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L Zhou

Publications and source records attributed to L Zhou.

At least 235 records · Page 13Linked to original sources
PubMed1999

Red nucleus neurons of Bcl-2 over-expressing mice are protected from cell death induced by axotomy.

The Bcl-2 proto-oncogene regulates apoptosis and prevents cell death. We studied the effect of Bcl-2 gene over-expression on the survival of axotomized red nucleus (RN) neurons after unilateral hemisection at cervical segment 4/5 (C4/5) in mice. Seventy-five percent of RN neurons survived in Bcl-2 over-expressing mice 1 or 2 months after surgery compared with only 55% of RN neurons in wild-type mice. However, Bcl-2 gene over-expression does not prevent lesion-induced shrinkage of RN neurons.

Animals↗
PubMed1999

Post-transcriptional regulation of the arginine transporter Cat-1 by amino acid availability.

The regulation of the high affinity cationic amino acid transporter (Cat-1) by amino acid availability has been studied. In C6 glioma and NRK kidney cells, cat-1 mRNA levels increased 3.8-18-fold following 2 h of amino acid starvation. The transcription rate of the cat-1 gene remained unchanged during amino acid starvation, suggesting a post-transcriptional mechanism of regulation. This mechanism was investigated by expressing a cat-1 mRNA from a tetracycline-regulated promoter. The cat-1 mRNA contained 1.9 kilobase pairs (kb) of coding sequence, 4.5 kb of 3'-untranslated region, and 80 base pairs of 5'-untranslated region. The full-length (7.9 kb) mRNA increased 5-fold in amino acid-depleted cells. However, a 3.4-kb species that results from the usage of an alternative polyadenylation site was not induced, suggesting that the cat-1 mRNA was stabilized by cis-acting RNA sequences within the 3'-UTR. Transcription and protein synthesis were required for the increase in full-length cat-1 mRNA level. Because omission of amino acids from the cell culture medium leads to a substantial decrease in protein synthesis, the translation of the increased cat-1 mRNA was assessed in amino acid-depleted cells. Western blot analysis demonstrated that cat-1 mRNA and protein levels changed in parallel. The increase in protein level was significantly lower than the increase in mRNA level, supporting the conclusion that cat-1 mRNA is inefficiently translated when the supply of amino acids is limited, relative to amino acid-fed cells. Finally, y(+)-mediated transport of arginine in amino acid-fed and -starved cells paralleled Cat-1 protein levels. We conclude that the cat-1 gene is subject to adaptive regulation by amino acid availability. Amino acid depletion initiates molecular events that lead to increased cat-1 mRNA stability. This causes an increase in Cat-1 protein, and y(+) transport once amino acids become available.

3' Untranslated Regions↗
PubMed1999

The quaking I-5 protein (QKI-5) has a novel nuclear localization signal and shuttles between the nucleus and the cytoplasm.

The mouse quaking (qk) gene is essential in both myelination and early embryogenesis. Its product, QKI, is an RNA-binding protein belonging to a growing protein family called STAR (signal transduction and activator of RNA). All members have an approximately 200-amino acid STAR domain, which contains a single extended heteronuclear ribonucleoprotein K homologue domain flanked by two domains called QUA1 and QUA2. We found that QKI isoforms could associate with each other, while one of the lethal mutations qkI(kt4) with a single amino acid change in QUA1 domain, leads to a loss of QKI self-interaction. This suggests that the QUA1 domain is responsible for QKI dimerization. Three QKI isoforms have different carboxyl termini and different subcellular localization. Here, using GFP fusion protein, we identified a 7-amino acid novel nuclear localization sequence in the carboxyl terminus of QKI-5, which is conserved in a subclass of STAR proteins containing SAM68 and ETLE/T-STAR. Thus, we name this motif STAR-NLS. In addition, the effects of active transcription, RNA-binding and self-interaction on QKI-5 localization were analyzed. Furthermore, using an interspecies heterokaryon assay, we found that QKI-5, but not another STAR protein ETLE, shuttles between the nucleus and the cytoplasm, which suggests that QKI-5 functions in both cell compartments.

3T3 Cells↗
PubMed1999

Drosophila presenilin is required for neuronal differentiation and affects notch subcellular localization and signaling.

Presenilins are a highly conserved family of proteins first identified as causative genes in early onset familial Alzheimer's disease. Recent studies have suggested a role for presenilins in the Notch-signaling pathway, but their specific function within this pathway remains unclear. Here, we have characterized the Drosophila presenilin gene and protein and studied their interaction with Notch in both mutants and transgenics. We find that the Drosophila presenilin protein is proteolytically cleaved and broadly expressed during development with the highest levels in neurons within the larval CNS. We also show that mutations in Drosophila presenilin (Dps) genetically interact with Notch and result in an early pupal-lethal phenotype characterized by defects in eye and wing development and incomplete neuronal differentiation within the larval CNS. Moreover, we find that processing of Notch in the Golgi by the furin protease is unaffected in Dps mutants and that Notch is present and may even accumulate on the plasma membrane of neuroblasts in the larval CNS of Dps mutants. In contrast, overexpression of Dps in transgenics causes Notch to accumulate in the cytoplasm. Taken together, these results indicate that Drosophila presenilin is required for proper neuronal differentiation and may regulate the subcellular localization of Notch proteins within cells, necessary for their accumulation and subsequent signaling capabilities.

Animals↗
PubMed1999

Mutagenesis of the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine DNA adduct in mammalian cells. Sequence context effects.

Site-specifically modified oligodeoxynucleotides were used to investigate the mutagenic properties of a major cooked food mutagen-derived DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (dG-C8-PhIP). dG-C8-PhIP-modified oligodeoxynucleotides were prepared by reacting an oligodeoxynucleotide containing a single dG (5'-TCCTCCTXGCCTCTC, where X = C, A, G, or T) with N-acetoxy-PhIP. The unmodified and dG-C8-PhIP-modified oligomers were inserted into single-stranded phagemid vectors. These single-stranded vectors were transfected into simian kidney (COS-7) cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. When dC was at the 5'-flanking position to dG-C8-PhIP, preferential incorporation of dCMP, the correct base, was observed opposite the dG-C8-PhIP. Targeted G --> T transversions were detected, along with lesser amounts of G --> A transitions and G --> C transversions. No mutations were detected for the unmodified vector. The influence of sequence context on the dG-C8-PhIP mutation frequency and spectrum was also explored. When the dC 5'-flanking base was replaced by dT, dA, or dG, the mutational spectra were similar to that observed with dC-flanking base. Higher mutational frequencies (28-30%) were observed when dC or dG was 5' to dG-C8-PhIP. A lower mutational frequency (13%) was observed when dA was at the 5' to the lesion. Single-base deletions were detected only when dG or dT flanked the adduct. We conclude that dG-C8-PhIP is mutagenic, generating primarily G --> T transversions in mammalian cells. The mutational frequency and specificity of dG-C8-PhIP vary depending on the neighboring sequence context.

Animals↗
PubMed1999

The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance.

The Slit gene encodes a secreted molecule essential for neural development in Drosophila embryos. Here we report the identification of three Slit homologues in the mouse. We demonstrate that the mouse SLIT1 protein can bind ROBO1, a transmembrane receptor implicated in axon guidance. Both whole-mount and section in situ hybridization studies reveal unique and complementary patterns of expression of the three mouse Slit genes and of Robo1, both within the central nervous system and in other developing tissues. The complementary expression patterns of Slit and Robo1 and their in vitro interaction suggest a ligand-receptor relationship. The expression of all three Slit genes in the floor plate suggests that they are likely to share the same functional properties with their Drosophila homologue in midline neural development and axon guidance. The complementary expression of Slit and Robo1 in different subdivisions of the somites suggests their possible function in axon pathfinding and neural crest cell migration. The unique expression pattern in limb and other organs indicates additional potential functions of the Slit gene family.

Amino Acid Sequence↗
PubMed1999

PPARgamma activation induces the expression of the adipocyte fatty acid binding protein gene in human monocytes.

The peroxisome-proliferator activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily of ligand activated transcription factors, plays a key role in the anti-diabetic actions of the thiazolidinediones (TZDs). PPARgamma induces the expression of many genes involved in lipid anabolism, including the adipocyte fatty acid binding protein (aP2), and is a key regulator of adipocyte differentiation. PPARgamma is also expressed in hematopoietic cells and is up-regulated in activated monocytes/macrophages. Activation of PPARgamma may play a role in the induction of differentiation of macrophages to foam cells that are associated with atherosclerotic lesions. We report that both natural and synthetic PPARgamma agonists induce time- and dose-dependent increases in aP2 mRNA in both primary human monocytes and the monocytic cell line, THP-1. These data suggest that PPARgamma activation may play a role in monocyte differentiation and function analogous to its well-characterized role in adipocytes.

Adipocytes↗
PubMed1999

Determinants of excitability at transition zones in Kv1.1-deficient myelinated nerves.

This study examines the role of K channel segregation and fiber geometry at transition zones of mammalian nerve terminals in the peripheral nervous system. Mutant mice that are deficient in Kv1.1, a fast Shaker K channel normally localized beneath the myelin sheath, display three types of cooling-induced abnormal hyperexcitability localized to regions before the transition zones of myelinated nerves. The first type is stimulus-evoked nerve backfiring that is absent at birth, peaks at postnatal day 17 (P17), and subsides in adults. The second type is spontaneous activity that has a more delayed onset, peaks at P30, and also disappears in older mice (>P60). TEA greatly amplifies this spontaneous activity with an effective dosage of approximately 0.7 mM, and can induce its reappearance in older mutant mice (>P100). These first two types of hyperexcitability occur only in homozygous mutants that are completely devoid of Kv1.1. The third type occurs in heterozygotes and represents a synergism between a TEA-sensitive channel and Kv1.1. Heterozygotes exposed to TEA display no overt phenotype until a single stimulation is given, which is then followed by an indefinite phase of repetitive discharge. Computer modeling suggests that the excitability of the transition zone near the nerve terminal has at least two major determinants: the preterminal internodal shortening and axonal slow K channels. We suggest that variations in fiber geometry create sites of inherent instability that is normally stabilized by a synergism between myelin-concealed Kv1.1 and a slow, TEA-sensitive K channel.

Animals↗
PubMed1999

Probing the conformational heterogeneity of the acetylaminofluorene-modified 2'-deoxyguanosine and DNA by 19F NMR spectroscopy.

19F NMR spectroscopy was used to probe the conformation of a DNA adduct derived from the carcinogen 7-fluoro-N-acetyl-2-aminofluorene (FAAF) in three structural contexts: as a monomer and incorporated into single- and double-stranded DNA. The 19F NMR spectrum of dG-C8-FAAF [N-(deoxyguanosin-8-yl)-N-acetyl-7-fluoro-2-aminofluorene] in methanol at -30 degrees C exhibited four interconvertible signals in a 11:52:26:11 ratio. Dynamic NMR analysis indicated that the four torsional isomers arise from restricted rotation about the amide (gamma) (14.4 kcal/mol) and the guanyl-nitrogen (alpha) bonds. The conformational heterogeneity persisted in a single strand FAAF-12-mer, d(CTTCTTG[FAAF]ACCTC), whose 19F NMR spectrum at 22 degrees C and pH 7.0 gave only two signals in a 40:60 ratio, instead of four. The two 19F signals followed a two-site exchange with the rotation barrier of 14.7 kcal/mol about the amide (gamma') bond. A similar conformational theme was observed in the FAAF-12-mer duplex, d(CTTCTTG[FAAF]ACCTC).d(GAGGTCAAGAAG), which revealed two 19F resonances in a 41:59 ratio at 22 degrees C and pH 7.0. According to solvent-induced isotope and magnetic anisotropy effects, the two duplex conformers adopt exclusively a base displacement structure, being different only in their relative acetyl group orientations, cis (gamma' approximately 180 degrees) or trans (gamma' approximately 0 degrees ). Dynamic NMR data indicated that the two conformers do not exchange over a wide range of temperatures. This contrasts with the nonacetylated counterpart, which exhibits an equilibrium between the "B-type" and "stacked" conformers [Zhou, L., et al. (1997) J. Am. Chem. Soc. 119, 5384-5389]. The exclusive stacked nature of the AAF adducts may provide insight into why AAF adducts are more mutagenic and prone to repair than the nonacetylated AF adducts.

2-Acetylaminofluorene↗
PubMed1999

Functional analysis of the two zinc fingers of SRE, a GATA-type factor that negatively regulates siderophore synthesis in Neurospora crassa.

Multiple GATA factors, zinc finger DNA binding proteins that recognize consensus GATA elements, exist in Neurospora crassa. One of them, SRE, is involved in controlling the iron metabolic pathway of N. crassa. In N. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. The siderophore synthesis pathway is negatively regulated by SRE; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. In the research presented here, the negative function of SRE was further confirmed by a heterokaryon test and by gene complementation. SRE was expressed as a GST fusion protein. In vitro EMSA revealed that SRE binds specifically to DNA molecules containing GATA sequence elements. Autoregulation of sre gene expression appears possible because the sre gene promoter itself contains GATA sequences. Mutations were introduced into sre that lead to amino acid substitutions in each of the zinc fingers that will disrupt their function. In vitro EMSA revealed that both N-terminal and C-terminal zinc fingers of SRE are involved in DNA binding. This feature is different from that found with the vertebrate two zinc finger GATA factors. Invivo tests, accomplished by transforming the mutant sre genes into sre rip mutant, showed that SRE with mutations in either or both zinc fingers still maintained its function under low-iron conditions. In contrast, these mutant SRE proteins fail to function under high-iron conditions. Our results predict the presence of other positive or negative regulators of the siderophore synthetic pathway.

Amino Acid Substitution↗
PubMed1999

Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons.

The olfactory bulb plays a central role in olfactory information processing through its connections with both peripheral and cortical structures. Axons projecting from the olfactory bulb to the telencephalon are guided by a repulsive activity in the septum. The molecular nature of the repellent is not known. We report here the isolation of vertebrate homologs of the Drosophila slit gene and show that Slit protein binds to the transmembrane protein Roundabout (Robo). Slit is expressed in the septum whereas Robo is expressed in the olfactory bulb. Functionally, Slit acts as a chemorepellent for olfactory bulb axons. These results establish a ligand-receptor relationship between two molecules important for neural development, suggest a role for Slit in olfactory bulb axon guidance, and reveal the existence of a new family of axon guidance molecules.

Animals↗
PubMed1999

Dissection of the molecular basis of pp60(v-src) induced gating of connexin 43 gap junction channels.

Suppression of gap-junctional communication by various protein kinases, growth factors, and oncogenes frequently correlates with enhanced mitogenesis. The oncogene v-src appears to cause acute closure of gap junction channels. Tyr265 in the COOH-terminal tail of connexin 43 (Cx43) has been implicated as a potential target of v-src, although v-src action has also been associated with changes in serine phosphorylation. We have investigated the mechanism of this acute regulation through mutagenesis of Cx43 expressed in Xenopus laevis oocyte pairs. Truncations of the COOH-terminal domain led to an almost complete loss of response of Cx43 to v-src, but this was restored by coexpression of the independent COOH-terminal polypeptide. This suggests a ball and chain gating mechanism, similar to the mechanism proposed for pH gating of Cx43, and K+ channel inactivation. Surprisingly, we found that v-src mediated gating of Cx43 did not require the tyrosine site, but did seem to depend on the presence of two potential SH3 binding domains and the mitogen-activated protein (MAP) kinase phosphorylation sites within them. Further point mutagenesis and pharmacological studies in normal rat kidney (NRK) cells implicated MAP kinase in the gating response to v-src, while the stable binding of v-src to Cx43 (in part mediated by SH3 domains) did not correlate with its ability to mediate channel closure. This suggests a common link between closure of gap junctions by v-src and other mitogens, such as EGF and lysophosphatidic acid (LPA).

Animals↗
PubMed1999

Fasting increases tissue uptake and interconversion of plasma unesterified linoleic acid in guinea pigs.

A large part of the arachidonic acid (20:4 n-6) pools in some extrahepatic tissues can be formed by local interconversion of linoleic acid (18:2 n-6) taken up as free fatty acid (FFA) from blood in both rats and guinea pigs. This study investigates the rate of uptake and interconversion of unesterified 14C-18:2 by different tissues in fasted guinea pigs. The initial half-life of 14C-18:2 in plasma was 5.8 s. The average concentration of plasma FFA was 551.3 nmol ml-1 and of plasma FFA-18:2 was 67.3 nmol ml-1. The total amount of 20:4 formed in the liver was 1.8 +/- 0.3 nmol min-1, which was lower than that in the gastrointestinal tract (3.1 nmol min-1), bone marrow (6.0 nmol min-1) and lung (2.1 nmol min-1). Due to the fast turnover and higher concentration of plasma FFA-18:2 in the fasting state, the retained 18:2 in tissue lipids was 5.8-25.6-fold higher than that in fed guinea pigs [L. Zhou et al. Biochim. Biophys. Acta 1349 (1997) 197-210]. The total delta 6-desaturase products both in liver and in extrahepatic tissues were also increased, 3.8-fold in liver, 7.2-fold in upper small intestine, 6.0-fold in colon, and 6.5-fold in bone marrow. The increased rate of tissue uptake of FFA during fasting is thus linked to an increased local interconversion of plasma FFA-18:2, which is an important source of 20:4 in some extrahepatic tissue in guinea pigs.

Animals↗
PubMed1999

Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms.

Methyl-n-amylnitrosamine (MNAN) induces esophageal cancer in rats, probably involving activation by cytochromes P450. We studied the metabolic depentylation of MNAN. [3H-4,5-pentyl]MNAN and [3H-2,3-pentyl]-MNAN were synthesized, purified, and incubated with rat esophageal microsomes (REM) or rat liver microsomes (RLM) to give [3H]pentaldehyde (depentylation), an indicator of MNAN activation. [3H]Pentaldehyde was determined by high-performance liquid chromatography of its 2,4-dinitrophenylhydrazone. Adding 5 mM semicarbazide to incubations increased the observed depentylation (except that due to CYP2E1) by >60%. MNAN depentylation by REM and uninduced and induced RLM showed Km values of 64, 610, and 170-330 microM, respectively (Vmax: 20, 220, and 160-1270 pmol/mg protein/min, respectively). The depentylation of 100 microM MNAN by REM was inhibited 98% by CO and 65% by coumarin preincubated for 15 min with REM (Ki, 120 microM) but was unaffected by antibodies inhibitory to various P450s. MNAN inhibited coumarin 7-hydroxylation by RLM and CYP2A6 (Ki, 3000 and 320 microM, respectively). REM showed slight coumarin 7-hydroxylase activity. MNAN depentylation by RLM was 41% inhibited by an antibody to CYP2C11. Km for rat CYP2E1, human CYP2E1, and human CYP2A6 was 210, 115, and 17 microM, respectively (Vmax: 900, 570, and 120 pmol/nmol P450/min, respectively). We conclude that MNAN activation by REM is probably due to a P450 related to CYP2A3, a rodent nasal P450.

Animals↗
PubMed1999

Oxidative stress affects cytoskeletal structure and cell-matrix interactions in cells from an ocular tissue: the trabecular meshwork.

The trabecular meshwork (TM) is a specialized eye tissue that regulates the aqueous humor outflow and controls intraocular pressure. Cells in this tissue are essential for maintenance of the outflow system. Disturbance of the TM cell status by insults such as oxidative stress may lead to elevation of the intraocular pressure and development of glaucoma. In the present study, we investigated the effect of oxidative stress on the adhesion of human TM cells to extracellular matrix (ECM) proteins. Treatment with 1 mM of H2O2 for 10 or 30 min did not affect cell viability, whereas the adhesion of TM cells to fibronectin, laminin, and collagen types I and IV was significantly reduced. Phalloidin and immunostaining also revealed reorganization of actin and vimentin structures. The level of integrins alpha5beta1, alphavbeta3, and beta1 was not altered, although the distribution of paxillin and focal adhesion kinase in focal contacts was reduced. Concomitantly, the level of transcription factor NF-kappaB was enhanced by the H2O2 treatment. Nuclear extracts of the treated cells also contained a heightened NF-kappaB binding activity. These changes persisted for up to 6 h after the H2O2 treatment but were partially recovered by 24 h. We concluded that under sublethal oxidative stress conditions, the TM cell adhesion to the ECM was impaired. The short-term loss of cell-matrix adhesiveness may be related to the rearrangement of cytoskeletal structures. Extensive and repeated oxidative stress in vivo may result in reduced TM cell adhesion, leading to cell loss, compromised TM integrity, and pathologic consequences.

Actins↗
PubMed1999

Molecule imprinting chiral stationary phase.

Molecule imprinting polymers (MIPs) with high chiral selectivity for N(alpha)-protected amino acids were synthesized in polar solvent using acrylamide (AM) or combined functional monomer methacrylic acid (MAA) + vinyl pyridine (VP). Factors that influence the chiral selectivity of MIPs and mechanisms of the chiral recognition process were investigated. It was found that the rigid structure and the polar functional group of the print molecule were very important for the chiral selectivity of MIPs. For MIPs made using a combined functional monomer, ionic and hydrophobic interaction may be responsible for the chiral recognition process in aqueous media. The number of binding sites on MIPs and dissociation constants for the complex of enantiomers and MIPs were determined by frontal chromatography analysis.

Acrylamide↗
PubMed1999

Inhibition of the CD40 pathway of monocyte activation by triazolopyrimidine.

Blockade of the CD40/CD40L pathway of monocyte/macrophage activation represents a promising strategy for the treatment of several inflammatory disorders. So far, most pharmacological agents developed for that purpose target CD40L (CD154) expressed on activated T cells. Herein, we provide evidence that triazolopyrimidine, a chemical compound primarily developed for the prevention of arterial thrombosis, strongly inhibits the response of human monocytes to CD40 ligation. First, we found that triazolopyrimidine inhibits the production of IL-12, TNF-alpha, and IL-6 by monocytes activated by coculture with fibroblasts transfected with the CD40L gene as well as the induction of procoagulant activity at their membrane. This was related to a decreased expression of CD40 on monocytes exposed to triazolopyrimidine, an effect that was already apparent at the mRNA level. Furthermore, the addition of triazolopyrimidine to monocytes cultured with IL-4 and GM-CSF prevented their differentiation into fully competent dendritic cells (DC) as DC differentiated in the presence of triazolopyrimidine expressed less CD40 at their surface and were profoundly deficient in the production of IL-12 upon exposure to CD40L transfectants. We conclude that triazolopyrimidine strongly inhibits the CD40 pathway of monocyte activation at least in part by downregulating the gene expression of CD40.

Blood Coagulation Factors↗