Biomedical subjects

W Zhu

Publications and source records attributed to W Zhu.

At least 109 records · Page 6Linked to original sources

PubMed2000

Lipopolysaccharide increases endogenous morphine levels in rat brain.

The present study was designed to determine whether whole body injection of lipopolysaccharide increases endogenous rat brain morphine levels. High performance liquid chromatography coupled to electrochemical detection and gas-chromatography mass spectrometry reveal that the resting brain morphine level is 7.0+/-3.2ng/g wet weight. In a time dependent manner, intraperitoneal injection of lipopolysaccharide (10 microg), leads to an increase of brain morphine level with a peak reaching after 36h post injection. After 72h following the lipopolysaccharide-injection, morphine levels increased to 66.0+/-5.4ng/g brain wet weight (P<0.001). Furthermore, 96h of fasting also increased the brain morphine level to 44.0+/-3. 6ng/g of brain wet weight. These results suggest that rat brain morphinergic processes respond to organismic challenges after a significant latent period.

Animals↗

PubMed2000

An androgen response element mediates LNCaP cell dependent androgen induction of the hK2 gene.

Human glandular kallikrein (hK2) is an androgen regulated protein primarily expressed in the prostate and recently identified as a novel prostate cancer marker. A 5 kb 5' flanking region of the hK2 gene was isolated and sequenced to characterize the regulatory mechanisms for the expression of hK2 in the androgen responsive prostate cell line, LNCaP. Using gene transfer, gel shift, and mutagenesis assays we have identified an ARE in the 5' far upstream promoter region of the hK2 gene that is crucial for its regulation in LNCaP cells. This study further demonstrated that the hK2 upstream ARE plays a predominant role in androgenic response. More interestingly, previously identified AREs in the prostate specific antigen promoter and the hK2 proximal promoter exert little activity in LNCaP cells. This study for the first time identifies a unique ARE that alone mediates the function of the androgen receptor in LNCaP cells in a cell dependent manner. This study also examines the activity of this ARE with 1alpha, 25 dihydroxy-vitamin D3 on the expression of the hK2 gene in LNCaP cells.

Androgens↗

PubMed2000

Capillary electrophoretic separation of the enantiomers of organophosphates with a phosphorus stereogenic center using the sodium salt of octakis(2,3-diacetyl-6-sulfo)-gamma-cyclodextrin as resolving agent.

The sodium salt of the single-isomer, chiral resolving agent, octakis(2,3-diacetyl-6-sulfo)-gamma-cyclodextrin (ODAS-gammaCD) has been used for the capillary electrophoretic separation of the enantiomers of alkylarylphosphates which carry a phosphorus-based stereogenic center. The effective mobilities and separation selectivities were measured at different ODAS-gammaCD and methanol concentrations to find the conditions under which the minor enantiomers could be adequately quantitated in samples obtained by chemical resolution of the racemic mixtures. This work extends the utility of ODAS-gammaCD to a hitherto unexplored field, the capillary electrophoretic separation of the enantiomers of organophosphorus compounds.

Cyclodextrins↗

PubMed2000

A CDC6 protein-binding peptide selected using a bacterial two-hybrid-like system is a cell cycle inhibitor.

Peptides or small molecules able to modulate protein-protein interactions hold promise as tools with which to probe and manipulate biological pathways. An important issue in this nascent field is to evaluate different methods with which to search libraries for molecules that modulate the function of specific target proteins. One strategy is to screen libraries for molecules that bind specifically to a protein known to be critical in the pathway of interest, with the expectation that the molecules isolated will recognize regions of the target protein important for its function and thereby exhibit biological activity. Here, a peptide library was screened using a two-hybrid-like system for molecules able to bind human CDC6 protein (CDC6p), required for the initiation of DNA replication in eukaryotic cells. From a collection of over a million peptides, a single species that exhibited good affinity and specificity for binding CDC6p was obtained. When expressed in human cells, the peptide inhibited cell cycle progression and exhibited other properties expected of a CDC6p inhibitor. This approach, which does not require detailed knowledge of the mechanism of action of a protein target, may be generally useful for isolating peptides capable of manipulating biological pathways.

Amino Acid Sequence↗

PubMed2000

Identification of morphine in the adrenal medullary chromaffin PC-12 cell line.

Morphine was identified in the adrenal medulla chromaffin PC-12 cell line by reversed-phase HPLC, following liquid and solid extraction. The morphine corresponding HPLC fractions (1.746+/-0.615 ng of morphine/million cells) were further analyzed by gas chromatography-mass spectrometry and found to be identical to synthetic morphine. Furthermore, using primers derived from the human neuronal mu 1 opiate receptor, we used RT-PCR to detect expression of mu transcripts from this cell line. The transcript was absent. The study conclusively proves morphine, but not a mu opiate receptor, is constitutively expressed in the adrenal medulla chromaffin PC-12 cell line.

Adrenal Gland Neoplasms↗

PubMed2000

Enantiomer separations by nonaqueous capillary electrophoresis using octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin.

The newest member of the single-isomer isomer sulfated cyclodextrin family, octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gamma-CD) was used for the first time as a resolving agent for the nonaqueous capillary electrophoretic separation of the enantiomers of 26 weak base pharmaceuticals in an acidic methanol background electrolyte. The solubility limit of ODAS-gamma-CD at room temperature proved to be 55 mM in this background electrolyte, which afforded good, fast enantiomer separations for most of the basic drugs tested. For all the bases studied, the effective mobilities and separation selectivities were found to follow the predictions of the charged resolving agent migration model of electrophoretic enantiomer separations. The effective mobilities of the weakly binding weak bases remained cationic throughout the entire 0 to 45 mM ODAS-gamma-CD concentration range; separation selectivities increased as the ODAS-gamma-CD concentration was increased. The effective mobilities of the moderately binding weak bases became anionic in the 2.5 to 45 mM ODAS-gamma-CD concentration range; separation selectivities first increased as the effective mobilities approached zero, then decreased again as the ODAS-gamma-CD concentration was increased further. The effective mobilities of the strongly binding weak bases became anionic in the 0 to 2.5 mM ODAS-gamma-CD concentration range; separation selectivities decreased as the ODAS-gamma-CD concentration was increased above 2.5 mM.

Cyclodextrins↗

PubMed2000

The virulence factor AvrXa7 of Xanthomonas oryzae pv. oryzae is a type III secretion pathway-dependent nuclear-localized double-stranded DNA-binding protein.

AvrXa7 is a member of the avrBs3 avirulence gene family, which encodes proteins targeted to plant cells by a type III secretion apparatus. AvrXa7, the product of avrXa7, is also a virulence factor in strain PXO86 of Xanthomonas oryzae pv. oryzae. Avirulence and virulence specificities are associated with the central repeat domain, which, in avrXa7, consists of 25.5 direct repeat units. Mutations in three C-terminal nuclear localization signal motifs eliminated avirulence and virulence activities in rice and severely reduced nuclear localization in a yeast assay system. Both pathogenicity functions and nuclear localization were restored on the addition of the sequence for the nuclear localization signal motif from SV40 T-antigen. The loss of avirulence activity because of mutations in the acidic transcriptional activation domain was restored by addition of the activation domain from the herpes simplex viral protein VP16. The activation domain was also required for virulence activity. However, the VP16 domain could not substitute for the endogenous domain in virulence assays. In gel shift assays, AvrXa7 bound double-stranded DNA with a preference for dA/dT rich sequences. The results indicate that products of the avrBs3-related genes are virulence factors targeted to host cell nuclei and have the potential to interact with the host DNA and transcriptional machinery as part of their mode of action. The results also suggest that the host defensive recognition mechanisms are targeted to the virulence factor site of action.

Amino Acid Motifs↗

PubMed2000

Frequency-encoding Thr17 phospholamban phosphorylation is independent of Ser16 phosphorylation in cardiac myocytes.

Both Ser(16) and Thr(17) of phospholamban (PLB) are phosphorylated, respectively, by cAMP-dependent protein kinase (PKA) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). PLB phosphorylation relieves cardiac sarcoplasmic reticulum Ca(2+) pump from inhibition by PLB. Previous studies have suggested that phosphorylation of Ser(16) by PKA is a prerequisite for Thr(17) phosphorylation by CaMKII and is essential to the relaxant effect of beta-adrenergic stimulation. To determine the role of Thr(17) PLB phosphorylation, we investigated the dual-site phosphorylation of PLB in isolated adult rat cardiac myocytes in response to beta(1)-adrenergic stimulation or electrical field stimulation (0. 1-3 Hz) or both. A beta(1)-adrenergic agonist, norepinephrine (10(-9)-10(-6) m), in the presence of an alpha(1)-adrenergic antagonist, prazosin (10(-6) m), selectively increases the PKA-dependent phosphorylation of PLB at Ser(16) in quiescent myocytes. In contrast, electrical pacing induces an opposite phosphorylation pattern, selectively enhancing the CaMKII-mediated Thr(17) PLB phosphorylation in a frequency-dependent manner. When combined, electric stimulation (2 Hz) and beta(1)-adrenergic stimulation lead to dual phosphorylation of PLB and exert a synergistic effect on phosphorylation of Thr(17) but not Ser(16). Frequency-dependent Thr(17) phosphorylation is closely correlated with a decrease in 50% relaxation time (t(50)) of cell contraction, which is independent of, but additive to, the relaxant effect of Ser(16) phosphorylation, resulting in hastened contractile relaxation at high stimulation frequencies. Thus, we conclude that in intact cardiac myocytes, phosphorylation of PLB at Thr(17) occurs in the absence of prior Ser(16) phosphorylation, and that frequencydependent Thr(17) PLB phosphorylation may provide an intrinsic mechanism for cardiac myocytes to adapt to a sudden change of heart rate.

Adenosine Triphosphatases↗

PubMed2000

Inhibition of spontaneous beta 2-adrenergic activation rescues beta 1-adrenergic contractile response in cardiomyocytes overexpressing beta 2-adrenoceptor.

Cardiac-specific overexpression of the human beta(2)-adrenergic receptor (AR) in transgenic mice (TG4) enhances basal cardiac function due to ligand-independent spontaneous beta(2)-AR activation. However, agonist-mediated stimulation of either beta(1)-AR or beta(2)-AR fails to further enhance contractility in TG4 ventricular myocytes. Although the lack of beta(2)-AR response has been ascribed to an efficient coupling of the receptor to pertussis toxin-sensitive G(i) proteins in addition to G(s), the contractile response to beta(1)-AR stimulation by norepinephrine and an alpha(1)-adrenergic antagonist prazosin is not restored by pertussis toxin treatment despite a G(i) protein elevation of 1.7-fold in TG4 hearts. Since beta-adrenergic receptor kinase, betaARK1, activity remains unaltered, the unresponsiveness of beta(1)-AR is not caused by betaARK1-mediated receptor desensitization. In contrast, pre-incubation of cells with anti-adrenergic reagents such as muscarinic receptor agonist, carbachol (10(-5)m), or a beta(2)-AR inverse agonist, ICI 118,551 (5 x 10(-7)m), to abolish spontaneous beta(2)-AR signaling, both reduce the base-line cAMP and contractility and, surprisingly, restore the beta(1)-AR contractile response. The "rescued" contractile response is completely reversed by a beta(1)-AR antagonist, CGP 20712A. Furthermore, these results from the transgenic animals are corroborated by in vitro acute gene manipulation in cultured wild type adult mouse ventricular myocytes. Adenovirus-directed overexpression of the human beta(2)-AR results in elevated base-line cAMP and contraction associated with a marked attenuation of beta(1)-AR response; carbachol pretreatment fully revives the diminished beta(1)-AR contractile response. Thus, we conclude that constitutive beta(2)-AR activation induces a heterologous desensitization of beta(1)-ARs independent of betaARK1 and G(i) proteins; suppression of the constitutive beta(2)-AR signaling by either a beta(2)-AR inverse agonist or stimulation of the muscarinic receptor rescues the beta(1)-ARs from desensitization, permitting agonist-induced contractile response.

Adrenergic beta-Agonists↗

PubMed2000

Regulation of TNF expression by multiple mitogen-activated protein kinase pathways.

Stimulating macrophages with bacterial endotoxin (LPS) activates numerous intracellular signaling pathways that lead to the production of TNF. In this study, we show that four mitogen-activated protein (MAP) kinase pathways are activated in LPS-stimulated macrophages: the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase, p38, and Big MAP kinase (BMK)/ERK5 pathways. Although specific activation of a single MAP kinase pathway produces only a modest effect on TNF promoter activation, activation of each MAP kinase pathway is important for full induction of the TNF gene. Interestingly, a dramatic induction of TNF promoter-driven gene expression was observed when all of the four MAP kinase pathways were activated simultaneously, suggesting a cooperative effect among these kinases. Unexpectedly, cis elements known to be targeted by MAP kinases do not play a major role in multiple MAP kinase-induced TNF gene expression. Rather, a 40-bp sequence harboring the TATA box, is responsible for the gene up-regulation induced by MAP kinases. The proximity of the MAP kinase-responsive element to the transcriptional initiation site suggested that MAP kinases regulate the transcriptional initiation complex. Utilizing alpha-amanitin-resistant RNA polymerase II mutants with or without a C-terminal domain (CTD) deletion, we found that deleting the CTD to 31 tandem repeats (Delta31) led to >90% reduction in MAP kinase-mediated TNF production. Thus, our data demonstrate coordination of multiple MAP kinase pathways in TNF production and suggest that the CTD of RNA polymerase II is required to execute MAP kinase signaling in TNF expression.

Animals↗

PubMed2000

Ca2+/calmodulin-dependent kinase II and calcineurin play critical roles in endothelin-1-induced cardiomyocyte hypertrophy.

Endothelin-1 (ET-1) induces cardiac hypertrophy. Because Ca(2+) is a major second messenger of ET-1, the role of Ca(2+) in ET-1-induced hypertrophic responses in cultured cardiac myocytes of neonatal rats was examined. ET-1 activated the promoter of the beta-type myosin heavy chain gene (beta-MHC) (-354 to +34 base pairs) by about 4-fold. This activation was inhibited by chelation of Ca(2+) and the blocking of protein kinase C activity. Similarly, the beta-MHC promoter was activated by Ca(2+) ionophores and a protein kinase C activator. beta-MHC promoter activation induced by ET-1 was suppressed by pretreatment with the calmodulin inhibitor, W7, the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitor, KN62, and the calcineurin inhibitor, cyclosporin A. beta-MHC promoter activation by ET-1 was also attenuated by overexpression of dominant-negative mutants of CaMKII and calcineurin. ET-1 increased the activity of CaMKII and calcineurin in cardiac myocytes. Pretreatment with KN62 and cyclosporin A strongly suppressed ET-1-induced increases in [(3)H]phenylalanine uptake and in cell size. These results suggest that Ca(2+) plays a critical role in ET-1-induced cardiomyocyte hypertrophy by activating CaMKII- and calcineurin-dependent pathways.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2000

Identification of drosophila bicoid-interacting proteins using a custom two-hybrid selection.

Bicoid directs pattern formation in the developing Drosophila embryo, and does so by performing two seemingly unrelated tasks; it activates transcription and represses translation. To understand how Bicoid carries out this dual role, we sought to identify Bicoid-ancillary proteins that might mediate Bicoid's function in transcription or translation. We used a customized version of the two-hybrid method and found two Bicoid-interacting proteins, Bin1 and Bin3, both of which interact with Bicoid in vitro. Bin1 is similar to a human protein (SAP18) involved in transcription regulation, and Bin3, described in this paper, is similar to a family of protein methyltransferases that modify RNA-binding proteins. Given that Bicoid's role as a translation regulator requires RNA binding, we suggest that the Bicoid-interacting methyltransferase might be important for that role. The custom two-hybrid method we used, in which Bicoid is bound to DNA via its own DNA binding domain, rather than via a fusion-protein tether, should be generally applicable to other DNA binding proteins.

Amino Acid Sequence↗

PubMed2000

Optimal treatment allocation in comparative biomedical studies.

In clinical trials involving several treatment protocols, there are usually several objectives of different importance. A general method for finding an optimal design which reflects this practical need is discussed. In particular, we show that the graphical method of Cook and Wong can be applied when there are two objectives. Several illustrative examples are discussed, including multiple-objective designs for comparing several treatment groups with a placebo group.

Humans↗

PubMed2000

A family of single-isomer, sulfated gamma-cyclodextrin chiral resolving agents for capillary electrophoresis. 1. Octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin.

The first member of the single-isomer, sulfated gamma-cyclodextrin family, the sodium salt of octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gamma CD) has been synthesized, analytically characterized, and used to separate, by capillary electrophoresis, a variety of neutral, acidic, basic, and amphoteric enantiomers in low pH background electrolytes. The anionic effective mobilities of the neutral and anionic analytes were found to increase with the concentration of ODAS-gamma CD. For weakly binding cationic analytes, the effective mobilities went from cationic high values, through zero, to increasingly larger anionic values as the concentration of ODAS-gamma CD was increased. For the strongly complexing cationic analytes, the effective mobilities became anionic even at very low ODAS-gamma CD concentrations and became smaller as the ionic strength of the background electrolyte increased with the increasing ODAS-gamma CD concentration. Separation selectivity followed the predictions of the charged resolving agent migration model: for neutral analytes it decreased as the concentration of ODAS-gamma CD was increased. For cationic analytes, selectivities were found to increase as the cationic effective mobilities approached zero, then decreased as the concentration of ODAS-gamma CD was increased further. The extent of peak resolution that could be realized with ODAS-gamma CD strongly depended on the magnitude of separation selectivity and the normalized electroosmotic flow mobility. ODAS-gamma CD proved to be a broadly applicable chiral resolving agent.

Cyclodextrins↗

PubMed2000

Identification of the soluble granulocyte-macrophage colony stimulating factor receptor protein in vivo.

On the basis of the finding of alternatively spliced mRNAs, the alpha-subunit of the receptor for GM-CSF is thought to exist in both a membrane spanning (tmGMRalpha) and a soluble form (solGMRalpha). However, only limited data has been available to support that the solGMRalpha protein product exists in vivo. We hypothesized that hematopoietic cells bearing tmGMRalpha would have the potential to also produce solGMRalpha. To test this hypothesis we examined media conditioned by candidate cells using functional, biochemical, and immunologic means. Three human leukemic cell lines that express tmGMRalpha (HL60, U937, THP1) were shown to secrete GM-CSF binding activity and a solGMRalpha-specific band by Western blot, whereas a tmGMRalpha-negative cell line (K562) did not. By the same analyses, leukapheresis products collected for autologous and allogeneic stem cell transplants and media conditioned by freshly isolated human neutrophils also contained solGMRalpha. The solGMRalpha protein in vivo displayed the same dissociation constant (Kd = 2-5 nmol) as that of recombinant solGMRalpha. A human solGMRalpha ELISA was developed that confirmed the presence of solGMRalpha in supernatant conditioned by the tmGMRalpha-positive leukemic cell lines, hematopoietic progenitor cells, and neutrophils. Furthermore, the ELISA demonstrated a steady state level of solGMRalpha in normal human plasma (36 +/- 17 pmol) and provided data suggesting that plasma solGMRalpha levels can be elevated in acute myeloid leukemias. (Blood. 2000;95:461-469)

Alternative Splicing↗

PubMed2000

Experimental verification of a predicted, hitherto unseen separation selectivity pattern in the nonaqueous capillary electrophoretic separation of weak base enantiomers by octakis (2,3-diacetyl-6-sulfato)-gamma-cyclodextrin.

The capillary electrophoretic separation of cationic enantiomers with single-isomer multivalent anionic resolving agents was reexamined with the help of the charged resolving agent migration model. Three general model parameters were identified that influence the shape of the separation selectivity and enantiomer mobility difference curves: parameter b, the binding selectivity (K(RCD)/K(SCD)), parameter s, the size selectivity (mu0(RCD)/mu0(SCD)), and parameter a, the complexation-induced alteration of the analyte's mobility (mu0(RCD)/mu0). In addition to the previously observed discontinuity in separation selectivity that occurs as mu(eff) of the less mobile enantiomer changes from cationic to anionic, a new feature, a separation selectivity maximum was predicted to occur in the resolving agent concentration range where both enantiomers migrate cationically provided that (i) K(RCD)/K(SCD) <1 and mu0(RCD)/mu0(SCD) >1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) > 1, or (ii) K(RCD)/K(SCD) >1 and mu0(RCD)/mu0(SCD) <1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) <1. This hitherto unseen separation selectivity pattern was experimentally verified during the nonaqueous capillary electrophoretic separation of the enantiomers of four weak base analytes in acidic methanol background electrolytes with octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gammaCD) as resolving agent.

Albuterol↗

PubMed2000

Experimental verification of a predicted, previously unseen separation selectivity pattern in the capillary electrophoretic separation of noncharged enantiomers by octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin.

The capillary electrophoretic separation of noncharged enantiomers with single-isomer anionic resolving agents is reexamined here with the help of the charged resolving agent migration model. Two general model parameters have been identified that influence the effective mobility, separation selectivity and mobility difference curves of the enantiomers: parameter b, called binding selectivity (K(RCD)/K(SCD)), and parameter s, called size selectivity (mu(o)RCD/mu(o)SCD). Analysis of the model in terms of these parameters indicates that in addition to the known, previously observed separation selectivity vs. resolving agent concentration patterns, a new pattern, increasing separation selectivity with increasing resolving agent concentration, is also possible provided that (i) K(RCD)/K(SCD)<1 and mu(o)RCD/mu(o)SCD>1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)>1, or (ii) K(RCD)/ K(SCD)>1 and mu(o)SCD/mu(o)SCD<1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)<1. This hitherto unseen separation selectivity pattern was experimentally verified during the capillary electrophoretic separation of the enantiomers of O-isopropyl p-nitrophenyl methylphosphonate with the single-isomer octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin as resolving agent.

Cyclodextrins↗

PubMed2000

Effects of L-THP on Ca2+ overload of cultured rat cardiomyocytes during hypoxia and reoxygenation.

The effects of L-tetrahydropalmatine (L-THP) on the cultured rat cardiomyocytes during hypoxia and reoxygenation and the mechanism of L-THP treating reperfusion-arrythmias were studied. The concentration of intracellular free calcium ([Ca2+]i) of single cultured ventricular myocyte was determined by using EPC-9 light-electricity measurement system. It was found that L-THP (100 mumol/L) could reduce the [Ca2+]i augmentation in single cultured ventricular myocyte during hypoxia and reoxygenation. Verapamil (10 mumol/L) had the similar effect. It was concluded that L-THP could inhibit the Ca2+ overload of cultured rat cardiomyocytes during hypoxia and reoxygenation.

Animals↗