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P Wu

Publications and source records attributed to P Wu.

At least 163 records · Page 9Linked to original sources
PubMed1997

Expression, purification, and characterization of human cAMP-specific phosphodiesterase (PDE4) subtypes A, B, C, and D.

Although four members (A, B, C, and D) of the cAMP-specific phosphodiesterase (PDE4) family have been cloned by different groups, no study comparing the characteristics of purified human PDE4 subtypes has been published. In this study, we have expressed human PDE4 A, B, C, and D in insect (SF9) cells by using the baculovirus expression system, purified the expressed proteins, and compared their characteristics. The recombinant PDE4 subtypes all showed catalytic activity for cAMP with a K(m) of 1-5 microM. V(max) values differed significantly among these subtypes with the following order: C > B > A > D. PDE4 A, B, C, and D showed a very similar Mg2+ dependence profile. PDE4 B and C showed similar pH profiles with the optimal pH being 8.0. The pH profiles of PDE4 A and D were very different from each other and from those of B and C, with the optimal pH being 6.5 and 7.5, respectively. Furthermore, although PDE4 A, B, C, and D were all inhibited by the standard PDE4 inhibitors rolipram, Ro20-1724, and etazolate, the inhibitory potency varied. Thus, by several criteria including kinetics, pH dependency, and inhibitor sensitivity, various PDE4 subtypes differ significantly from one another.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Reconstitution of mitogen-activated protein kinase phosphorylation cascades in bacteria. Efficient synthesis of active protein kinases.

Mitogen-activated protein (MAP) kinase pathways include a three-kinase cascade terminating in a MAP kinase family member. The middle kinase in the cascade is a MAP/extracellular signal-regulated kinase (ERK) kinase or MEK family member and is highly specific for its MAP kinase target. The first kinase in the cascade, a MEK kinase (MEKK), is characterized by its ability to activate one or more MEK family members. A two-plasmid bacterial expression system was employed to express active forms of the following MEK and MAP kinase family members: ERK1, ERK2, alpha-SAPK, and p38 and their upstream activators, MEK1, -2, -3, and -4. In each kinase module, the upstream activator, a constitutively active mutant of MEK1 or MEKK1, was expressed from a low copy plasmid, while one or two downstream effector kinases were expressed from a high copy plasmid with different antibiotic resistance genes and origins of replication. Consistent with their high activity, ERK1 and ERK2 were doubly phosphorylated on Tyr and Thr, were recognized by an antibody specific to the doubly phosphorylated forms, and were inactivated by either phosphoprotein phosphatase 2A or phosphotyrosine phosphatase type 1. Likewise, activated p38 and alpha-stress-activated protein kinase could also be inactivated by either phosphatase, and alpha-stress-activated protein kinase was recognized by an antibody specific to the doubly phosphorylated forms. These three purified, active MAP kinases have specific activities in the range of 0.6-2.3 micromol/min/mg. Coexpression of protein kinases with their substrates in bacteria is of great value in the preparation of numerous phosphoproteins, heretofore not possible in procaryotic expression systems.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation.

Development of the lymphoid system is dependent on the activity of zinc finger transcription factors encoded by the Ikaros gene. Differences between the phenotypes resulting from a dominant-negative and a null mutation in this gene suggest that Ikaros proteins act in concert with another factor with which they form heterodimers. Here we report the cloning of Aiolos, a gene which encodes an Ikaros homologue that heterodimerizes with Ikaros proteins. In contrast to Ikaros--which is expressed from the pluripotent stem cell to the mature lymphocyte--Aiolos is first detected in more committed progenitors with a lymphoid potential and is strongly up-regulated as these differentiate into pre-T and pre-B cell precursors. The expression patterns of Aiolos and Ikaros, the relative transcriptional activity of their homo- and heteromeric complexes, and the dominant interfering effect of mutant Ikaros isoforms on Aiolos activity all strongly suggest that Aiolos acts in concert with Ikaros during lymphocyte development. We therefore propose that increasing levels of Ikaros and Aiolos homo- and heteromeric complexes in differentiating lymphocytes are essential for normal progression to a mature and immunocompetent state.

3T3 Cells↗
PubMed1997

An automated high throughput filtration assay: application to polymerase inhibitor identification.

A high throughput filtration assay was developed and automated for screening compounds and natural product extracts using 96-well filterplates. The selection of a hydrophobic membrane and appropriate capture conditions enabled us to perform the enzymatic reaction, capture of products, vacuum filtration, and detection in a single filtration microplate, without any transfer step. The hydrophobic membrane has the advantage of preventing any loss of aqueous solution during the enzymatic reaction. The results are comparable with those obtained in a solid plate format followed by transferring the reaction mixture to a traditional hydrophilic filterplate. This assay is being used to screen for microbial RNA polymerase inhibitors as potential new drugs for treatment of emerging antibiotic-resistant bacterial and fungal infections. The filtration setup can be applied to a variety of assay systems.

Cytidine Triphosphate↗
PubMed1997

cDNA sequence analysis and expression of kappa-bungarotoxin from Taiwan banded krait.

The cDNAs encoding kappa-bungarotoxin was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus by reverse transcription-polymerase chain reaction. A high degree of nucleotide sequence homology was observed between kappa-bungarotoxin and other kappa-neurotoxins. The kappa-bungarotoxin was subcloned into the expression vector pET32a(+) and transformed into BL21(DE3) E. coli strain. The recombinant toxin was expressed as a fusion protein. Recombinant kappa-bungarotoxin was separated from the fused protein by cleavage with CNBr and purified by reversed phase high performance liquid chromatography. In addition to kappa-bungarotoxin, the cDNA fragment encoding kappa3-bungarotoxin was also found in the cDNA mixtures prepared from the cellular RNA of the venom glands of the same snake. This result suggests that the venom glands of Taiwanese B. multicinctus should secrete at least two kinds of kappa-neurotoxins.

Amino Acid Sequence↗
PubMed1997

T cell cytokine pattern in the joints of patients with Lyme arthritis and its regulation by cytokines and anticytokines.

OBJECTIVE: To investigate whether type 1 helper (Th1) or Th2 cytokines are found in the joints of patients with Lyme arthritis, and whether the cytokine pattern can be modulated by cytokines or anticytokines. METHODS: The cytokine pattern in the joints of 10 patients with Lyme arthritis was investigated. Expression of interferon-gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), interleukin-4 (IL-4), and IL-10 was measured by enzyme-linked immunosorbent assay (ELISA), after stimulation of synovial fluid mononuclear cells (SFMC) with Borrelia burgdorferi (Bb) in the supernatant. Expression of cytokine messenger RNA and protein in synovial membrane (SM) and nonstimulated SFMC was studied using semiquantitative reverse transcriptase-polymerase chain reaction and immunohistologic techniques. The effects of recombinant cytokines or neutralizing anticytokine antibodies on cytokine production in Bb-stimulated SFMC were investigated by ELISA. RESULTS: SFMC produced high amounts of IFN gamma and TNF alpha, but little or no IL-4, upon stimulation with Bb antigen, indicating a Th1-type cytokine pattern. In SM, IFN gamma was detectable in all patients, while the other cytokines were less frequently found. Serial sections of SM revealed that all cytokines were located in the same area. The Th1 response, especially the production of TNF alpha, could be down-regulated in vitro by both endogenous and exogenous IL-10, but not by IL-4 or anti-IL-12. CONCLUSION: A Th1-type cytokine pattern was found in the joints of patients with Lyme arthritis. This Th1 response could be down-regulated by IL-10, suggesting insufficient IL-10 production in vivo.

Adolescent↗
PubMed1997

Crucial role of interleukin-10/interleukin-12 balance in the regulation of the type 2 T helper cytokine response in reactive arthritis.

OBJECTIVE: To investigate whether a predominant type 1 T helper (Th1) or Th2 cytokine pattern is present in the joints of patients with reactive arthritis (ReA), and whether the cytokine pattern can be modulated by cytokines or anticytokines. METHODS: Eleven patients with ReA following infection with either Chlamydia trachomatis, Yersinia enterocolitica, or Salmonella enteritidis were investigated for the presence of Th1/Th2 cytokines in the joints. Release of the bacteria-specific cytokines interferon-gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), interleukin-10 (IL-10), and IL-4 was measured in synovial fluid mononuclear cells (SFMC) using enzyme-linked immunosorbent assay and polymerase chain reaction. In the synovial membrane, secretion of IFN gamma and IL-4 was determined by immunohistologic analysis. Cytokine regulation was studied by adding cytokines and anticytokines to the cultures. RESULTS: Upon stimulation with specific bacteria, SFMC secreted low amounts of IFN gamma and TNF alpha, but high amounts of IL-10. IL-10 was responsible for the suppression of IFN gamma and TNF alpha, as judged by the effect of adding either anti-IL-10 antibodies or exogenous IL-10 to these cultures. The addition of neutralizing anti-IL-12 to the cultures completely abolished the effects of anti-IL-10, suggesting that inhibition of the Th1-like cytokines by IL-10 is mediated through suppression of IL-12 synthesis. Exogenous IL-12 clearly enhanced IFN gamma and TNF alpha secretion. In the synovial membrane, a higher number of cells were positive for the Th2 cytokine IL-4, compared with the amount of IFN gamma-secreting cells. CONCLUSION: These data indicate that a Th2 cytokine pattern predominates in the joints of patients with ReA. Since Th1 cytokines are necessary for the elimination of ReA-associated bacteria, Th2 cytokines might contribute to bacterial persistence in the joint. Therefore, the IL-10/IL-12 balance appears to be crucial for regulation of the cytokine pattern in the joints of patients with ReA.

Adolescent↗
PubMed1997

Assessment of postural differences in regional pulmonary perfusion in man by single-photon emission computerized tomography.

1. With the advent of single-photon emission computerized tomography, controversy has arisen with regard to the significance of gravitational influences on regional pulmonary perfusion (Qr) in the supine versus prone postures. We investigated the dorsal-ventral distributions of Qr in prone (n = 5) and supine (n = 5) normal subjects, as assessed by single-photon emission computerized tomography after intravenous injection of technetium-99m-labelled macroaggregated albumin at end-tidal expiration. Reconstructed serial (one pixel thickness) coronal sections were traced on a computer screen, to yield the encompassed radioactive counts and number of pixels per each image. Coronal section data (expressed as mean radioactive counts/pixel) were expressed in a 'profile' and normalized to the maximum coronal section of each lung (% maximum). 2. Coefficients of variation and linear regression slopes for the prone versus supine profiles for left and right lungs were not statistically different (unpaired Student's t-test). The coronal section with maximum Qr was identified in the more dependent lung regions and, hence, affected by gravity. 3. We conclude that, in contrast to previous canine models, which have suggested postural differences in dorsal-ventral perfusion gradients, in normal man gravity primarily determines the non-dependent to dependent distribution of Qr. We speculate that interspecies differences in physiology may be teleological and related to the different perfusion demands of the quadruped compared with upright man.

Adult↗
PubMed1997

Distribution of hepatitis C virus (HCV) RNA in whole blood and blood cell fractions: plasma HCV RNA analysis underestimates circulating virus load.

Previous experiments using a cationic surfactant to detect hepatitis C virus (HCV) RNA in whole blood (WB) suggested that WB was a more plentiful source of viral RNA than was plasma. The relative HCV RNA titers in WB, plasma, peripheral blood mononuclear cells (PBMC), neutrophils, and red blood cells (RBC)/platelets from 10 patients with chronic HCV infection were compared. WB contained significantly more HCV RNA than plasma, which contained more HCV RNA than PBMC, neutrophils, or RBC/platelets (P < .001). To determine if this increased sensitivity was clinically relevant, results of WB and plasma HCV RNA assays were compared with commercial quantitative and qualitative plasma HCV RNA assay results obtained for patients receiving interferon therapy. WB was significantly more sensitive than commercial plasma reverse transcription-polymerase chain reaction for detecting HCV RNA (P < .005). These data indicate that a significant proportion of HCV RNA in peripheral blood is not identified by standard plasma RNA detection methods.

Blood Cells↗
PubMed1997

Surreptitious hepatitis C virus (HCV) infection detected in the majority of patients with cryptogenic chronic hepatitis and negative HCV antibody tests.

Reverse transcription-polymerase chain reaction was used to identify hepatitis C virus (HCV) RNA in peripheral whole blood (WB) and plasma samples from 15 patients with chronic, unexplained hepatitis. These patients were serologically negative for hepatitis A, B, and C and were classified as having chronic non-A, non-B, non-C hepatitis (NANBNC). HCV RNA was repeatedly detected in WB samples from 10 (67%). In contrast, plasma samples from only 5 were intermittently positive. Statistically, HCV RNA detection in WB was significantly more sensitive than in plasma. Nucleic acid hybridization and HCV genotypic analysis confirmed the specificity of the HCV RNA assay. Liver biopsies from these patients suggested histopathologic differences between HCV RNA-positive and -negative groups. These data demonstrate that HCV infection is present in patients with unexplained chronic hepatitis more frequently than previously believed. Additionally, WB HCV RNA detection is more sensitive than plasma assays in identifying antibody-negative HCV infection.

Adult↗
PubMed1997

Nop2p is required for pre-rRNA processing and 60S ribosome subunit synthesis in yeast.

To investigate the function of the nucleolar protein Nop2p in Saccharomyces cerevisiae, we constructed a strain in which NOP2 is under the control of a repressible promoter. Repression of NOP2 expression lengthens the doubling time of this strain about fivefold and reduces steady-state levels of 60S ribosomal subunits, 80S ribosomes, and polysomes. Levels of 40S subunits increase as the free pool of 60S subunits is reduced. Nop2p depletion impairs processing of the 35S pre-rRNA and inhibits processing of 27S pre-rRNA, which results in lower steady-state levels of 25S rRNA and 5.8S rRNA. Processing of 20S pre-rRNA to 18S rRNA is not significantly affected. Processing at sites A2, A3, B1L, and B1S and the generation of 5' termini of different pre-rRNA intermediates appear to be normal after Nop2p depletion. Sequence comparisons suggest that Nop2p may function as a methyltransferase. 2'-O-ribose methylation of the conserved site UmGm psi UC2922 is known to take place during processing of 27S pre-rRNA. Although Nop2p depletion lengthens the half-life of 27S pre-RNA, methylation of UmGm psi UC2922 in 27S pre-rRNA is low during Nop2p depletion. However, methylation of UmGm psi UC2922 in mature 25S rRNA appears normal. These findings provide evidence for a close interconnection between methylation at this conserved site and the processing step that yields the 25S rRNA.

Amino Acid Sequence↗
PubMed1997

Prolonged reduction of high blood pressure with an in vivo, nonpathogenic, adeno-associated viral vector delivery of AT1-R mRNA antisense.

To produce a prolonged decrease in blood pressure, we have developed a nonpathogenic adeno-associated viral vector (AAV) with the antisense DNA for AT1-R. AAV has many advantages over other viral vectors. AAV does not stimulate inflammation or immune reaction. AAV enters nondividing cells and does not replicate. Therefore, it is an appropriate choice for gene therapy. Recombinant AAV was prepared with a cassette containing a cytomegalovirus promoter and the cDNA for the AT1 receptor inserted in the antisense direction. The cassette was packaged in the virion. Stable transfection of NG108-15 cells with the PAAV-AS (plasmid AAV) antisense to AT1-R produced a significant reduction in AT1 receptors. A single injection of the rAAV-AS (viral vector) was made in adult spontaneously hypertensive rats, either directly in the hypothalamus (1 microL) or in the lateral ventricles (5 microL). The result shows that there is a significant decrease of blood pressure (approximately 23 +/- 2 mm Hg) for up to 9 weeks after injection. Control injections of mock vector produced no change in blood pressure during the same time period in age-matched controls. In young spontaneously hypertensive rats (3 weeks), a single intracardiac injection of recombinant rAAV-AS reduced blood pressure and slowed the development of hypertension compared with controls (P < .01). The results suggest that a prolonged reduction in high blood pressure can be achieved with AAV vectors delivering antisense to inhibit AT1 receptors with a single administration.

Animals↗
PubMed1997

Transcription of the rat sarcoplasmic reticulum Ca2+ adenosine triphosphatase gene is increased by 3,5,3'-triiodothyronine receptor isoform-specific interactions with the myocyte-specific enhancer factor-2a.

Thyroid hormone (T3) increases the transcription of the sarcoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase) gene (SERCA 2) through three thyroid hormone response elements. The existence of repetitive cis elements with different configurations is likely to serve specific functions such as interactions with nuclear transcription factors. In addition, the presence of different T3 receptor isoforms (T3Rs) may contribute to another level of complexity in providing specificity for T3 action. In this study, we investigated T3R alpha 1-vs. T3R beta 1-specific interactions with the myocyte enhancer-specific factor-2 (MEF-2) on the expression of the SERCA 2 gene in transient transfection assays in embryonal heart-derived H9c2 cells. MEF-2a in combination with either T3R alpha 1 or T3R beta 1 isoforms resulted in a 2.5-fold increase in SERCA 2 transgene expression in the absence of T3. Addition of T3 did not induce any further increase in SERCA 2 expression when T3R alpha 1 and MEF-2a expression vectors were cotransfected. In contrast, in the presence of T3R beta 1 and MEF-2, the addition of T3 increased chlorampenicol acetyltransferase activity by an additional 2.2-fold to a total 5.5-fold increase. The interaction between MEF-2a and T3R is transcription factor specific because another factor that binds to MEF-2 consensus sites (heart factor 1b) was not able to interact with T3R. In addition, MEF-2a failed to interact with other nuclear factors (cAMP response element-binding protein and Egr-1) that stimulate SERCA 2 gene transcription. In addition, we found that a single homologous thyroid hormone response element is not able to mediate the interactions between MEF-2a and T3Rs to increase SERCA 2 gene transcription. Our findings point to T3R isoform-specific interactions with a cell type-specific transcription factor (MEF-2) in the regulation of SERCA 2 gene expression.

Animals↗
PubMed1997

[Splenic artery embolization for the treatment of traumatic splenic rupture].

We used splenic artery gelfoam embolization to save the spleen Seldinger's method in 20 patients with traumatic rupture of the spleen. All patients had the spleen reserved by single embolizing hemostasis satisfactorily. We delineate the method, clinical value, indication and experience of embolism-hemostasis in detail.

Adult↗
PubMed1997

[An experimental study on tissue renin-angiotensin-aldosterone system].

Aldosterone biosynthesis in kidney has been proved by means of kidney perfusion in vitro, high performance liquid chromatography, radioimmunoassay and TR-PCR, indicating that aldosterone biosynthesis is possible in tissues other than adrenal. Bilateral nephrectomy was carried out in male Wistar rats and the plasma renin activity would disappear after 30 hours. However, RT-PCR showed that the vasculature in the nephrectomized rats was still able to express renin mRNA. It means that the vasculature is different from the heart which depends on taking up renin from the circulation, while the vasculature takes renin produced locally in its own tissue to initiate the renin-angiotensin-aldosterone system. Perindopril, an angiotensin converting enzyme inhibitor (ACEI), inhibits not only the production of angiotensin II, but also the synthesis of aldosterone in the vasculature. ACEI reversion of vascular remodeling is probably related with its inhibition of aldosterone synthesis in the vasculature.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1996

Direct cytotoxicity of HIV-1 envelope protein gp120 on human NT neurons.

A new in vitro system comprising a pure population of neurons, human NT cells, was used to characterize the direct neurotoxic effect of HIV-1 envelope protein gp120. Cytotoxicity was monitored by a quantitative assay after exposure to recombinant gp120 in the presence or absence of other reagents. Treatment of mature NT neurons with various doses of gp120 for 24 h caused a decrease of up to 27% in the number of viable cells. This neurotoxicity was abolished by co-treatment with either D-2-amino-5-phosphonopentanoic acid (APV), MK801 or nimodipine, or by culturing cells in a Ca(2+)-free environment. Taken together, these data indicate that gp120 exerts a direct neurotoxic effect by acting through NMDA receptors and Ca2+ channels.

2-Amino-5-phosphonovalerate↗