Biomedical subjects

H Wu

Publications and source records attributed to H Wu.

At least 217 records · Page 12Linked to original sources

PubMed2001

[Homologous recombination in Streptomyces lincolnensis B48].

To study frequency and mechanism of homologous recombination in Streptomyces, an E. coli plasmid which cannot replicate in Streptomyces was transformed into Streptomyces lincolnensis B48. After homologous recombination between delta lincomycin biosynthetic genes inactivated by thiostrepton resistant gene (tsr) carried on pYYE04al and homologous sequences on the chromosome, S. lincolnensis YY1 and S. lincolnensis, YY2 were obtained on SMA with low thiostrepton concentration. Hybridization of chromosomal DNA samples of S. lincolnensis YY1, S. lincolnensis YY2, standard S. lincolnensis and S. lincolnensis YYc digested with SmaI with the probe of tsr gene gave signal corresponding to a fragment of 1.5 kb in the former two; Nevertheless, hybridization of chromosomal DNA digested with Hind III and Sma I using the probe of delta lacZ' gene resulted in positive fragment of 4.4 kb only in S. lincolnensis YY2. Southern hybridizations indicate that S. lincolnensis YY1 is the result of homologous exchange while S. lincolnensis YY2 comes from bomologous recombination. To prove the existence of E. coli replicon and ampicillin resistant gene on the chromosome of S. lincolnensis YY2, its DNA digested with SphI was ligated and then transformed into E. coli JM83 competent cell. Two transformants named pSLE1 grew on the plate containing ampicillin. It's confirmed that pSLE1 is a part of pYYE04a1 from its digestion with different enzymes.

Chromosomes, Bacterial↗

PubMed2001

[Effect of rhubarb on intestinal immune associated secretion in healthy mice and in burn mice].

OBJECTIVE: To explore the therapeutic mechanism of rhubarb (sennosides) in protecting intestinal muco-membranous barrier by observing the change of intestinal immune associated secretion (IIAS) in mice before and after burn and the enhancing effect of rhubarb on it in healthy and in burn mice. METHODS: Bal b/c mice were divided into 4 groups. Group A (n = 8), untreated healthy mice; Group B (n = 11), healthy mice treated with 10% rhubarb decoction 12 ml/kg, once every 6 hrs; Group C (n = 7), the mice with 30% back full-thickness burn and no treatment was given; Group D (n = 9), the mice with 30% back full-thickness burn and treated with 10% rhubarb decoction 12 ml/kg, once every 6 hrs. The animals were killed 24 hrs after burn, the intestinal juice was collected after intestinal lavage, and centrifuged for determination of IgA, total protein, C3 and high density lipoprotein. RESULTS: IgA content in intestinal juice of Group A, B, C and D were 93.5 +/- 13.7 mg/L, 150.8 +/- 44.6 mg/L, 43.4 +/- 21.5 mg/L and 59.8 +/- 19.3 mg/L respectively, suggesting that it was significantly reduced by burn (P < 0.05), while rhubarb decoction could increase it in either healthy or burn mice (P < 0.05). The other immune associated substance, such as total protein, C3 and high density lipoprotein in intestinal juice were also increased significantly after rhubarb decoction treatment. CONCLUSION: Rhubarb could increase the reduced intestinal juice IgA content in mice caused by burn, it may be an important mechanism of rhubarb in protecting muco-membranous barrier.

Adjuvants, Immunologic↗

PubMed2001

[Effects of calcium channel antagonist on gastrin-induced proliferation of HT29 colon carcinoma cells].

OBJECTIVE: This study aimed to assess the effects of nifedipine on gastrin-induced proliferation of HT29 colon carcinoma cells and inquire into the possible mechanisms. METHODS: Flow cytometry was used to monitor the cytoplasmic free calcium; MTT colorimetry was used to determine the proliferation of HT29 cells. RESULTS: The results showed that 2.5 x 10(-6) mol/L pentagastrin (PG) induced a quick rise of intracellular free calcium ([Ca2+]i) (P < 0.01). 10(-5) mol/L nifedipine can significantly inhibited the rise of [Ca2+]i induced by pentagastrin (P < 0.01), in parallel, at growth assay we demonstrated that 10(-5)-10(-6) mol/L nifedipine could obviously block the increase in cell number elicited by 2.5 x 10(-6) mol/L PG. CONCLUSION: These data indicate that nifedipine can stop the influx of Ca2+ and hence inhibit the pentagastrin-induced proliferation.

Biological Transport, Active↗

PubMed2001

[Operative outcome of patients with left ventricular giant aneurysm: mid-term follow-up in 49 patients].

OBJECTIVE: To assess the mid-term outcome of the patients with left ventricular aneurysm repaired, and analyze the factors influencing early and late mortality and morbidity. METHODS: From January 1992 to November 1998, 58 patients underwent surgical repair for postischemic left ventricular aneurysm. In the 56 survivals, 49 patients (87.5%) were followed-up for an average of 47.6 months (the longest time was 90.0 months). RESULTS: Early mortality rate was 3.4% (2 patients). In the follow-up period (mean 47.6 +/- 22.4 months), 10 patients died. The 5-year survival rates were 63.7%. The left ventricular end-diastolic diameter and left ventricular ejection fraction were improved significantly 29.0 months after operation. There was significant improvement in mean NYHA (from class 2.5 +/- 0.7 to 1.3 +/- 0.5). Of the survivors, 43 (87.8%) were free from angina, and 7 had arrhythmias. Multivariate analysis showed that left ventricular end-diastolic diameter larger than 70 mm and left ventricular ejection fraction lower than 35% were independent risk factors. 33% patients were free from drugs. CONCLUSION: Left ventricular aneurysm repair is an important therapeutic intervention and can be performed successfully. Patients who could benefit from the operation are those with a normal postoperative contraction pattern. Cardiac surgical procedures;

Adult↗

PubMed2000

Effect of preintervention plaque burden on subsequent intimal hyperplasia in stented coronary artery lesions.

We sought to determine if axial and circumferential distribution of plaque before stenting determines the axial and circumferential distribution of subsequent intimal hyperplasia (IH). We studied 22 patients with a single Palmaz-Schatz stent implanted in a native coronary artery, who underwent intravascular ultrasound (IVUS) imaging before intervention, after stenting, and at 6-month follow-up. For each lesion, 7 locations were analyzed: proximal and distal reference, proximal and distal edge of the stent, proximal and distal location within the body of the stent, and the articulation. Pre- and postintervention and follow-up image slices were precisely aligned and analyzed for pre- and postintervention plaque area and follow-up IH area and thickness. The location of maximal IH area was at or adjacent to the location of maximal preintervention plaque in 17 of 22 of the patients (77%). Similiarly, the circumferential distribution of IH at follow-up paralleled the eccentricity pattern of the native plaque burden in 69% (24 of 35 slices). Using multivariant analysis, the strongest predictor of IH was preintervention plaque area (p = 0.001). IH accumulates axially and circumferentially preferentially at the site of maximal preintervention plaque.

Angioplasty, Balloon, Coronary↗

PubMed2000

Identification of a novel cis element required for cell density-dependent down-regulation of insulin-like growth factor-2 P3 promoter activity in Caco2 cells.

The activity of the exogenous, full-length insulin-like growth factor-2 (IGF-2) P3 promoter is significantly up-regulated during the logarithmic growth phase but rapidly declines in confluent CaCo2 cells undergoing differentiation. Nuclear run-on assays confirmed cell density-dependent regulation of endogenous P3 promoter. To identify regulatory elements in the P3 promoter that may be required for regulating cell density-dependent transcriptional activity, we used the methods of promoter truncation, electrophoretic mobility shift assay, DNase footprinting, and mutation analysis. The relative activity of the full-length (-1229/+140) and truncated (-1090/+140) promoter was identical, being approximately 19, 27, 7, and 3% of pSV-luc activity on days 3, 5, 7, and 9 of cell culture, respectively. However, truncation to -1048 resulted in complete loss of cell density-dependent down-regulation of P3 promoter activity on days 7 and 9, suggesting the presence of regulatory elements between -1091 and -1048 sequence. Further stepwise truncation to -515 did not change promoter activity. Truncation to -138/+140 resulted in complete loss of promoter activity, suggesting that the core promoter was within the -515/-138 segment. A 14-base pair footprint (-1084/-1070) was identified by DNase footprinting within the distal -1091/-1048 segment. Electrophoretic mobility shift assay with wild type and mutant probes confirmed the presence of a novel 7-base pair (CGAGGGC) (-1084/-1078) cis element (P3-D); its mutation abolished binding. Functionality of P3-D cis element was confirmed by measuring the activity of core P3 promoter ligated to distal P3 segment containing either the mutant or wild type P3-D element. We have, therefore, identified a novel cis element, P3-D, that appears to play a critical role in regulating IGF-2 P3 promoter activity in a cell density/differentiation-dependent manner.

Base Sequence↗

PubMed2000

Sequencing oligonucleotides with blocked termini using exonuclease digestion and electrospray mass spectrometry.

A method for sequencing ODNs with both termini blocked using mass spectrometry (MS) is reported. The ladder sequencing method is based on our investigation and understanding of critical factors affecting snake venom phosphodiesterase (SVP) digestion of such ODNs. To produce sequence ladders suitable for MS analysis, digestion conditions such as SVPs from three snake species and pH values of digestion buffer were investigated. SVP of Crotalus duressus terrificus (SVP I) was found to be the most suitable for sequencing ODNs with both termini blocked. The pH value of 9.4, which is optimal for SVP digestion of unmodified ODNs, was found to be unsuitable for ladder sequencing ODNs with both termini blocked. Instead, digestion in a wide range of pH values (pH 5-8), including rarely used acidic conditions, was found to be necessary to obtain otherwise unobtainable sequence information. With digestion buffer of desired pH values, sequence ladders which are recorded as MWs of truncated ODNs from SVP digestion are obtained. Examples of sequencing ODNs up to 26 bases long with both termini blocked are demonstrated in this work.

Animals↗

PubMed2000

Human RNase III is a 160-kDa protein involved in preribosomal RNA processing.

A human RNase III gene encodes a protein of 160 kDa with multiple domains, a proline-rich, a serine- and arginine-rich, and an RNase III domain. The expressed purified RNase III domain cleaves double-strand RNA and does not cleave single-strand RNA. The gene is ubiquitously expressed in human tissues and cell lines, and the protein is localized in the nucleus of the cell. The levels of transcription and translation of the protein do not change during different phases of the cell cycle. However, a significant fraction of the protein in the nucleus is translocated to the nucleolus during the S phase of the cell cycle. That this human RNase III is involved in processing of pre-rRNA, but might cleave at sites different from those described for yeast RNase III, is shown by antisense inhibition of RNase III expression. Inhibition of human RNase III expression causes cell death, suggesting an essential role for human RNase III in the cell. The antisense inhibition technique used in this study provides an effective method for functional analysis of newly identified human genes.

Amino Acid Sequence↗

PubMed2000

Desensitization and resensitization of adrenomedullin-sensitive receptor in rat mesangial cells.

Adrenomedullin is a potent adenylate cyclase activator and a vasodilatory peptide, that has anti-proliferative and apoptotic effects in rat mesangial cells. The present study was designed to determine the mechanisms of desensitization and resensitization of adrenomedullin-sensitive receptor in mesangial cells. Adrenomedullin caused a rapid desensitization of cAMP response evident within 5 min that was almost complete by 1 h of treatment. Pretreatment of cells with forskolin, that activates protein kinase-A by direct activation of adenylate cyclase, also caused adrenomedullin receptor desensitization. In addition, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿], a potent protein kinase-A inhibitor inhibited adrenomedullin-induced desensitization of cAMP response. Adrenomedullin also caused desensitization of isoproterenol- and epinephrine-mediated cAMP accumulation. Furthermore, adrenomedullin induced cross-desensitization of endothelin-stimulated inositol phosphate accumulation. The attenuated cAMP response of adrenomedullin was restored to original levels within 2 h of agonist removal. This resensitization response was blocked by treatment with okadaic acid, a protein phosphatase (protein phosphatase-1/protein phosphatase-2A) inhibitor, during the 2 h resensitization period, indicating that protein phosphatase-1/protein phosphatase-2A may be involved in the resensitization of the adrenomedullin-sensitive receptor. We demonstrate for the first time in rat mesangial cells that the adrenomedullin-sensitive receptor undergoes heterologous desensitization and resensitization, and that it likely involves protein kinase-A and protein phosphatase-1/protein phosphatase-2A, respectively.

Adrenomedullin↗

PubMed2000

Assessing a strategy of initial stand-alone extractional atherectomy followed by staged stent placement in degenerated saphenous vein graft lesions.

To assess whether a staged strategy (initial stand alone transluminal extraction atherectomy and coumadin therapy followed by stenting six weeks later) could reduce ischemic complications in degenerated saphenous vein graft (SVG) interventions, we studied 72 patients undergoing percutaneous interventions of degenerated SVG. Patients were divided into two groups; 28 were treated with a staged strategy (group I) and 44 with similar lesion characteristics were treated with a definitive initial procedure with transluminal extraction atherectomy +/- adjunctive balloon angioplasty and stenting (group II). Procedural success, major in-hospital complications (death, Q-wave myocardial infarction, and emergent coronary bypass surgery), and incidence of distal embolization were compared between the 2 groups. Procedural success was lower (92% vs 100%, p = 0.14) and major in-hospital complications were higher (0% vs 11%, p = 0.14) in group II. Distal embolization occurred in 11% of the patients in group I compared with 23% of the patients in group II (p = 0.19). At 6 week follow-up (group I), 9 patients (33%) had negative symptoms, 11 (41%) underwent stent implantation, 3 (11%) did not require any further therapy (without significant stenosis), and 4 (14%) had total occlusions. We therefore conclude that this staged strategy in degenerated SVG appears to reduce distal embolization but most importantly avoids major in-hospital complications, including any deaths either at the time of initial procedure or during the 6-week follow-up period.

Aged↗

PubMed2000

Intramolecular interactions regulate SAP97 binding to GKAP.

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Autocrine gastrins in colon cancer cells Up-regulate cytochrome c oxidase Vb and down-regulate efflux of cytochrome c and activation of caspase-3.

Suppression of the gastrin gene in human colon cancer cells by stably expressing antisense (AS) gastrin RNA results in significant growth suppression of AS cells. To understand mechanisms mediating the growth effects of autocrine gastrins, differential expression of transcripts by AS and control (C) clones of a representative cell line (HCT-116) was analyzed to identify target genes of autocrine gastrins. Six differentially expressed transcripts were confirmed and sequenced. Of these, the RNA and protein levels of cytochrome c oxidase (COX) Vb were significantly higher in C versus AS cells. The expression of COX Vb by colon cancer cells was proportional to the expression of gastrin. Higher levels of COX Vb coprecipitated with cytochrome c in the mitochondria of C versus AS cells. Treatment of mitochondria with digitonin resulted in a 2-fold higher release of cytochrome c from AS versus C mitochondria. As a corollary, the cytosolic levels of cytochrome c were significantly higher in AS versus C cells, which correlated with approximately 2- and approximately 3-fold higher activation of caspase-9 and -3, respectively, in AS versus C cells in response to camptothecin. Thus, autocrine gastrins may support growth/survival of cells by up-regulating COX Vb, which may decrease the sensitivity of the cancer cells to apoptotic stimuli by increasing retention of cytochrome c in mitochondria.

Colonic Neoplasms↗

PubMed2000

Accessory cell defect in unresponsiveness of neonates and aged to polysaccharide vaccines.

T independent antigens elicit antibody responses in the absence of carrier specific T helper cells but require signals from accessory cells (macrophages and dendritic cells) or specific cytokines. They are further subdivided into TI-1 and TI-2 categories based on the ability of TI-1 but not TI-2 antigens to elicit immune responses from neonates. Most bacterial polysaccharides including the pneumococcal polysaccharide vaccines belong to the TI-2 class. It is hypothesized that defects in accessory cell function play a critical role in the failure of neonates to respond to such TI-2 antigens. Immune responses to these TI-2 stimuli are also reduced in the aged, also due to a quantitative deficiency in accessory cells. Agents that can stimulate accessory cell function may provide an alternative strategy to improve the immunogenicity of the polysaccharide vaccines in the neonates and the aged.

Aged↗

PubMed2000

Solution structure of BmP01 from the venom of scorpion Buthus martensii Karsch.

From the venom of scorpion Buthus martensii Karsch,a short peptide (BmP01, 29 amino acid residues) was isolated and characterized as previously reported (Lebren, R. R., et al. (1997) Eur. J. Biochem. 245, 457-464). It was shown to reduce 33% outward K(+) channel (hippocampal neurons) currents at 10 microM. The solution structure of BmP01 was determined by 2D (1)H NMR spectroscopy. The NOEs, coupling constants, and H-D exchange obtained from NMR spectroscopy were used in structural calculations. The conformation of BmP01 is composed of a short alpha-helix (Cys 3-Thr 12) and a two-stranded antiparallel beta-sheet (Ala 15-Asp 20 and Lys 23-Pro 28). There are three disulfide bridges (Cys 3-Cys 19, Cys 6-Cys 24 and Cys 10-Cys 26) connecting the alpha-helix and beta-sheet. Asp 20 to Lys 23 form a type II turn linking the two strands. Structural and electrostatic potential comparison between BmP01 and its analogues are also presented.

Amino Acid Sequence↗

PubMed2000

p55CDC/hCDC20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase.

Eukaryotic cells have evolved a mechanism that delays the progression of mitosis until condensed chromosomes are properly positioned on the mitotic spindle. We have been studying genes that regulated the spindle checkpoint in human cells. Enforced expression of human BUBR1, but not a BUBR1 mutant allele, enhances accumulation of mitotic cells. Yeast two-hybrid system and GST-pulldown analyses show that p55CDC/hCdc20, a protein known to link spindle checkpoint components such as MAD2 to anaphase promoting complex (APC), interacts with BUBR1. In addition, p55CDC is capable of pulling down BUBR1 in sf-9 cells infected with both p55CDC and His6-BUBR1 recombinant baculoviruses but not in the cells infected with p55CDC baculoviruses or with the baculoviral vector alone. Moreover, immunoprecipitation followed by Western blot analyses confirmed that native p55CDC is associated with BUBR1 in HeLa cells. Spindle checkpoint activation by nocodazole treatment enhances the association between p55CDC and His6-BUBR1. In nocodazole-arrested mitotic cells, both CDC16 and hyperphosphorylated CDC27, two APC components, preferentially associate with His6-BUBR1 resins, but not the control resins. Furthermore, BUBR1 phosphorylates p55CDC in vitro, and the phosphorylation of p55CDC by BUBR1 appears to be correlated with spindle checkpoint activation. Together, our studies strongly suggest that BUBR1 may target APC via p55CDC.

Alleles↗

PubMed2000

Molecular basis for CD40 signaling mediated by TRAF3.

Tumor necrosis factor receptors (TNFR) are single transmembrane-spanning glycoproteins that bind cytokines and trigger multiple signal transduction pathways. Many of these TNFRs rely on interactions with TRAF proteins that bind to the intracellular domain of the receptors. CD40 is a member of the TNFR family that binds to several different TRAF proteins. We have determined the crystal structure of a 20-residue fragment from the cytoplasmic domain of CD40 in complex with the TRAF domain of TRAF3. The CD40 fragment binds as a hairpin loop across the surface of the TRAF domain. Residues shown by mutagenesis and deletion analysis to be critical for TRAF3 binding are involved either in direct contact with TRAF3 or in intramolecular interactions that stabilize the hairpin. Comparison of the interactions of CD40 with TRAF3 vs. TRAF2 suggests that CD40 may assume different conformations when bound to different TRAF family members. This molecular adaptation may influence binding affinity and specific cellular triggers.

Amino Acid Sequence↗

PubMed2000

D-Alanine substitution of teichoic acids as a modulator of protein folding and stability at the cytoplasmic membrane/cell wall interface of Bacillus subtilis.

The extracytoplasmic folding of secreted proteins in Gram-positive bacteria is influenced by the microenvironment of the compartment into which they are translocated, namely the negatively charged matrix of the cell wall polymers. In this compartment, the PrsA lipoprotein facilitates correct post-translocational folding or prevents misfolding of secreted proteins. In this study, a secretion mutant of B. subtilis (prsA3) encoding a defective PrsA protein was mutagenized and screened for restored secretion of the AmyQ alpha-amylase. One mini-Tn10 insertion, which partially suppressed the secretion deficiency, was found to interrupt dlt, the operon involved in the d-alanylation of teichoic acids. The inactivation of dlt rescued the mutant PrsA3 protein from degradation, and the increased amount of PrsA3 was shown to enhance the secretion of PrsA-dependent proteins. Heterologous or abnormal secreted proteins, which are prone to degradation after translocation, were also stabilized and secreted in increased quantities from a dlt prsA(+) strain. Furthermore, the dlt mutation partially suppressed the lethal effect of PrsA depletion, suggesting that the dlt deficiency also leads to stabilization of an essential cell wall protein(s). Our results suggest that main influence of the increased net negative charge of the wall caused by the absence of d-alanine is to increase the rate of post-translocational folding of exported proteins.

Alanine↗

PubMed2000

Molecular cloning and analysis of a group of genes differentially expressed in cells which overexpress the Hoxa-1 homeobox gene.

The homeobox gene Hoxa-1 is transcriptionally regulated by retinoic acid (RA) and encodes a transcription factor which has been shown to play important roles in cell differentiation and embryogenesis. In order to clone and characterize target genes of Hoxa-1, we utilized differential hybridization screening and cDNA subtractive hybridization methods to identify genes which are differentially expressed in F9-10, a murine F9 teratocarcinoma stem cell line which expresses high levels of exogenous Hoxa-1, compared to F9 wild-type stem cells, which do not express endogenous Hoxa-1 mRNA in the absence of RA. Twenty-eight candidate genes were identified; these genes encode very diverse proteins, including signaling molecules such as BMP-4, the enzyme superoxide dismutase, the cell adhesion molecule cadherin-6, proteins involved in gene transcription such as HMG-1 and SAP18, homeodomain-containing proteins Gbx-2 and Evx-2, and cell cycle regulatory proteins such as the retinoblastoma binding protein-2. Clone 104 encodes a novel protein; the expression of the clone 104 mRNA is also regulated in a fashion very similar to that of the exogenous Hoxa-1 gene in another F9 cell line, called F9-tet-Hoxa1-8, in which the exogenous Hoxa-1 mRNA expression is tightly regulated by a Tet-off gene expression system. These data strongly suggest that clone 104 is a direct downstream target of the transcription factor Hoxa-1. The cDNA sequence of clone 104 is related to that of human ubiquitin carboxyl-terminal hydrolase T. Further characterization of these putative Hoxa-1 target genes will aid in delineating the functions of the Hoxa-1 protein in the differentiation processes which occur during embryogenesis.

Animals↗