Search PubMed⌕ Search

Biomedical subjects

L Wang

Publications and source records attributed to L Wang.

At least 1,207 records · Page 67Linked to original sources
PubMed1998

Antisense to MDR1 mRNA reduces P-glycoprotein expression, swelling-activated C1- current and volume regulation in bovine ciliary epithelial cells.

Native ciliary epithelial cells from the ciliary epithelium of the eye exhibit anti-P-glycoprotein (P-gp) immunofluorescence. We have used an antisense 'knock-down' approach to investigate the relationship between P-gp and the volume-activated chloride current (IC1,swell) and its role in volume regulation. An antisense oligonucleotide to the human multidrug resistance (MDR1) gene, taken up by the cells in a dose-dependent manner, reduced P-gp immunofluorescence, inhibited IC1,swell and significantly increased the latency of activation of IC1,swell. Increasing the hypotonic stress did not result in an increased activation of ICl,swell. MDR1 antisense 'knock-down' also reduced the ability of the cells to volume regulate following a hypotonic challenge. These cells are known to express at least two volume-activated chloride channels, and the data suggest that P-gp is involved in the activation pathway of a subset of channels that contribute to whole-cell IC1,swell and participate in volume regulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Werner helicase is localized to transcriptionally active nucleoli of cycling cells.

Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS), a "caricature of aging." We have localized the Werner protein (WRNp) to the nucleoli of replicating mammalian cells, where its appearance is associated with transcriptional activity. A dramatic reduction of the nucleolar signal and of [3H]uridine incorporation occurred when cultures were made quiescent or were exposed to 4-nitroquinoline-1-oxide (4NQO), to which WS cells are particularly susceptible. Total cellular levels of WRNp, however, did not change, and virtually all WRNp was in the nuclear fractions, consistent with translocation to the nucleoplasm and/or masking of the epitopes. The 4NQO-induced altered state of WRNp was prevented by Na3VO4, but not by okadaic acid, suggesting that WRNp localization/function is partially regulated by kinases/phosphatases for Tyr substrates on WRNp or interacting proteins. The repression of rDNA transcription by 4NQO was not reversed by Na3VO4. We suggest that physiological states and genotoxic agents modulate the interaction of WRNp with rDNA, consistent with a role of WRNp in rDNA transcription.

4-Nitroquinoline-1-oxide↗
PubMed1998

Structure and function of the human Werner syndrome gene promoter: evidence for transcriptional modulation.

The Werner syndrome (WS) is an autosomal recessive segmental progeroid syndrome caused by mutations in a novel member ( WRN ) of the RecQ family of helicases. Somatic WS cells are hypermutable and have elongated S phases, suggesting possible defects in DNA replication and/or repair. As an initial approach to the investigation of how this locus might be responsive to DNA damage, we determined the structure of the human WRN promoter. The WRN promoter region has two transcription initiation sites and exhibits several features characteristic of so-called constitutive promoters, including the absence of TATA and CAAT boxes. A luciferase reporter assay revealed that the upstream promoter was used 2-10-fold less frequently than the downstream promoter, the variation being a function of cell type. The activity of the WRN promoter was dramatically reduced in cells from WS patients. The reduction of activity was not seen in three other promoters tested, including one TATA-less promoter and one TATA-containing promoter. This is consistent with the presence of a positive regulatory mechanism of WRN expression.

Base Sequence↗
PubMed1998

Expression of the O antigen gene cluster is regulated by RfaH through the JUMPstart sequence.

O antigen genes are clustered, with a JUMPstart sequence located upstream. JUMPstart is a 39-bp sequence, present upstream of many polysaccharide gene clusters and also upstream of haemolysin and F factor gene clusters. RfaH is known to regulate the expression of E. coli group II capsule, haemolysin, F factor and the outer core of lipopolysaccharide all of which have the JUMPstart sequence, and has been shown to function as a transcriptional antiterminator in some cases. Using lacZ fusions to genes in the O antigen gene cluster of Salmonella enterica serovar Typhimurium, we found that RfaH also regulates the expression of O antigen. We showed that RfaH enhances expression of the 18-kb O antigen gene cluster, with promoter-distal genes affected more dramatically. We also showed that the JUMPstart sequence was required for RfaH function.

Antigens, Bacterial↗
PubMed1998

Preclinical characterization of an anti-tat ribozyme for therapeutic application.

A hammerhead ribozyme retroviral construct, denoted RRz2, targeting the coding region of the human immunodeficiency virus type 1 (HIV-1) tat gene, has shown itself to be effective in a range of test systems. Inhibition of the replication of HIV-1 IIIB and primary drug-resistant strains in pooled transduced CEMT4 cells was consistently found to be more than 80% compared with the control-vector transduced cells, whereas a mutant RRz2 gave approximately 45% inhibition. A multiple HIV-1 passage assay showed the absence of emergence of mutations within the specific viral RNA ribozyme target sequences. This lack of generation of ribozyme "escape mutants" occurred despite the almost complete disappearance of a HIV-1 quasi-species in the testing virus. When RRz2 was tested in peripheral blood lymphocytes (PBLs) from HIV-1-infected patients, paired analysis showed that cell viability in the ribozyme-transduced HIV-1-infected PBLs was significantly higher than that in the vector-transduced cells. This difference in viability (vector versus RRz2) was not observed in PBLs from non-HIV-1-infected donors. Taken together, these results indicate that the transfer of an anti-HIV-1 ribozyme gene into human T lymphocytes could have major impact on viral replication and T cell viability in the HIV-1-infected individual.

Base Sequence↗
PubMed1998

Intracellular calcium distribution in apoptosis of HL-60 cells induced by harringtonine: intranuclear accumulation and regionalization.

Harringtonine (HT), an anticancer drug with high chemotherapeutic efficiency to human chronic granulocytic/myelomonocytic leukemia, has been reported to rapidly induce apoptosis in HL-60 cells in a wide scope/range of dosage by investigators from our lab and others. In the present studies, by using video enhancement contrast (VEC) microscopy, we dynamically analyzed changes in intracellular calcium distribution in a single HL-60 cell over the period from the initiation of apoptosis to the obvious appearance of chromatin condensation. The results from this paper demonstrated the striking distinction of intracellular calcium distribution at different time points after treatment with HT. Before treatment in normal HL-60 cells the highest [Ca2+]i accumulation was observed in the peri-nuclear area and the lowest was observed in the nucleus; after treatment with 1 microg/ml HT for 30 min intracellular calcium diffused all over the cell compartments, while intranuclear calcium increased comparatively and significantly. The phenomenon of intranuclear calcium accumulation was further confirmed by using laser scanning confocal microscopy (LSCM). In addition, co-localization of the highest calcium region with condensed chromatin in apoptotic HL-60 cells was also observed by LSCM. Our results suggest that two sequential alterations of intracellular calcium distribution occurred in apoptotic HL-60 cells induced by HT, i.e. (a) accumulation of calcium in the nucleus and (b) regionalization in a specific nuclear region.

Antineoplastic Agents↗
PubMed1998

Complementation studies with co-expressed fragments of human red cell band 3 (AE1): the assembly of the anion-transport domain in xenopus oocytes and a cell-free translation system.

We examined the assembly of the membrane domain of the human red cell anion transporter (band 3; AE1) by co-expression of recombinant N- and C-terminal fragments in Xenopus oocytes and in cell-free translation with canine pancreatic microsomes. Co-immunoprecipitation was performed in non-denaturing detergent solutions using antibodies directed against the N- and C-termini of the membrane domain. Eleven of the twelve fragments were expressed stably in oocytes in the presence or absence of their respective partners. However, the fragment containing from putative span nine to the C-terminus could be detected in oocytes only when co-expressed with its complementary partner containing the first eight spans. Co-expression of pairs of fragments divided in the first, second, third and fourth exofacial loops and in the fourth cytoplasmic loop resulted in a concentration-dependent association, but a pair of fragments divided in the sixth cytoplasmic loop did not co-immunoprecipitate. When two complementary fragments were translated separately in the cell-free system and the purified microsomes were then mixed, co-immunoprecipitation was observed only if the membranes were first fused using polyethylene glycol. This shows that co-immunoprecipitation results from specific interactions within the membrane and is not an artefact of detergent solubilization or immunoprecipitation. We demonstrate that band 3 assembly can occur within the membrane after translation, insertion and initial folding of the individual fragments have been completed. We conclude that most band 3 fragments contain the necessary information to fold in the membrane and adopt a structure that is sufficiently similar to the native protein that it permits correct assembly with its complementary partner.

Animals↗
PubMed1998

Oligoribonucleotide circularization by 'template-mediated' ligation with T4 RNA ligase: synthesis of circular hammerhead ribozymes.

Circular hammerhead ribozymes were synthesized from linear oligoribonucleotides using T4 RNA ligase. Some of the precursors could not be efficiently circularized under standard conditions. For these molecules, the use of a DNA template allowed their efficient circularization. The template was designed to prevent the precursor from folding into an unsuitable structure. The template allowed circular ribozymes as small as 15 nucleotides in length to be efficiently synthesized at concentrations as high as 50 microM in the ligation reaction. The circular products retained their biological activity.

Bacteriophage T4↗
PubMed1998

Crystal structure of apo-cellular retinoic acid-binding protein type II (R111M) suggests a mechanism of ligand entry.

The crystal structure of unliganded mutant R111M of human cellular retinoic acid-binding protein type II (apo-CRABPII (R111M)) has been determined at 2.3 A and refined to a crystallographic R-factor of 0. 18. Although the mutant protein has lower affinity for all-trans-retinoic acid (RA) than the wild-type, it is properly folded, and its conformation is very similar to the wild-type. apo-CRABPII (R111M) crystallizes in space group P1 with two molecules in the unit cell. The two molecules have high structural similarity except that their alpha2 helices differ strikingly. Analyses of the molecular conformation and crystal packing environment suggest that one of the two molecules assumes a conformation compatible with RA entry. Three structural elements encompassing the opening of the binding pocket exhibit large conformational changes, when compared with holo-CRABPII, which include the alpha2 helix and the betaC-betaD and betaE-betaF hairpin loops. The alpha2 helix is unwound at its N terminus, which appears to be essential for the opening of the RA-binding pocket. Three arginine side-chains (29, 59, and 132) are found with their guanidino groups pointing into the RA-binding pocket. A three-step mechanism of RA entry has been proposed, addressing the opening of the RA entrance, the electrostatic potential that directs entry of RA into the binding pocket, and the intramolecular interactions that stabilize the RA.CRABPII complex via locking the three flexible structural elements when RA is bound.

Apoproteins↗
PubMed1998

Synthetic signal peptides specifically recognize SecA and stimulate ATPase activity in the absence of preprotein.

Although it is known that virtually all exported proteins require a signal peptide, it is not clearly understood how the signal peptide interfaces with the translocation machinery to achieve transport. In this study we document a direct interaction between the signal peptide and SecA, a primary component of the translocase in Escherichia coli, and show that the signal peptide itself can stimulate SecA-lipid ATPase activity. Using synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and two model sequences, we find that the extent of stimulation of SecA ATPase activity by the different peptides parallels the hierarchy of results found for in vivo function (Izard, J. W., Doughty, M. B., and Kendall, D. A. (1995) Biochemistry 34, 9904-9912). The peptide-induced activity requires a lipid to protein molar ratio of at least 300:1 and liposomes enriched in negatively charged phospholipids. Furthermore, specific binding of the signal peptide to SecA was demonstrated using chemical cross-linking and competition with unlabeled peptides.

Adenosine Triphosphatases↗
PubMed1998

Antibacterial agents that inhibit two-component signal transduction systems.

A class of antibacterials has been discovered that inhibits the growth of Gram-positive pathogenic bacteria. RWJ-49815, a representative of a family of hydrophobic tyramines, in addition to being a potent bactericidal Gram-positive antibacterial, inhibits the autophosphorylation of kinase A of the KinA::Spo0F two-component signal transduction system in vitro. Analogs of RWJ-49815 vary greatly in their ability to inhibit growth of bacteria and this ability correlates directly with their activity as kinase A inhibitors. Compared with the potent quinolone, ciprofloxacin, RWJ-49815 exhibits reduced resistance emergence in a laboratory passage experiment. Inhibition of the histidine protein kinase::response regulator two-component signal transduction pathways may present an opportunity to depress chromosomal resistance emergence by targeting multiple proteins with a single inhibitor in a single bacterium. Such inhibitors may represent a class of antibacterials that potentially may represent a breakthrough in antibacterial therapy.

Anti-Bacterial Agents↗
PubMed1998

Fos expression in the brain induced by peripheral injection of CCK or leptin plus CCK in fasted lean mice.

We previously reported a synergistic interaction between leptin and cholecystokinin (CCK) to reduce food intake through CCK-A receptors in lean mice fasted for 24 h. To identify the activated neuronal pathways, we investigated changes in Fos expression in brain nuclei 2 h after single or combined intraperitoneal (i.p.) injections of leptin (120 microg/kg) and sulfated CCK-8 (3.5 microg/kg) in male lean mice (C57BL/6) fasted for 24 h using immunohistochemistry for Fos, the protein product of the early gene, c-fos. Leptin did not increase Fos expression in the brain compared with vehicle-treated mice. CCK increased the numbers of Fos-positive neurons in the nucleus of the solitary tract (NTS)/area postrema (AP), central nucleus of the amygdala (CeA) and, to a smaller extent, in the paraventricular nucleus of the hypothalamus (PVN) (5.2-, 2.3- and 0. 3-fold respectively). Injections of leptin-CCK further enhanced Fos expression by 40% in the PVN compared with that induced by CCK alone, but not in the other nuclei. Devazepide (a CCK-A receptor antagonist, 1 mg/kg, i.p.) prevented the increase in Fos expression induced by leptin-CCK in the PVN and by CCK alone in the PVN, CeA and NTS/AP. These results indicate that in fasted mice, i.p. injection of CCK increases Fos expression in specific brain nuclei through CCK-A receptors while leptin alone had no effect. Leptin in conjunction with CCK selectively enhanced Fos expression in the PVN. The PVN may be an important site mediating the synergistic effect of leptin-CCK to regulate food intake.

Adipose Tissue↗
PubMed1998

Transforming growth factor-beta1 stimulates protein kinase A in mesangial cells.

We recently demonstrated that transforming growth factor-beta (TGF-beta) stimulates phosphorylation of the type I inositol 1,4, 5-trisphosphate receptor (Sharma, K., Wang, L., Zhu, Y., Bokkala, S., and Joseph, S. (1997) J. Biol. Chem. 272, 14617-14623), possibly via protein kinase A (PKA) activation in murine mesangial cells. In the present study, we evaluated whether TGF-beta stimulates PKA activation. Utilizing a specific PKA kinase assay, we found that TGF-beta increases PKA activity by 3-fold within 15 min of TGF-beta1 treatment, and the enhanced kinase activity was completely reversed by the inhibitory peptide for PKA (PKI; 1 microM). In mesangial cells transfected with a PKI expression vector, enhanced PKA activity could not be demonstrated with TGF-beta1 treatment. TGF-beta1 was also found to stimulate translocation of the alpha-catalytic subunit of PKA to the nucleus by Western analysis of nuclear protein as well as by confocal microscopy. TGF-beta1-mediated phosphorylation of cAMP response element-binding protein was completely reversed by H-89 (3 microM), a specific inhibitor of PKA. Stimulation of fibronectin mRNA by TGF-beta1 was also attenuated in cells overexpressing PKI. We thus conclude that TGF-beta stimulates the PKA signaling pathway in mesangial cells and that PKA activation contributes to TGF-beta stimulation of cAMP response element-binding protein phosphorylation and fibronectin expression.

Animals↗
PubMed1998

Homologous DNA pairing domain peptides of RecA protein: intrinsic propensity to form beta-structures and filaments.

The 20 amino acid residue peptides derived from RecA loop L2 have been shown to be the pairing domain of RecA. The peptides bind to ss- and dsDNA, unstack ssDNA, and pair the ssDNA to its homologous target in a duplex DNA. As shown by circular dichroism, upon binding to DNA the disordered peptides adopt a beta-structure conformation. Here we show that the conformational change of the peptide from random coil to beta-structure is important in binding ss- and dsDNA. The beta-structure in the DNA pairing peptides can be induced by many environmental conditions such as high pH, high concentration, and non-micellar sodium dodecyl sulfate (6 mM). This behavior indicates an intrinsic property of these peptides to form a beta-structure. A beta-structure model for the loop L2 of RecA protein when bound to DNA is thus proposed. The fact that aromatic residues at the central position 203 strongly modulate the peptide binding to DNA and subsequent biochemical activities can be accounted for by the direct effect of the aromatic amino acids on the peptide conformational change. The DNA-pairing domain of RecA visualized by electron microscopy self-assembles into a filamentous structure like RecA. The relevance of such a peptide filamentous structure to the structure of RecA when bound to DNA is discussed.

Amino Acid Sequence↗
PubMed1998

Inhibitors of farnesyl protein transferase. 4-Amido, 4-carbamoyl, and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]- cyclohepta[1,2-b]pyridin-11-yl)piperazine and 1-(3-bromo-8-chloro-6,11- dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperazine.

The synthesis of a variety of novel 4-amido, 4-carbamoyl and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl) piperazine to explore the SAR of this series of FPT inhibitors is described. This resulted in the synthesis of the 4- and 3-pyridylacetyl analogues 45a and 50a, respectively, both of which were orally active but were found to be rapidly metabolized in vivo. Identification of the principal metabolites led to the synthesis of a variety of new compounds that would be less readily metabolized, the most interesting of which were the 3- and 4-pyridylacetyl N-oxides 80a and 83a. Novel replacements for the pyridylacetyl moiety were also sought, and this resulted in the discovery of the 4-N-methyl and 4-N-carboxamidopiperidinylacetyl derivatives 135a and 160a, respectively. All of these derivatives exhibited greatly improved pharmacokinetics. The synthesis of the corresponding 3-bromo analogues resulted in the discovery of the 4-pyridylacetyl N-oxides 83b (+/-) and 85b [11S(-)] and the 4-carboxamidopiperidinylacetamido derivative 160b (+/-), all of which exhibited potent FPT inhibition in vitro. All three showed excellent oral bioavailability in vivo in nude mice and cynomolgus monkeys and exhibited excellent antitumor efficacy against a series of tumor cell lines when dosed orally in nude mice.

3T3 Cells↗
PubMed1998

Engineering the independent folding of the subtilisin BPN' pro-domain: correlation of pro-domain stability with the rate of subtilisin folding.

The 77-amino acid pro-domain greatly accelerates the in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In this complex the pro-domain has a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helixes. When isolated from subtilisin, however, the pro-domain is 97% unfolded even under optimal folding conditions. The instability of the isolated pro-domain suggests that there may be a thermodynamic linkage between the stability of the pro-domain and its ability to facilitate subtilisin folding. On the basis of the X-ray crystal structure of the pro-domain subtilisin complex, we have designed stabilizing mutations in three areas of the pro-domain: alpha-helix 23-32 (E32Q), beta-strands 35-51 (Q40L), and alpha-helix 53-61 (K57E). These amino acid positions were selected because they do not contact subtilisin in the complex and because they appear to be in regions of the structure which are not well packed in the wild type pro-domain. Since none of the mutations directly contact subtilisin, their effects on the folding of subtilisin are linked to whether or not they stabilize a conformation of the pro-domain which promotes subtilisin folding. By sequentially introducing the three stabilizing mutations, the equilibrium for folding the pro-domain was shifted from 97% unfolded to 65% folded. By measuring the ability of these mutants to fold subtilisin, we are able to establish a correlation between the stability of the pro-domain and its ability to accelerate subtilisin folding. As the pro-domain is stabilized, the folding reaction becomes faster and distinctly biphasic. A detailed mechanism was determined for the double mutant, Q40L-K57E, which is 50% folded: P + Su if (30 800 M-1 s-1, 0.04 s-1) PSI if (0.07 s-1, <0.005 s-1) PS. PSI is an intermediate complex which accumulates in the course of the reaction, and PS is the fully folded complex. The more stable the pro-domain, the faster the folding reaction up to the point at which the isomerization of the intermediate into the fully folded complex becomes the rate-limiting step in the folding process.

Cloning, Molecular↗
PubMed1998

Critical residues for histone acetylation by Gcn5, functioning in Ada and SAGA complexes, are also required for transcriptional function in vivo.

Several previously known transcription cofactors have been demonstrated in vitro recently to be histone acetyltransferases and deacetyltransferases, suggesting that remodeling of chromatin through histone acetylation plays a fundamental role in gene regulation. Clear evidence has not yet been obtained, however, to demonstrate that histone acetylation is required for gene activation in vivo. In this study we performed an alanine-scan mutagenesis through the HAT (histone acetyltransferase) domain identified previously by deletion mapping in recombinant yeast Gcn5. We identified multiple substitution mutations that eliminated completely Gcn5's ability to potentiate transcriptional activation in vivo. Strikingly, each of these mutations was also critical for free and nucleosomal histone acetylation by Gcn5 functioning within the native yeast HAT complexes, Ada, and SAGA. Moreover, the growth phenotypes of these mutations as measured by colony size and liquid growth assay closely tracked transcription and HAT activities. In contrast, mutations that did not affect in vivo function of Gcn5 were able to acetylate histones. These data argue strongly that acetylation is required for gene regulation by Gcn5 in vivo, and support previous arguments that nucleosomal histones are among the physiological substrates of acetylation by Gcn5.

Acetylation↗