung inflammation and suggesting that animal models exhibiting endogenous increases in adenosine will be useful for the analysis of adenosine function. Adenosine deaminase (ADA) is a purine catabolic enzyme responsible for regulating the levels of adenosine in tissues and cells. ADA-deficient mice develop lung inflammation and damage reminiscent of that seen in asthma in association with elevated adenosine levels. In the current study, we investigated the status of mast cells in ADA-deficient lungs. ADA-deficient mice exhibited extensive lung mast cell degranulation concurrent with elevated adenosine levels. ADA enzyme therapy prevented the accumulation of lung adenosine as well as mast cell degranulation, suggesting that this process was dependent on elevated lung adenosine levels. Consistent with this, treatment of ADA-deficient mice with broad spectrum adenosine receptor antagonists attenuated degranulation by 30 to 40%, supporting the involvement of adenosine receptor signaling. Moreover, these studies demonstrate the ability of endogenously generated adenosine to influence lung mast cell degranulation in a receptor-mediated manner and establish ADA-deficient mice as a model system to investigate the specific adenosine receptor responses involved in the degranulation of lung mast cells.

Adenosine↗

PubMed2001

[A gene analysis of the low density lipoprotein receptor in Chinese with homozygous familial hyperchol-esterolemia].

OBJECTIVE: To investigate low density lipoprotein receptor(LDLR) gene mutations in Chinese with familial hyperchol-esterolemia(FH). METHODS: Genomic DNA was extracted from five unrelated children with clinical diagnosis of homozygous FH, together with their family members. Promoter and all of the 18 exons of LDLR gene were amplified by polymerase chain reaction (PCR) and were analyzed by single strand conformation polymorphism (SSCP). The PCR products of abnormal patterns shown by SSCP were sequenced directly. RESULTS: Two point mutations (A606T, C263R) were found in two families respectively. The two mutations were only found in China, and C263R is a novel mutation. CONCLUSION: The two mutations of LDLR gene are the cause of FH and there may be LDLR gene mutations only carried by FH patients in China.

Asian People↗