Biomedical subjects

S Zhuo

Publications and source records attributed to S Zhuo.

15 recordsLinked to original sources

PubMed2001

Modulation of human cytomegalovirus immediate-early gene enhancer by mitogen-activated protein kinase kinase kinase-1.

The immediate-early (IE) promoter of human cytomegalovirus (HCMV) constitutes a primary genetic switch, which determines the progression of viral infection. Earlier reports by others have shown mitogen-activated protein kinase kinase kinase-1 (MEKK1) to be able to up-regulate HCMV-IE promoter through downstream mitogen-activated protein kinase (MAPK) pathways. However, we noticed that the activation of the HCMV-IE promoter by constitutively active MEKK1 (MEKK1-TRU) might not be through the MAPK pathways. Using a HCMV-IE enhancer/promoter (- 522 to + 72) driving a luciferase reporter, we demonstrated that the downstream MAPK activation actually repressed the up-regulation of the promoter by MEKK1 in CHO-K1 and human 293 cells. We further found that the up-regulation of HCMV-IE promoter by MEKK1 could be in great extent suppressed by over-expression of IkappaBalpha. Deletion of the NFkappaB/rel sites in the HCMV-IE enhancer region by mutagenesis proportionally reduced the transcriptional activation by MEKK1-TRU, whereas deletion of the ATF/CREB binding sites or cyclic AMP response elements (CRE) had no effects. Furthermore, the NFkappaB/rel deletion mutant also showed repression on the basic transcription activity of the HCMV-IE promoter. Our results indicate that the NFkappaB/rel sites are not only responsible for the modulation of HCMV-IE enhancer activity by MEKK1 but also control the basic transcription activity of the HCMV-IE promoter. On the other hand, the four consensus CRE sites were found to have no function in the activation of the promoter by MEKK1.

Animals↗

PubMed2000

Nonproteolytic role for the urokinase receptor in cellular migration in vivo.

The urokinase receptor (uPAR) binds and localizes urokinase activity at cellular surfaces, facilitating fibrinolysis and cellular migration at sites of tissue injury. uPAR also participates in cellular signaling and regulates integrin-dependent adhesion and migration in vitro. We now report evidence that uPAR occupancy regulates cellular migration in vivo in the absence of functional urokinase. Recombinant murine KC (1.5 microg), a potent neutrophil chemoattractant, was delivered to the lungs of wild-type, urokinase-deficient or uPAR-deficient mice 18 h after intraperitoneal injection of 200 microg human immunoglobulin G (IgG) or a fusion protein composed of an amino-terminal receptor-binding fragment of urokinase and a human IgG Fc fragment (GFD-Fc). Whole lung lavage for recovery of leukocytes was performed 4 h later. KC treatment resulted in a 100-fold increase in lavage neutrophils. GFD-Fc injection resulted in >50% reduction in neutrophil influx in both wild-type and urokinase-deficient animals but had no effect on uPAR -/- mice. A concomitant reduction in alveolar protein leakage but no change in numbers of circulating neutrophils accompanied this attenuated inflammatory response. The reduction in neutrophil influx induced by GFD-Fc is thus related to uPAR occupancy and yet not due to disruption of uPAR-mediated proteolysis. These observations verify that protease-independent functions of uPAR operate in vivo and identify uPAR as a potential target for regulation of inflammatory processes characterized by neutrophil-mediated injury.

Administration, Intranasal↗

PubMed1999

Urokinase receptor antagonists: discovery and application to in vivo models of tumor growth.

Urokinase receptor antagonists based on the growth factor domains of both human and murine urokinase which show sub-nanomolar affinities for their homologous receptors have been expressed as recombinant proteins. Further modification of these molecules by preparing fusions with the constant region of human IgG has led to molecules with high affinities and long in vivo half-lives. Smaller peptidic inhibitors have been obtained by a combination of bacteriophage display and peptide analog synthesis. All of these molecules inhibit the binding of the growth factor domain of uPA to the uPA receptor and enhance binding of the uPA receptor to vitronectin. Protein uPA receptor antagonists were tested in an in vivo tumor model using the human breast carcinoma MDAmb231 in immunodeficient mice. Both human and murine receptor antagonists showed significant inhibition of primary tumor growth, demonstrating that in vivo, both tumor and stromal cell uPA receptor dependent plasminogen activation can modulate tumor growth.

Amino Acid Sequence↗

PubMed1999

[FPMULTI--a software for multi-layer composition and thickness analysis and its applications].

Main features of FPMULTI, a software for analyzing composition and thickness of multi-layer samples simultaneously using XRF, are briefly described in this paper. Based on fundamental parameter method, the software has the capability of determining multi-layer samples containing up to 10 layers and 25 elements. Calibration standards can be bulk or multi-layer of pure element or multi-element standards. Application examples for tin-plates, hot dip galvanized zinc-plates and 'fingerprint-resistant' plates were given. FPMULTI is used to predict the relationship between intensities of different X-ray lines and the thickness first and then to analyze those samples. When using only few calibration standards, the results from FPMULTI are much better than those from linear regression method, and this reflects the advantages of the fundamental parameter approach.

English Abstract↗

PubMed1997

Effects of sulfhydryl regents on the activity of lambda Ser/Thr phosphoprotein phosphatase and inhibition of the enzyme by zinc ion.

Sulfhydryl reagents, such as dithiothreitol (DTT), affected the activity of Ser/Thr phosphoprotein phosphatases. Addition of DTT to the assay buffer increased the affinity of lambda Ser/Thr phosphoprotein phosphatase (lambda-PPase) for its Mn2+ cofactor. On the other hand, the enzyme was found to be inactivated simply by dilution in Tris buffer. The inactivation could be completely prevented by the presence of DTT or Mn2+ in the buffer. Further studies showed that oxidation or reduction of cysteine residues in lambda-PPase may not be the cause of the change in the enzyme activity. Without exception, mutation of all cysteine residues in lambda-PPase to serine did not convert the enzyme into a thiol-insensitive mutant. By careful examination of the effects of different sulfhydryl reagents, metal ion cofactors and substrates on lambda-PPase, it was found that the role of sulfhydryl reagents was the chelation of small amounts of inhibitory metal ions, which were present in plastic laboratory ware, such as disposable cuvets and tubes, with prevention of the enzyme from inactivation. One of the main contaminants found in plastic cuvets was Zn2+, which is a potent inhibitor of lambda-PPase. The inhibition of lambda-PPase by Zn2+ was characterized. Pre-treatment of the enzyme (1-4 nM) with 1 microM of ZnCl2 almost completely inhibited the enzymatic activity in response to 2 mM Mn2+. However, no significant inhibition was found when the enzyme was added to the assay mixture containing 1 microM Zn2+ and 2 mM Mn2+ . This confirms the sensitivity of the holoenzyme to inhibitory metal ions in vitro. The kinetic analysis indicated that the inhibitory metal ion might compete with Mn2+ to bind to the active site of lambda-PPase. This was further supported by the mutation of metal cofactor binding amino acid residues of the enzyme. Mutants which have less affinity for Mn2+ are also less sensitive to Zn2+. Our results suggest that inhibitory metal ions may induce a different structural conformation for lambda-PPase.

Animals↗

PubMed1996

Effects of depletion of intracellular tetrahydrobiopterin in murine erythroleukemia cells.

The biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH4) in murine erythroleukemia (MEL) cells is almost completely inhibited by 10 mM, 2,4-diamino-6-hydroxypyrimidine (DAHP), which targets GTP cyclohydrolase. The inhibition results in dephosphorylation of the retinoblastoma gene product, prolongation of the G1-phase in the cell cycle, and subsequent commitment to terminal differentiation of MEL cells. Reversal of the processes by repletion of cellular BH4 with biopterin-related compounds including BH4, 7,8-dihydrobiopterin (7,8-BH2), sepiapterin, and 7,8-dihydroneopterin has generated complicated results. Low micromolar exogenous pterin compounds had little or no effect. At 300 microM or higher, the synthesis of hemoglobin by DAHP-induced MEL cells is significantly inhibited by 7,8-dihydrobiopterin and sepiapterin. However, further cell cycle analysis shows that the inhibition of cell differentiation by 7,8-BH2 and sepiapterin may not be due to the reversal of cell proliferation. Inhibition of BH4 biosynthesis in MEL cells by inhibitors of sepiapterin reductase has also been studied. None of the inhibitors that were tested, including N-chloroacetyl-dopamine and N-acetylserotonin, which are specific for sepiapterin reductase, can block MEL cells in G1-phase or induce the cells to commit to terminal differentiation. Furthermore, inhibitors of sepiapterin reductase are found to reduce or to abolish hemoglobin synthesis in differentiating MEL cells induced by hexamethylene bisacetamide. The mechanism for this is not clear. Not all of the effects caused by the depletion of BH4 synthesis can be rescued by repletion of BH4. These results suggest that BH4 may not regulate proliferation or differentiation of MEL cells as previously thought. Its function in MEL cells is still not clear.

Acetamides↗

PubMed1996

Field studies on the preventive effect of oral artemether against schistosomal infection.

OBJECTIVE: To study the preventive effect of oral artemether (Art) against schistosomal infection in the field. METHODS: In Minglang District of Yiyang City, Hunan Province, there is an islet with embankment type endemic area in the southern Dongting Lake region. From August to October 1994 the residents who frequently contacted the infested water were selected for study and allocated to the Art group and the control group. About one month before the preventive Art administration, all the residents understudied were examined by stool hatching technique, and then treated orally with praziquantel at a single dose of 40 mg/kg in stool egg-negative residents and 50 mg/kg in stool egg-positive ones. In the Art group, the first dose of 6 mg/kg was given at the end of August, followed by 3 repeated doses every 15 days. Placebo (starch) was given to the control group at the same time as in the Art group. The preventive efficacy was evaluated by stool examination 25-32 days after the last medication. RESULTS: In the Art group, 20 out of 365 studied residents became stool positive with an infection rate of 5.5%, while in the control group, 51 out of 376 studied residents were stool positive with an infection rate of 13.6%. The egg count per gram of feces (EPG) determined by the Kato-Katz method was 122 +/- 79 in the Art group and 681 +/- 909 in the control group. Meanwhile, two cases of acute schistosomiasis were found in the control group, but none was observed in the Art group. No apparent adverse side effect was seen during the treatment with Art. CONCLUSION: Oral Art exhibited apparent preventive effect on the residents who contacted the infested water in schistosomiasis endemic area.

Administration, Oral↗

PubMed1995

Study of the role of retinoblastoma protein in terminal differentiation of murine erythroleukemia cells.

Hexamethylenebisacetamide-induced terminal differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells can be inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. The inhibition is shown to be correlated with prevention of dephosphorylation of retinoblastoma protein (pRB) in cells and bypass of G1 prolongation in the cell cycle. These results suggest that pRB-mediated G1 prolongation is necessary for MEL cells to commit to terminal differentiation. However, further experiments demonstrate that the simple cell cycle exit is not sufficient for commitment to terminal differentiation. Induction of dephosphorylation of pRB and subsequent G1 prolongation by forskolin does not lead MEL cells to differentiate. Additional pRB has been expressed in MEL cells by transfection with a neo-resistant plasmid containing RB cDNA under the control of a cytomegalovirus promoter. Exogenously expressed pRB is hyperphosphorylated in logarithmically growing MEL cells without any noticeable change in growth rate between the transfected cell line and the parental cell line. This result suggests that pRB in MEL cells is regulated by protein kinases and protein phosphatases and not by transcription.

Acetamides↗

PubMed1995

[Field studies on preventive effect of artemether against infection with Schistosoma japonicum].

From August to October 1994, a field trial of preventive treatment with artemether (Art) was carried out in Minglang District of Yiyang City, Hunan Province, an islet with embankment type endemic area nearby southern Donting Lake region. The residents who frequently contacted with the infested water were selected for study and allocated to the Art group and the control group in reference to paired methods of randomization table. About one month before administration of Art, all residents under study were examined by stool hatching technique after nylon-bag concentration to determine the infection rate of each group, and then treated with praziquantel at a single dose of 40 mg/kg in stool egg-negative residents and 50 mg/kg in stool egg-positive ones. In Art group, the first dose of 6 mg/kg was given in late August, followed by repeated dosing every 15 days for 3 times. Placebo (starch) was given to the study residents in the control group at the same time as in Art group. The efficacy was evaluated by stool examination 25-32 days after the last medication. The results showed that after the preventive administration of Art, 20 out of 365 study residents in Art group revealed stool positive with an infection rate of 5.5%, while in the control group 51 out of 376 study residents showed stool positive with an infection rate of 13.6%. The difference between the two groups was statistically significant. Meantime, two cases of acute schistosomiasis were seen in the control group, but none was observed in Art group. The egg per gram of feces (EPG) determined with the Kato-Katz method was 122 +/- 79 (range 12-192) in Art group and 681 +/- 909 (range 12-2,760) in the control group. No apparent adverse side effect was seen during the treatment with Art, and no abnormal change in liver and renal function was detected after the last medication. The preliminary study indicates that application of the preventive administration of Art in the endemic area during the transmission season may effectively reduce both the infection rate and intensity of schistosomiasis.

Adolescent↗

PubMed1994

Mutational analysis of a Ser/Thr phosphatase. Identification of residues important in phosphoesterase substrate binding and catalysis.

The Ser/Thr phosphoprotein phosphatases (PPases) display similarities in amino acid sequence and biochemical properties. Most members of this family require transition metal ions for activity. The smallest family member, the bacteriophage lambda PPase (lambda-PPase), has been successfully overexpressed in Escherichia coli, purified, and characterized (Zhuo, S., Clemens, J.C., Hakes, D.J., Barford, D., and Dixon, J. E. (1993) J. Biol. Chem. 268, 17754-17761). Site-directed mutagenesis has now been employed to define amino acid residues in lambda-PPase required for metal ion binding and catalysis. Conservative amino acid substitutions at residues Asp20, His22, Asp49, His76, and Glu77 affected lambda-PPase catalysis and metal ion binding, whereas substitutions at residues Arg53 and Arg73 affected catalysis and substrate binding. Each of these residues is invariant in all phosphoprotein phosphatases, suggesting that these residues may play important roles in binding and catalysis in all of the PPases. Computer-assisted sequence alignment further revealed that lambda-PPase residues Asp20, His22, Asp49, His76, Arg53, and Arg73 lie within three larger regions of PPase sequence identity with the consensus sequence (DXH-(approximately 25)-GDXXD-(approximately 25)-GNHD/E). This motif can be found in a wide variety of phosphoesterases unrelated to the PPases and defines structural and catalytic features utilized by a diverse group of enzymes for the hydrolysis of phosphate esters.

Amino Acid Sequence↗

PubMed1993

Expression, purification, crystallization, and biochemical characterization of a recombinant protein phosphatase.

A protein phosphatase (PPase) from the bacteriophage lambda was overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity yielding approximately 17 mg of enzyme from a single liter of bacterial culture. Biochemical characterization of the enzyme showed that it required Mn2+ or Ni2+ as an activator. The recombinant enzyme was active toward serine, threonine, and tyrosine phosphoproteins and phosphopeptides. Surprisingly, the bacterial histidyl phosphoprotein, NRII, was also dephosphorylated by the lambda-PPase. The lambda-PPase shares a number of kinetic and structural properties with the eukaryotic Ser/Thr phosphatases, suggesting that the lambda-PPase will serve as a good model for structure-function studies. Crystallization of the recombinant purified lambda-PPase yielded monoclinic crystals. The crystals diffract to 4.0 A when exposed to synchrotron x-ray radiation.

Amino Acid Sequence↗

PubMed1993

Photoinactivation of the bovine heart mitochondrial F1-ATPase by [14C]dequalinium cross-links phenylalanine-403 or phenylalanine-406 of an alpha subunit to a site or sites contained within residues 440-459 of a beta subunit.

Synthesis of [14C]dequalinium, 1,1'-(1,10-[1,10-14C]decanediyl)bis[4-amino-2-methylquinolinium ], is described, which photoinactivates the bovine heart mitochondrial F1-ATPase (MF1). Maximal photoinactivation occurs on incorporation of about 1.5 mol of [14C]dequalinium/mol of MF1. Three radioactive species were resolved when photoinactivated enzyme was submitted to polyacrylamide gel electrophoresis at pH 4.0 in the presence of tetradecyltrimethylammonium bromide, which correspond to the alpha and beta subunits and a cross-linked species with an M(r) of 116,000. Fractionation of a tryptic digest of photoinactivated enzyme by high-performance liquid chromatography led to isolation of a radioactive peptide which contains residues 399-420 of a alpha subunit. Two fragments containing equal amounts of radioactivity were obtained on fractionation of an endoproteinase Asp-N digest of the isolated radioactive tryptic peptide by high-performance liquid chromatography. Amino acid sequence analysis showed that both fragments contained residues 399-408 of the alpha subunit, but one was missing Phe-alpha 403 and the other was lacking Phe-alpha 406. Fractionation of a cyanogen bromide digest of photoinactivated enzyme followed by trypsin digestion of partially purified cyanogen bromide fragments and fractionation of the resulting radioactive tryptic fragments yielded several radioactive species comprised of residues 399-420 of the alpha subunit cross-linked to residues 440-459 of the beta subunit and a radioactive fragment containing residues 399-420 of the alpha subunit. Partial sequence analyses of the cross-linked fragments suggest that Phe-alpha 403 and Phe-alpha 406 participate in cross-links, whereas no information was obtained on the site or sites of cross-linking in the beta subunit fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Irradiation of the bovine mitochondrial F1-ATPase previously inactivated with 5'-p-fluorosulfonylbenzoyl-8-azido-[3H]adenosine cross-links His-beta 427 to Tyr-beta 345 within the same beta subunit.

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p'-fluorosulfonylbenzoyl-8-azidoadenosine (8-N3-FSBA) with an apparent Kd of 0.47 mM at pH 8.0 and 23 degrees C in the absence of light. Irradiation of dark-inactivated enzyme with long-wavelength UV light produced cross-linked dimers and, to a lesser extent, trimers made up of alpha and beta subunits. Two major radioactive peptides were resolved by high-performance liquid chromatography from tryptic digests of MF1 which had been inactivated with 8-N3-FSB[3H]A at pH 8.0 in the dark. Sequence analysis revealed that one contained Tyr-beta 368 and the other contained His-beta 427 which were labeled in the ratio of 18:15. Sequence analysis of radioactive tryptic peptides isolated from digests of irradiated MF1 derivatized with 8-N3-FSB[3H]A showed that photolysis induced cross-linking of His-427 to Tyr-345 within the same beta subunit in high yield. When MF1 derivatized with 8-N3-FSB[3H]A was irradiated in the presence of beta-mercaptoethanol, alpha-beta cross-links were eliminated, whereas those between His-beta 427 and Tyr-beta 345 were unaffected. Analysis of radioactive peptides in tryptic digests of MF1 derivatized with 8-N3-FSB[3H]A and then irradiated in the presence or absence of beta-mercaptoethanol showed that the nitrene generated from reagent attached to Tyr-beta 368 participates in formation of alpha-beta cross-links in the absence of beta-mercaptoethanol. Therefore, the nitrene generated from reagent tethered to His-beta 427 is shielded from solvent and reacts with the side chain of Tyr-beta 345. In contrast, the nitrene generated from reagent attached to Tyr-beta 368 is exposed to solvent, but in the absence of scavengers reacts with side chains present in the alpha subunit. Irradiation of MF1, partially inactivated with 8-N3-FSBA, led to loss of residual ATPase activity without affecting residual ITPase activity. The amount of photoinactivation was greater when partial dark inactivation was performed at pH 6.9, where modification of His-beta 427 predominates, than when performed at pH 8.0, where modification of Tyr-beta 368 predominates. This suggests that cross-linking of His-beta 427 to Tyr-beta 345, and not cross-linking of alpha and beta subunits, is responsible for the augmented inactivation induced by irradiation.

Adenosine↗

PubMed1992

Functional sites in F1-ATPases: location and interactions.

This review focuses on the location and interaction of three functional sites in F1-ATPases. These are catalytic sites which are located in beta subunits, noncatalytic nucleotide-binding sites which are located at interfaces of alpha and beta subunits and modulate the hydrolytic activity of the enzyme, and a site that binds inhibitory amphipathic cations which is at an interface of alpha and beta subunits. The latter site may participate in transmission of conformational signals between catalytic sites in F1 and the proton-conducting apparatus of F0 in the intact ATP synthases.

Amino Acid Sequence↗

PubMed1988

Inhibition and photoinactivation of the bovine heart mitochondrial F1-ATPase by the cytotoxic agent, dequalinium.

The bovine heart mitochondrial F1-ATPase is inhibited in the dark by the amphipathic cation, dequalinium, with a I0.5 of about 12 microM at pH 7.5. When illuminated at 350 nm in the presence of 1.7 microM dequalinium, the F1-ATPase is inactivated with a pseudo-first order rate constant of 7.9 X 10(-3) min-1. The apparent Kd of the dequalinium-enzyme complex was estimated to be about 12.5 microM by examining the rate of inactivation of the ATPase with 1.7-16.7 microM dequalinium. ATP, ADP, Pi, and Mg2+, singly or in combination, protected the ATPase against photoinactivation, with Mg2+ plus Pi being the most effective.

Adenosine Triphosphate↗