Biomedical subjects

H Zhu

Publications and source records attributed to H Zhu.

At least 181 records · Page 10Linked to original sources

PubMed2000

Activation of transcription factors activator protein-1 and nuclear factor-kappaB by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin), the prototype agonist of the aromatic hydrocarbon (Ah) receptor, is a potent tumor promoter as well as a complete liver carcinogen that produces an oxidative stress response in rodents and in cultured cell lines. It has been proposed that TCDD promotes neoplastic transformation through oxidative signal transduction pathways, which results in activation of immediate-early response transcription factors. To set the stage for a test of this hypothesis, we evaluated the effect of TCDD treatment on the activation of several transcription factors, including those in the nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) families, which are activated by changes in the redox state of cells. In an extension of prior results, we found that TCDD treatment produced a sustained overexpression of AP-1 for at least 72 hr in wild-type mouse hepatoma Hepa-1 cells, but not in the Ah receptor-deficient derivative c35 or in cytochrome P450-1A1 (CYP1A1)-negative c37 cells. In addition, TCDD treatment caused a significant increase in the DNA binding activity of NF-kappaB, but not in the activities of the other transcription factors tested. AP-1 and NF-kappaB activation were blocked by the thiol antioxidant N-acetylcysteine and by nordihydroguaiaretic acid, an antioxidant and lipooxygenase inhibitor and an inhibitor of the epoxygenase activity of CYP1A1, and did not take place in c35, c37, or in Ah nuclear translator-deficient c4 cells. Hence, sustained activation of these two transcription factors by TCDD is likely to result from a CYP1A1-dependent and Ah receptor complex-dependent oxidative signal. Electrophoretic mobility supershift analyses with specific antibodies showed that most of the increase in NF-kappaB binding activity could be accounted for by increases in p50/p50 complexes. Since these complexes are known to repress NF-kappaB-dependent gene transcription, our results delineate a second molecular mechanism, in addition to the recently found block of tumor necrosis factor-alpha-mediated p50/p65 activation, that may be responsible for the immunosuppresive effects of TCDD.

Animals↗

PubMed2000

X-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): a dimer-dimer model of LYS7-SOD association and copper delivery.

Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD. The 1.55 A crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods. The monomeric structure reveals an eight-stranded Greek key beta-barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the beta-barrel come together to bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested. In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2. The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer model over the heterodimer model are enumerated.

Computer Simulation↗

PubMed2000

[Study on plasma lipids, apolipoprotein A1, apolipoprotein B100 in simple obese children].

To investigate the disorder of lipid metabolism and explore the possible mechanism of increasing risk of atherosclerosis(AS) in simple childhood obesity, serum lipids and apolipoprotein were examined. 49 obese children were selected and another 49 non-obese children were selected as control. Their serum lipids (TG, TC, VLDL-ch, HDL-ch and LDL-ch) and apolipoprotein(apoA1 and apoB100) were measured. The results showed TG, VLDL-ch, apoB100, artherosclerosis index (AI) and LDL-ch/HDL-ch ratio in childhood obesity were significantly higher than that in normal children (P < 0.05), while HDL-ch, apoA1, HDL-ch/TC, apoA1/apoB100 ratio in obese children were obviously lower than that in normal ones(P < 0.05). The results suggested that metabolic disorder of lipid and apolipoprotein took place in obese children. The disorder which may increase the risk of AS should be paid great attention.

Adolescent↗

PubMed2000

The food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine activates S-phase checkpoint and apoptosis, and induces gene mutation in human lymphoblastoid TK6 cells.

The mutagenic heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-blpyridine (PhIP) is formed at parts per billion levels when meat is cooked. It is efficiently absorbed from cooked food and extensively activated to its genotoxic N-hydroxy derivative by human cytochrome P4501A enzymes. It is also a rodent carcinogen. To better understand the genetic toxicity of PhIP, we have examined its effect on the cell cycle and gene mutation frequency using human lymphoblastoid cells (TK6) as a model. Because TK6 cells are unable to activate PhIP, we have cultured the cells in the presence of irradiated Chinese hamster XEMh1A2-MZ cells that have been genetically engineered to express human CYP1A2. Asynchronized TK6 cells were harvested at various times after treatment with PhIP (1.25-10 microg/ml), fixed and stained with propidium iodide for the examination of cell cycle by fluorescence-activated flow cytometry. After 20 h of PhIP treatment, a slight S-phase delay of the cell cycle was observed. Normal cell cycle recovered after the cells were washed and further cultured in the absence of PhIP for 5 days. However, PhIP treatment for 40 h induced a more pronounced S-phase arrest that was accompanied by a decrease in the level of cyclin A, an S-phase cyclin. This was followed by the appearance of a sub-G1 population (indicative of apoptotic cell death), range from 13 to 54% with PhIP concentrations from 1.25 to 10 microg/ml, compared with 5% in the vehicle control. A concomitant increase of mutation frequency at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, assessed by colony formation assay in the presence of 6-thioguanine, was detected after 40 h-range, 16 to 45 x 10(-6) compared with 12 x 10(-6) in cultures without PhIP. In G1-enriched cell populations (synchronized culture), although PhIP induced S-phase delay, the induction of sub-G1 cells was substantially decreased. Our studies show that in TK6 cells, PhIP activates S-phase checkpoint, yet eludes G1 and G2-M checkpoints, and is accompanied by increased apoptosis and gene mutation. If treatment with PhIP induces similar cellular reactions in vivo, then activation of S-phase checkpoint with avoidance of G1 and G2-M checkpoints could be important factors in PhIP-induced genetic damage and neoplastic disease.

Animals↗

PubMed2000

A fragment of recombinant GABA(A) receptor alpha1 subunit forming rosette-like homo-oligomers.

The type A gamma-aminobutyric acid (GABA(A)) receptor plays a major role in inhibitory synaptic transmission in the central nervous system. A fragment consisting of residues Cys166 to Leu296 of the alpha1 subunit of the GABA(A) receptor was overexpressed in Escherichia coli and was found to have stable beta-rich structures. Here, results from laser scattering, gel electrophoresis and electron microscopy demonstrated that this recombinant protein formed rosette-like homo-oligomers, mainly pentamers in solution. Therefore, the fragment apparently provides a valuable model system for studying the pentameric holoreceptor assembly. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of the fragment showed that disulfide bonds formed between monomers contributed to the oligomerization of the fragment. The fact that this fragment alone could form pentamers in vitro strongly suggests that amino acid residues located within the Cys166-Leu296 region of the alpha1 subunit may contribute to the oligomerization of GABA(A) receptor in vivo.

Animals↗

PubMed2000

The Xenopus clock gene is constitutively expressed in retinal photoreceptors.

Many aspects of normal retinal physiology are controlled by a retinal circadian clock. In Xenopus laevis, the photoreceptor cells within the retina contain a circadian clock that controls melatonin release. In this report we present the cloning and characterization of the Xenopus homolog of the Clock gene, known to be critical for normal circadian behavioral rhythms in the mouse. The Xenopus Clock gene is expressed primarily in photoreceptors within the eye and is expressed at constant levels throughout the day. Analysis of other tissues revealed that, as in other species, the Xenopus Clock gene is widely expressed. This characterization of the Clock gene provides a useful tool for further exploration of the role of the circadian clock in normal retinal function.

Amino Acid Sequence↗

PubMed2000

Presenilin-1 mutation increases neuronal vulnerability to focal ischemia in vivo and to hypoxia and glucose deprivation in cell culture: involvement of perturbed calcium homeostasis.

Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Studies of cultured neural cells suggest that PS1 mutations result in perturbed cellular calcium homeostasis and may thereby render neurons vulnerable to apoptosis. In light of evidence that metabolic impairment plays a role in AD, that cerebral ischemia may be a risk factor for AD, and that individuals with AD have increased morbidity and mortality after stroke, we examined the impact of a PS1 mutation on neuronal vulnerability to ischemic injury. We report that the extent of brain injury after focal cerebral ischemia reperfusion is increased, and behavioral outcome is worsened, in PS1 mutant knock-in mice compared to wild-type mice. Cultured cortical neurons from PS1 mutant mice exhibit increased vulnerability to glucose deprivation and chemical hypoxia compared to their wild-type counterparts. Calcium imaging studies demonstrated enhanced elevation of intracellular calcium levels after glucose deprivation and chemical hypoxia in neurons from PS1 mutant mice. Agents that block calcium release from IP(3)- and ryanodine-sensitive stores (xestospongin and dantrolene, respectively) protected against the endangering action of the PS1 mutation. Our data suggest that presenilin mutations may promote neuronal degeneration in AD by increasing the sensitivity of neurons to age-related ischemia-like conditions. The data further suggest that drugs that stabilize endoplasmic reticulum calcium homeostasis may prove effective in suppressing the neurodegenerative process in AD patients.

Alzheimer Disease↗

PubMed2000

Cloning and functional identification of a neuronal glutamine transporter.

Glutamine is the preferred precursor for the neurotransmitter pool of glutamate, the major excitatory transmitter in the mammalian central nervous system. We have isolated a complementary DNA clone (designated GlnT) encoding a plasma membrane glutamine transporter from glutamatergic neurons in culture, and its properties have been examined using the T7 vaccinia system in fibroblasts. When GlnT is transfected into CV-1 cells, L-glutamine is the preferred substrate. Transport is Na(+)-dependent and inhibited by alpha-methylaminoisobutyric acid, a specific inhibitor of neutral amino acid transport system A. Kinetic analysis of glutamine uptake by GlnT is saturable, with a Michaelis constant (K(m)) of 489 +/- 88 microM at pH 7.4. Glutamine uptake mediated by GlnT is pH-sensitive with a 5-fold greater efficiency of uptake at pH 8.2 than at pH 6.6. Only the maximal velocity of transport increases without a significant change in K(m). The distribution of GlnT mRNA and protein in the central nervous system is widespread and is expressed on neurons that use glutamate as their neurotransmitter. In cultured cerebellar granule cells, GlnT is expressed only on neurons and is absent from astrocytes. GlnT expression increases concomitantly with the morphologic and functional differentiation of these cells in vitro, consistent with its role of supplying glutamatergic neurons with their neurotransmitter precursor. GlnT is the first member of the system A family of neutral amino acid transporters with 11 putative membrane-spanning domains and is a potential target to modulate presynaptic glutamatergic function.

Amino Acid Transport Systems, Basic↗

PubMed2000

The first 28 N-terminal amino acid residues of human heart muscle carnitine palmitoyltransferase I are essential for malonyl CoA sensitivity and high-affinity binding.

Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30-100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues. All of the deletions except Delta83 were active. Mitochondria from the yeast strains expressing Delta28 and Delta39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding. The high-affinity site (K(D1), B(max1)) for binding of malonyl CoA to M-CPTI was completely abolished in the Delta28, Delta39, Delta51, and Delta72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme. Delta18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity malonyl CoA binding similar to the wild type. Replacement of the N-terminal domain of L-CPTI with the N-terminal domain of M-CPTI does not change the malonyl CoA sensitivity of the chimeric L-CPTI, suggesting that the amino acid residues responsible for the differing sensitivity to malonyl CoA are not located in this N-terminal region. These results demonstrate that the N-terminal residues critical for activity and malonyl CoA sensitivity in M-CPTI are different from those of L-CPTI.

Amino Acid Sequence↗

PubMed2000

[Study on genotypes of cystathionine beta-synthase in neural tube defects].

Mildly elevated maternal plasma homocysteine (Hcy) levels (hyperhomocysteinemia) have recently been observed in some neural tube defects(NTDs) pregnancies. The aetiology of NTDs is also known to have both genetic and nutritional components. The frequency of two relatively common mutations in the enzyme of cystathionine beta-synthase (CBS), which is one of the main enzymes that controls Hcy level, was examined. Among 76 nonpregnant women in the study, 40 of whom had given birth to infants with NTDs and 36 control women previously had normal offspring. The results showed that no significant difference was found between NTDs mothers and non-NTDs mothers for the frequency of T833-C and the G919-A mutations in the CBS gene. The data suggests that the mother's genotype is not the direct factor involved in the pathogenesis of NTDs.

Adult↗

PubMed2000

CD44 interaction with tiam1 promotes Rac1 signaling and hyaluronic acid-mediated breast tumor cell migration.

In this study we have explored the interaction between CD44 (the hyaluronic acid (HA)-binding receptor) and Tiam1 (a guanine nucleotide exchange factor) in metastatic breast tumor cells (SP1 cell line). Immunoprecipitation and immunoblot analyses indicate that both the CD44v3 isoform and the Tiam1 protein are expressed in SP1 cells and that these two proteins are physically associated as a complex in vivo. Using an Escherichia coli-derived calmodulin-binding peptide-tagged Tiam1 fragment (i.e. the NH(2)-terminal pleckstrin homology (PHn) domain and an adjacent protein interaction domain designated as PHn-CC-Ex, amino acids 393-738 of Tiam1) and an in vitro binding assay, we have detected a specific binding interaction between the Tiam1 PHn-CC-Ex domain and CD44. Scatchard plot analysis indicates that there is a single high affinity CD44 binding site in the PHn-CC-Ex domain of Tiam1 with an apparent dissociation constant (K(d)) of 0.2 nM, which is comparable with CD44 binding (K(d) = approximately 0.13 nM) to intact Tiam1. These findings suggest that the PHn-CC-Ex domain is the primary Tiam1-binding region for CD44. Most importantly, the binding of HA to CD44v3 of SP1 cells stimulates Tiam1-catalyzed Rac1 signaling and cytoskeleton-mediated tumor cell migration. Transfection of SP1 cells with Tiam1cDNA promotes Tiam1 association with CD44v3 and up-regulates Rac1 signaling as well as HA/CD44v3-mediated breast tumor cell migration. Co-transfection of SP1 cells with PHn-CC-Ex cDNA and Tiam1 cDNA effectively inhibits Tiam1 association with CD44 and efficiently blocks tumor behaviors. Taken together, we believe that the linkage between CD44v3 isoform and the PHn-CC-EX domain of Tiam1 is required for HA stimulated Rac1 signaling and cytoskeleton-mediated tumor cell migration during breast cancer progression.

Adjuvants, Immunologic↗

PubMed2000

Loss of in vitro metal ion binding specificity in mutant copper-zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis.

The presence of the copper ion at the active site of human wild type copper-zinc superoxide dismutase (CuZnSOD) is essential to its ability to catalyze the disproportionation of superoxide into dioxygen and hydrogen peroxide. Wild type CuZnSOD and several of the mutants associated with familial amyotrophic lateral sclerosis (FALS) (Ala(4) --> Val, Gly(93) --> Ala, and Leu(38) --> Val) were expressed in Saccharomyces cerevisiae. Purified metal-free (apoproteins) and various remetallated derivatives were analyzed by metal titrations monitored by UV-visible spectroscopy, histidine modification studies using diethylpyrocarbonate, and enzymatic activity measurements using pulse radiolysis. From these studies it was concluded that the FALS mutant CuZnSOD apoproteins, in direct contrast to the human wild type apoprotein, have lost their ability to partition and bind copper and zinc ions in their proper locations in vitro. Similar studies of the wild type and FALS mutant CuZnSOD holoenzymes in the "as isolated" metallation state showed abnormally low copper-to-zinc ratios, although all of the copper acquired was located at the native copper binding sites. Thus, the copper ions are properly directed to their native binding sites in vivo, presumably as a result of the action of the yeast copper chaperone Lys7p (yeast CCS). The loss of metal ion binding specificity of FALS mutant CuZnSODs in vitro may be related to their role in ALS.

Amino Acid Substitution↗

PubMed2000

Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta.

Apoptosis, or cellular suicide, is important for normal development and tissue homeostasis, but too much or too little apoptosis can also cause disease. The family of cysteine proteases, the so- called caspases, are critical mediators of programmed cell death, and thus far 14 family members have been identified. Some of these, such as caspase-8, mediate signal transduction downstream of death receptors located on the plasma membrane. Others, such as caspase-9, mediate apoptotic signals after mitochondrial damage. Stress in the endoplasmic reticulum (ER) can also result in apoptosis. Here we show that caspase-12 is localized to the ER and activated by ER stress, including disruption of ER calcium homeostasis and accumulation of excess proteins in ER, but not by membrane- or mitochondrial-targeted apoptotic signals. Mice that are deficient in caspase-12 are resistant to ER stress-induced apoptosis, but their cells undergo apoptosis in response to other death stimuli. Furthermore, we show that caspase-12-deficient cortical neurons are defective in apoptosis induced by amyloid-beta protein but not by staurosporine or trophic factor deprivation. Thus, caspase-12 mediates an ER-specific apoptosis pathway and may contribute to amyloid-beta neurotoxicity.

Alzheimer Disease↗

PubMed2000

An association study of polymorphisms in the alpha-antichymotrypsin gene for Alzheimer disease in Han-Chinese.

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing were employed to screen the coding region of the alpha-antichymotrypsin (AACT) gene in Han-Chinese population for polymorphism possibly associated with Alzheimer's disease (AD). Consequently, seven polymorphic sites including 25A>G, 39G>A, 370C>T, 662T>G, 892C>T, 923T>C and 1332A>G were detected. Of them, the 25A>G was reported previously and the others are all novel. We subsequently focused on the 25A>G and the 39G>A polymorphism that were of interest to us and conducted an association study of them by another scanning of 246 controls that matched the AD patients. Statistic test showed that both genotype (p=0.0378, Fisher's exact, two tailed) and allele frequency (p=0.0382, Fisher's exact, two tailed) of 39G>A are different between AD patients and the controls. As for 25A>G, lain only the heterozygous genotype A/G associates with AD (p=0.0220, chi(2)), but not the A allele frequency (p=0.1141, chi(2)).

Aged↗

PubMed2000

CD4+ T cell apoptosis induced by anti-CD4 antibodies.

To explore the inhibitory effects of anti-CD4 human/murine chimeric antibodies on lymphocyte proliferation, CD4+ T cell apoptosis induced by anti-CD4 antibodies was examined. Annexin-V-FITC and PI double stain method was employed to qualitatively and quantitatively determined CD4+ T cell apoptosis induced by anti-CD4 antibodies. Our results showed that anti-CD4 chimeric antibodies could specifically induce CD4+ T cell apoptosis. The ability of anti-CD4 chimeric antibodies to induce CD4+ T cell apoptosis was related with the presence of monocytes. It is concluded that the further cross-linking of anti-CD4 antibodies is important for inducing CD4+ T cell apoptosis.

Animals↗

PubMed2000

Screening of novel epilepsy-related genes and isolation and identification of cDNAs.

Twenty cDNA differential fragments were isolated from the hippocampus of rats in epileptic state using mRNA differential display technique. Four fragments were sequenced and compared with the known sequences in the Genebank, which showed that ERG8, ERG11, ERG12 had no significant identity to any known sequences; ERG14 had 64%-69% identity to microtubulin-associated protein of the rat. Because the differential expression of these genes was caused by epilepsy inducer coriaria lactone (CL) and anti-epilepsy drug MK-801 and ERG8 might be a novel candidate epilepsy gene; ERG11 and ERG12 might be novel candidate anti-epilepsy genes. Since the microtubulin-associated protein is closely associated with the collateral sprouting of mossy fibers in the hippocampus of seizured rat, the high expression of ERG14 in the early stage of epilepsy might predict the growth of axon and formation of synapse.

Animals↗

PubMed2000

Construction and analysis of three-dimensional graphic model of single-chain Fv derived from an anti-human placental acidic isoferritin monoclonal antibody by computer.

A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (MAb) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station. The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the inker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

Amino Acid Sequence↗

PubMed2000

The role of TGF-beta 1 in mice hepatic fibrosis by Schistosomiasis Japonica.

To investigate the role of transforming growth factor-beta 1 (TGF-beta 1) in mice with hepatic fibrosis caused by Schistosomiasis Japonica, ELISA, VG staining and multimedia color hieroglyph quantitative analysis were used to study the change of the serum TGF-beta 1, liver collagen fiber and reticular fiber in mice. The level of serum TGF-beta 1 in experimental group was significantly higher than that in control group (P < 0.01 or P < 0.05) 8, 10, 12 weeks after infected by schistosomiasis. After infection, the level of liver collagen fiber and reticular fiber, and that of TGF-beta 1 increased over time (P < 0.01 or P < 0.05). In mice infected by Schistosomiasis Japonica, the level of TGF-beta 1 increased with prolongation of infection time, and with the increase of liver collagen fiber and reticular fiber. TGF beta 1 plays an important role of immunomodulation in hepatic fibrosis formation caused by Schistosomiasis Japonica.

Animals↗