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Biomedical subjects

H Zhu

Publications and source records attributed to H Zhu.

At least 253 records · Page 14Linked to original sources
PubMed1999

A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation.

The TGF-beta superfamily of proteins regulates many different biological processes, including cell growth, differentiation and embryonic pattern formation. TGF-beta-like factors signal across cell membranes through complexes of transmembrane receptors known as type I and type II serine/threonine-kinase receptors, which in turn activate the SMAD signalling pathway. On the inside of the cell membrane, a receptor-regulated class of SMADs are phosphorylated by the type-I-receptor kinase. In this way, receptors for different factors are able to pass on specific signals along the pathway: for example, receptors for bone morphogenetic protein (BMP) target SMADs 1, 5 and 8, whereas receptors for activin and TGF-beta target SMADs 2 and 3. Phosphorylation of receptor-regulated SMADs induces their association with Smad4, the 'common-partner' SMAD, and stimulates accumulation of this complex in the nucleus, where it regulates transcriptional responses. Here we describe Smurf1, a new member of the Hect family of E3 ubiquitin ligases. Smurf1 selectively interacts with receptor-regulated SMADs specific for the BMP pathway in order to trigger their ubiquitination and degradation, and hence their inactivation. In the amphibian Xenopus laevis, Smurf1 messenger RNA is localized to the animal pole of the egg; in Xenopus embryos, ectopic Smurf1 inhibits the transmission of BMP signals and thereby affects pattern formation. Smurf1 also enhances cellular responsiveness to the Smad2 (activin/TGF-beta) pathway. Thus, targeted ubiquitination of SMADs may serve to control both embryonic development and a wide variety of cellular responses to TGF-beta signals.

Animals↗
PubMed1999

Activity and conformation changes of Chinese hamster dihydrofolate reductase in reverse micelles.

Changes in the activity and conformation of Chinese hamster dihydrofolate reductase (ch-DHFR) were studied in reverse micelles of cetyltrimethylammonium bromide (CTAB) and dodecylammonium butyrate (DAB) at various water contents and denaturant concentrations. The ch-DHFR entrapped in CTAB and DAB reverse micelles shows very low activity at a lower ratio of water to surfactant (omega(0)). The activity was enhanced in the presence of low concentrations of guanidine hydrochloride. Emission fluorescence spectra of ch-DHFR in aqueous medium and in reverse micelles in the presence of low concentrations of denaturants were compared. Only a slight change in emission intensity of ch-DHFR accompanies enzyme activation in the presence of low concentrations of guanidine hydrochloride in CTAB and DAB reverse micelles. There was no detectable change in the average diameters of CTAB and DAB reverse micelles in the presence of low concentrations of denaturants measured by laser light scattering, indicating that the enzyme activation in the presence of denaturant is not due to a size change in the reverse micelles. Our results suggest that the reduced activity of ch-DHFR in reverse micelles at low omega(0) is due to the restrictions in enzyme conformation caused by an environment with low water content. The reduction in enzyme activity was restored by the presence of low concentrations of denaturant or increased water content, indicating that conformation flexibility is important for full expression of enzyme activity.

Amines↗
PubMed1999

A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents.

Exposure of human tumor cell lines to different chemotherapeutic drugs, ionizing radiation, and differentiating agents induced morphological, enzymatic, and ploidy changes resembling replicative senescence of normal cells. Moderate doses of doxorubicin induced this senescence-like phenotype (SLP) in 11 of 14 tested cell lines derived from different types of human solid tumors, including all of the lines with wild-type p53 and half of p53-mutated cell lines. SLP induction seemed to be independent from mitotic cell death, the other major effect of drug treatment. Among cells that survived drug exposure, SLP markers distinguished those cells that became terminally growth-arrested within a small number of cell divisions from the cells that recovered and resumed proliferation. SLP induction in breast carcinoma cells treated with retinoids in vitro or in vivo was found to correlate with permanent growth inhibition under the conditions of minimal cytotoxicity, suggesting that this response may be particularly important for the antiproliferative effect of differentiating agents. The senescence-like program of terminal proliferation arrest may provide an important determinant of treatment outcome and a target for augmentation in cancer therapy.

Adenocarcinoma↗
PubMed1999

Detection of mitochondria-derived reactive oxygen species production by the chemilumigenic probes lucigenin and luminol.

Both lucigenin and luminol have widely been used as chemilumigenic probes for detecting reactive oxygen species (ROS) production by various cellular systems. Our laboratory has previously demonstrated that lucigenin localizes to the mitochondria of rat alveolar macrophages and that lucigenin-derived chemiluminescence (CL) appears to reflects superoxide O2(-.) production by mitochondria in the unstimulated macrophages. In this study, we further examined the ability of lucigenin- and luminol-derived CL to assess O2(-.) and H2O2 formation, respectively, by isolated intact mitochondria. Mitochondria were isolated from monocytes/macrophages differentiated from monoblastic ML-1 cells. Incubation of the substrate-supported mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondria in the presence of horseradish peroxidase (HRP). The lucigenin-derived CL was diminished extensively by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6, 6-tetramethylpiperidine-N-oxyl and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. On the other hand, luminol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect mitochondrial respiration exhibited corresponding effects on both lucigenin- and luminol-derived CL. Taken together, our results demonstrate that with isolated mitochondria lucigenin-derived CL monitors intramitochondrial O2(-.) production by the mitochondrial electron transport chain, whereas the luminol-derived CL detects H2O2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS production by mitochondria.

Acridines↗
PubMed1999

Imidazoline receptor antisera-selected cDNA clone and mRNA distribution.

A novel cDNA, designated Imidazoline Receptor Antisera-Selected cDNA-1 (iras-1), encodes a 167-kD protein. Two of its predicted peptides (42-43 kD) are immunologically consistent with a previously reported 1(1)-imidazoline binding protein. In the present study, two forms of iras mRNA (6.0 and 9.5 kb) were quantified across fresh rat tissues. Highest levels were found in brain (almost exclusively 6.0 kb in size), followed by liver and lung (9.5 > or = 6.0 kb iras mRNA), kidney (6.0 > 9.5 kb), heart (6.0 kb), spleen (6.0 > or = 9.5 kb), testes (6.0 > 9.5 kb), and skeletal muscle (6.0 > 9.5 kb). A correlation exists (p = 0.71, p = 0.05) between total (6.0 + 9.5 kb) iras mRNA and I1 BMAX values across rat tissues, corrected for housekeeping gene expression. Thus, total iras mRNA appears to be roughly proportional to the density of I1-imidazoline binding sites.

Animals↗
PubMed1999

Plasma agmatine and platelet imidazoline receptors in depression.

Plasma agmatine concentrations are elevated significantly in depressed patients compared to healthy controls. Treatment with the antidepressant bupropion normalized plasma agmatine levels. Correlational evidence is presented that a change in plasma agmatine levels may lead to similar changes in platelet I1 imidazoline receptors.

Agmatine↗
PubMed1999

A single amino acid change (substitution of glutamate 3 with alanine) in the N-terminal region of rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA inhibition and high affinity binding.

We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.

Alanine↗
PubMed1999

Oxygen sensing and signaling: impact on the regulation of physiologically important genes.

A growing number of physiologically relevant genes are regulated in response to changes in intracellular oxygen tension. It is likely that cells from a wide variety of tissues share a common mechanism of oxygen sensing and signal transduction leading to the activation of the transcription factor hypoxia-inducible factor 1 (HIF-1). Besides hypoxia, transition metals (Co2+, Ni2+ and Mn2+) and iron chelation also promote activation of HIF-1. Induction of HIF-1 by hypoxia is blocked by the heme ligands carbon monoxide and nitric oxide. There is growing, albeit indirect, evidence that the oxygen sensor is a flavoheme protein and that the signal transduction pathway involves changes in the level of intracellular reactive oxygen intermediates. The activation of HIF-1 by hypoxia depends upon signaling-dependent rescue of its alpha-subunit from oxygen-dependent degradation in the proteasome, allowing it to form a heterodimer with HIF-1beta (ARNT), which then translocates to the nucleus and impacts on the transcription of genes whose cis-acting elements contain cognate hypoxia response elements.

Animals↗
PubMed1999

A novel method for increasing the transformation efficiency of Escherichia coli-application forbacterial artificial chromosome library construction.

Bacterial artificial chromosome (BAC) libraries play a pivotal role in genomics studies. A crucial step in BAC library construction is the transformation of Escherichia coli by electroporation. Absolute efficiency (cfu/microgram DNA) is affected by a number of factors including the topological form and treatment of DNA samples. Here we report a simple new protocol using tRNA assisted precipitation that increased transformation efficiency by 70-fold for BAC ligations and up to 400-fold for plasmid ligations. The mechanism may involve altering or stabilizing the topographical form of the DNA molecules.

Chromosomes, Bacterial↗
PubMed1999

Role of ERAB/L-3-hydroxyacyl-coenzyme A dehydrogenase type II activity in Abeta-induced cytotoxicity.

Endoplasmic reticulum-associated amyloid beta-peptide (Abeta)-binding protein (ERAB)/L-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer's disease (AD)-affected brain, binds Abeta, and contributes to Abeta-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a Km of approximately 68 microM and a Vmax of approximately 430 micromol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Abeta-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant beta-amyloid precursor protein (betaAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and betaAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2-C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17beta-estradiol. Addition of micromolar levels of synthetic Abeta(1-40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (Ki approximately 1.6 microM), as well as oxidation of 17beta-estradiol (Ki approximately 3.2 microM) and (-)-2-octanol (Ki approximately 2.6 microM). Because micromolar levels of Abeta were required to inhibit ERAB/HADH II activity, whereas Abeta binding to ERAB/HADH II occurred at much lower concentrations (Km approximately 40-70 nM), the latter more closely simulating Abeta levels within cells, Abeta perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and betaAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or betaAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and betaAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Abeta-rich environment.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1999

Region specific expression of NMDA receptor NR1 subunit mRNA in hypothalamus and pons following chronic morphine treatment.

The NMDA receptor has been implicated in opioid tolerance and physical dependence. Using in situ hybridization techniques, the effects of chronic morphine treatment on the expression of mRNAs encoding the NMDA receptor subunits NRI, NR2A, and NR2B were investigated. A significant increase in the level of the NR1 subunit mRNA was found in the locus coeruleus and the hypothalamic paraventricular nucleus following 3 days of intracerebroventricular (i.c.v.) morphine infusion (26 nmol microl(-1) h(-1)) through osmotic minipumps. No changes were detected in expression of the NRI mRNA in the frontal cortex, caudate-putamen, nucleus accumbens, amygdala, CA1, CA2, and the dentate gyrus of the hippocampus, and in the central grey after morphine treatment. The expression of NR2A and NR2B subunit mRNAs did not change after morphine treatment in any brain region. These results suggest that changes in gene expression of the NRI subunit of the NMDA receptor are involved in the development of morphine tolerance and dependence.

Analgesics, Opioid↗
PubMed1999

Autoradiography of [3H]glutamate binding during pentobarbital tolerance and withdrawal in the rat.

The influence of centrally administered pentobarbital on [3H]glutamate receptor binding in the rat brain was examined. Animals were rendered tolerant by intracerebroventricular (i.c.v.) infusion through osmotic minipumps with pentobarbital (300 microg/10 microl/h, for 6 days), and dependent, by 24 h after withdrawal from pentobarbital. Pentobarbital tolerant rats have significant increases in [3H]glutamate binding in the cortex and hippocampus area. Pentobarbital withdrawal produced increases in glutamate binding in many regions, e.g., the cortex, hippocampus area, thalamus, and cerebellum. These results show that chronic i.c.v. infusion with pentobarbital increases N-methyl-D-aspartate (NMDA) displaceable [3H]glutamate binding, suggesting that an increase in NMDA binding sites may play an important role in the development of tolerance to and withdrawal from pentobarbital.

Animals↗
PubMed1999

Rho-kinase (ROK) promotes CD44v(3,8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells.

Metastatic breast tumor Met-1 cells express CD44v(3,8-10), a major adhesion receptor that binds extracellular matrix components at its extracellular domain and interacts with the cytoskeletal protein, ankyrin, at its cytoplasmic domain. In this study, we have determined that CD44v(3,8-10) and RhoA GTPases are physically associated in vivo, and that CD44v(3,8-10)-bound RhoA displays GTPase activity, which can be inhibited by botulinum toxin C3-mediated ADP-ribosylation. In addition, we have identified a 160 kDa Rho-Kinase (ROK) as one of the downstream targets for CD44v(3,8-10)-bound RhoA GTPase. Specifically, RhoA (complexed with CD44v(3, 8-10)) stimulates ROK-mediated phosphorylation of certain cellular proteins including the cytoplasmic domain of CD44v(3,8-10). Most importantly, phosphorylation of CD44v(3,8-10) by ROK enhances its interaction with the cytoskeletal protein, ankyrin. We have also constructed two ROK cDNA constructs that encode for proteins consisting of 537 amino acids [designated as the constitutively active form of ROK containing the catalytic domain (CAT, also the kinase domain)], and 173 amino acids [designated as the dominant-negative form of ROK containing the Rho-binding domain (RB)]. Microinjection of the ROK's CAT domain into Met-1 cells promotes CD44-ankyrin associated membrane ruffling and projections. This membrane motility can be blocked by CD44 antibodies and cytochalasin D (a microfilament inhibitor). Furthermore, overexpression of a dominant-negative form of ROK by transfection of Met-1 cells with ROK's Rho-binding (RB) domain cDNA effectively inhibits CD44-ankyrin-mediated metastatic behavior (e.g., membrane motility and tumor cell migration). These findings support the hypothesis that ROK plays a pivotal role in CD44v(3,8-10)-ankyrin interaction and RhoA-mediated oncogenic signaling required for membrane-cytoskeleton function and metastatic tumor cell migration.

Amino Acid Sequence↗
PubMed1999

Alterations in rabbit kidney protein expression following lead exposure as analyzed by two-dimensional gel electrophoresis.

It was recently reported that low blood lead levels impaired kidney function in men. To develop a set of molecular markers of renal lead exposure and effect, we investigated changes in renal protein expression while approximating occupational lead exposure at subchronic, low blood levels. Lead was administered to male Dutch Belted rabbits as a lead acetate solution adjusted weekly to achieve and maintain the target blood lead levels of 0, 20, 40, and 80 microg/dL for 15 weeks. Lead exposure did not affect kidney or body weights. The effect of increasing blood lead on protein expression was evaluated in rabbit kidney by large-scale two-dimensional electrophoresis (2-DE). Significant quantitative changes (p < 0.05) occurred in a dose-related manner in 12 proteins at 20 microg/dL exposure, 25 at 40 microg/dL, and 102 at 80 microg/dL. At a higher level of significance (p < 0.001), 40 microg/dL blood lead resulted in one protein alteration and 80 microg/dL affected 14 proteins. A set of quantitatively altered charge variants was tentatively identified as glutathione-S-transferase (GST), based on similar observations in rodents subjected to short-term, very high lead exposure. The significance of the protein alterations observed as markers of toxicity awaits their conclusive identification. Investigation of the kidney 2-DE profile in lead-exposed rabbit may be useful in understanding the mechanism of lead nephrotoxicity in humans.

Animals↗
PubMed1999

Tumor necrosis factor increases mitochondrial oxidant production and induces expression of uncoupling protein-2 in the regenerating mice [correction of rat] liver.

The growth-stimulatory actions of tumor necrosis factor alpha (TNF-alpha) after partial hepatectomy (PH) are difficult to reconcile with its well-established role in the genesis of liver injury. The lethal actions of TNF are thought to involve the induction of oxidant production by mitochondria. It is not known if TNF initiates mitochondrial oxidant production after PH. Furthermore, if this potentially toxic response follows PH, it is not clear how hepatocytes defend themselves sufficiently so that replication, rather than death, occurs. These studies test the hypothesis that TNF does increase mitochondrial oxidant production after PH but that these oxidants primarily promote the induction of antioxidant defenses in regenerating hepatocytes. Consistent with this concept, H2O2 production by liver mitochondria increases from 5 minutes to 3 hours after PH, beginning before the transient inductions of hepatic NF kB activity (which peaks at 30 minutes post-PH) and uncoupling protein-2 (UCP-2) (which begins around 30 minutes and peaks from 6-24 hours post-PH). Pretreatment with neutralizing anti-TNF antibodies, which inhibits hepatocyte DNA synthesis after PH, also reduces post-PH hepatic mitochondrial oxidant production by 80% and inhibits NF kappaB activation and UCP-2 induction by 50% and 80%, respectively. In contrast, pretreatment with D609, an agent that inhibits phosphatidylcholine-specific phospholipase C, neither inhibits regenerative induction of mitochondrial oxidant production, UCP-2 expression, nor hepatocyte DNA synthesis, although it inhibits NF kappaB activation by 50%. Given published evidence that NF kappaB is antiapoptotic and that UCP-2 may decrease mitochondrial oxidant production in some cells, these results suggest that TNF-dependent increases in oxidant production by liver mitochondria promote the induction of antioxidant defenses in the regenerating liver.

Animals↗
PubMed1999

Different role of antioxidants in endotoxin-induced late myocardial protection.

Previous studies have proposed that endogenous antioxidants play a protective role against cardiac ischemia-reperfusion injury in endotoxin pretreatment. However, the mechanism underlying this effect remains elusive. We therefore evaluated the role of endogenous antioxidants in delayed myocardial protection after different doses of endotoxin administration using cultured rat neonatal cardiomyocytes. Myocytes were treated with normal saline (control) or lipopolysaccharide (Escherichia coli, serotype O111) at doses of 40 and 80 microg/ml (ET40 and ET80). Also, antisense oligodeoxyribonucleotide (1.5 micromol/L) to manganese superoxide dismutase (Mn-SOD) and 3-amino-1,2,4-triazole (25 mg/ml) were added along with a 40 or 80 microg/ml endotoxin pretreatment in the IET40 and IET80 groups. Twenty-four hours later, Cells were subjected to hypoxia (pO2 < 1 kPa, 3 h) and reoxygenation (pO2: 19 kPa, 1 h). Compared with controls, cell viability enhanced significantly (65.3 +/- 5.9, 63.8 +/- 4.6, and 69.7 +/- 5.2% vs 47.2 +/- 4.3%, P < 0.05) and creatine kinase release decreased (7.34 +/- 1.76, 7.11 +/- 1.49, and 6.27 +/- 1.24 U/mg protein vs 11.23 +/- 2.49 U/mg protein, P < 0. 05) in ET40, IET40, and ET80 groups following reoxygenation. No statistically significant difference was found between the control and the IET80 groups. Furthermore, the levels of Mn-SOD (1.12 +/- 0. 31 vs 0.75 +/- 0.15 U/mg. protein, P < 0.05) and catalase activity (1265 +/- 109 vs 996 +/- 85 U/mg. protein, P < 0.05) were higher only in the ET80 group. The results suggest that at a dose of 40 microg/ml, cells were protected by mechanisms other than the augmentation of endogenous antioxidant activity which were more evident at a dose of 80 microg/ml. It seems that different doses of endotoxin pretreatment may induce delayed myocardial protection through various mechanisms.

Animals↗