Search PubMed⌕ Search

Biomedical subjects

H Wu

Publications and source records attributed to H Wu.

At least 343 records · Page 19Linked to original sources
PubMed1999

Cloning and characterization of the gene for a new epithelial beta-defensin. Genomic structure, chromosomal localization, and evidence for its constitutive expression.

Mammalian beta-defensins are endogenous cysteine-rich peptide antibiotics that are produced either by epithelial cells lining the respiratory, digestive, and urogenital tracts or by granulocytes and macrophages. A growing body of evidence has implicated these peptides in host defense, particularly mucosal innate immunity. We previously reported the cloning of the full-length cDNA for a porcine beta-defensin (pBD-1), which was found to be expressed throughout the airway and oral mucosa. Here, we provide the structural organization of the pBD-1 gene, showing that the entire gene spans approximately 1.9 kilobases with two short exons separated by a 1.5-kilobase intron. Fluorescence in situ hybridization mapped the pBD-1 gene to porcine chromosome 15q14-q15. 1 within a region of conserved synteny to the chromosomal locations of human and mouse alpha- and beta-defensins. We also provide several independent lines of evidence showing that the pBD-1 gene is expressed constitutively during inflammation and infection, despite its resemblance to many inducible epithelial beta-defensins in amino acid sequence, genomic structure, and sites of expression. First, stimulation of primary porcine tongue epithelial cells with lipopolysaccharide, tumor necrosis factor-alpha, and interleukin (IL)-1beta failed to up-regulate the expression of pBD-1 mRNA. Second, pBD-1 gene expression was not enhanced in either digestive or respiratory mucosa of pigs following a 2-day infection with Salmonella typhimurium or Actinobacillus pleuropneumoniae. Last, direct transfection of the pBD-1 gene promoter into NIH/3T3 cells showed no difference in reporter gene activity in response to stimulation by lipopolysaccharide and IL-1beta. The constitutive expression of pBD-1 in airway and oral mucosa, which is consistent with a lack of consensus binding sites for nuclear factor-kappaB or NF-IL-6 in its promoter region, suggests that it may play a surveillance role in maintaining the steady state of microflora on mucosal surfaces.

3T3 Cells↗
PubMed1999

Quantitative angiographic analysis of stent restenosis in the Scripps Coronary Radiation to Inhibit Intimal Proliferation Post Stenting (SCRIPPS) Trial.

To identify luminal dimension changes occurring within the stent alone and within the stent + margin segment, we reviewed the quantitative angiographic results obtained from the Scripps Coronary Radiation to Inhibit Proliferation Post Stenting (SCRIPPS) trial, a prospective randomized trial assessing the effect of iridium-192 (Ir-192) on the prevention of stent restenosis. Fifty-five patients were randomly assigned to receive Ir-192 or placebo sources after successful intervention. Procedural and 6-month follow-up cineangiograms were quantitatively reviewed in 52 patients to identify changes within the stent and the stent + margin segment. The percent diameter stenosis was lower within the stent than within the stent + margin segment after the procedure (6 +/- 22% vs 21+/- 15%, p <0.0001) and at follow-up (28 +/- 29% vs 42 +/- 21%, p <0.0001). As a result, a lower restenosis rate was found within the stent than within the stent + margin (25% vs 37%, p <0.0001); isolated stent margin restenosis occurred in 11.5% of lesions. Treatment with Ir-192 reduced restenosis within the stent (8% vs 39%; p = 0.010) and within the stent + margin segment (17% vs 54%; p = 0.010); the reduction in restenosis at the margin only (8.3% vs 14.3%, p = 0.503) was not significant. The lowest relative risk for restenosis resulting from Ir-192 occurred within the stent (0.21; 95% confidence interval [CI] 0.05 to 0.86) compared with the stent + margin segment (0.31; 95% CI 0.12 to 0.81) or the stent margin (0.58; 95% CI 0.12 to 2.91). In the SCRIPPS trial, 32% of restenosis occurred at the stent margins. Treatment with Ir-192 reduced restenosis primarily within the stent rather than the margin. Whether extending the treatment length to fully include the stent margins will further reduce restenosis requires further study.

Brachytherapy↗
PubMed1999

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases.

BACKGROUND: Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate and CO2 with the concomitant reduction of NAD(P)+ to NAD(P)H. They are widely distributed in nature and have important biological functions. Human mitochondrial NAD(P)+-dependent malic enzyme (mNAD-ME) may have a crucial role in the metabolism of glutamine for energy production in rapidly dividing cells and tumors. Moreover, this isoform is unique among malic enzymes in that it is a cooperative enzyme, and its activity is controlled allosterically. RESULTS: The crystal structure of human mNAD-ME has been determined at 2.5 A resolution by the selenomethionyl multiwavelength anomalous diffraction method and refined to 2.1 A resolution. The structure of the monomer can be divided into four domains; the active site of the enzyme is located in a deep cleft at the interface between three of the domains. Three acidic residues (Glu255, Asp256 and Asp279) were identified as ligands for the divalent cation that is required for catalysis by malic enzymes. CONCLUSIONS: The structure reveals that malic enzymes belong to a new class of oxidative decarboxylases. The tetramer of the enzyme appears to be a dimer of dimers. The active site of each monomer is located far from the tetramer interface. The structure also shows the binding of a second NAD+ molecule in a pocket 35 A away from the active site. The natural ligand for this second binding site may be ATP, an allosteric inhibitor of the enzyme.

Amino Acid Sequence↗
PubMed1999

Long-term follow-up after percutaneous transluminal coronary angioplasty was not performed based on intravascular ultrasound findings: importance of lumen dimensions.

BACKGROUND: Angiography is limited in determining the anatomic severity of coronary artery stenoses. Clinical decision-making in patients with symptoms and intermediate lesions remains challenging. METHODS AND RESULTS: The current analysis included 300 patients (357 intermediate native artery lesions) in whom intervention was deferred based on intravascular ultrasound (IVUS) findings. Standard clinical, angiographic, and IVUS parameters were collected. Patients were followed for >1 year. Events occurred in 24 patients (8%). They included 2 cardiac deaths, 4 myocardial infarctions, and 18 target-lesion revascularizations (TLR; 12 percutaneous transluminal coronary angiographies and 6 coronary artery bypass grafts; only 3 TLRs occurred within 6 months after the IVUS study). All significant univariate clinical, angiographic, and IVUS parameters (P<0.05) were tested in multivariate models. These included diabetes mellitus, IVUS lesion lumen area, maximum lumen diameter, minimum lumen diameter, plaque area, plaque burden, and area stenosis (AS). No angiographic measurement was significant at P<0.05. The only independent predictors of an event (death, myocardial infarction, or TLR) were IVUS minimum lumen area and AS. The only independent predictors of TLR were diabetes mellitus, IVUS minimum lumen area, and AS. In 248 lesions with a minimum lumen area >/=4.0 mm(2), the event rate was only 4.4% and the TLR rate 2.8%. CONCLUSIONS: Long-term follow-up after IVUS-guided deferred interventions in patients with de novo intermediate native artery lesions showed a low event rate. In patients with a minimum lumen area >/=4.0 mm(2), the event rate was especially low. IVUS imaging is an acceptable alternative to physiological assessment in these patients.

Angioplasty, Balloon, Coronary↗
PubMed1999

In situ electrochemical oxygen generation with an immunoisolation device.

The viability and function of transplanted tissue encapsulated in immunobarrier devices is subject to oxygen transport limitation. In this study, we have designed and used an in situ electrochemical oxygen generator which decomposes water electrolyticaly to provide oxygen to the adjacent planer immunobarrier diffusion chamber. The rate of oxygen generation, which increases linearly with electrical current, was accurately controlled. A theoretical model of oxygen diffusion was also developed and was used to calculate the oxygen profiles in some of the experimental systems. In vitro culture experiments were carried out with beta TC3 cells encapsulated in titanium ring devices. The growth and viability of cells with or without in situ oxygen generation was studied. We found that under otherwise similar culturing conditions, the thickness of the cell layer and the viability of cells was the highest in devices cultured in stirred media with oxygen generation, even though the thickness had not reached the theoretically predicted value, and lowest in those unstirred and without oxygen generation.

Animals↗
PubMed1999

PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway.

To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten-/- ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G1/S transition was accompanied by down-regulation of p27(KIP1), a major inhibitor for G1 cyclin-dependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosage-dependent increases in phosphorylation and activation of Akt/protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten-/- cell survival. Our studies suggest that PTEN regulates the phosphatidylinositol 3,4, 5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.

Animals↗
PubMed1999

Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation.

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.

Amino Acid Sequence↗
PubMed1999

Structural basis for self-association and receptor recognition of human TRAF2.

Tumour necrosis factor (TNF)-receptor-associated factors (TRAFs) form a family of cytoplasmic adapter proteins that mediate signal transduction from many members of the TNF-receptor superfamily and the interleukin-1 receptor. They are important in the regulation of cell survival and cell death. The carboxy-terminal region of TRAFs (the TRAF domain) is required for self-association and interaction with receptors. The domain contains a predicted coiled-coil region that is followed by a highly conserved TRAF-C domain. Here we report the crystal structure of the TRAF domain of human TRAF2, both alone and in complex with a peptide from TNF receptor-2 (TNF-R2). The structures reveal a trimeric self-association of the TRAF domain, which we confirm by studies in solution. The TRAF-C domain forms a new, eight-stranded antiparallel beta-sandwich structure. The TNF-R2 peptide binds to a conserved shallow surface depression on one TRAF-C domain and does not contact the other protomers of the trimer. The nature of the interaction indicates that an SXXE motif may be a TRAF2-binding consensus sequence. The trimeric structure of the TRAF domain provides an avidity-based explanation for the dependence of TRAF recruitment on the oligomerization of the receptors by their trimeric extracellular ligands.

Amino Acid Sequence↗
PubMed1999

Investigation of frontal lobe subregions in first-episode schizophrenia.

The evidence for frontal lobe structural abnormalities in schizophrenia using magnetic resonance (MR) imaging has been mixed, but most studies used either single slice measures or total volumes of a single frontal region, neither of which is sensitive to potential volume differences in more specific subregions. This study employed reliable methods for parcellation of the frontal lobes from MR images based on the sulcal anatomy. Following a cytoarchitectonic theory that distinguishes dorsomedial (archicortically derived) from ventrolateral (paleocortically derived) frontal subregions, we measured the superior frontal gyrus, anterior cingulate gyrus, and orbital frontal region in 19 first-episode schizophrenia patients and 26 healthy comparison subjects. Results indicated that male patients had significantly larger right orbital frontal volume compared to their left orbital frontal volume and compared to healthy men. Among male patients larger right orbital frontal volume was significantly correlated with smaller right 'archicortical' (i.e. anterior cingulate and superior frontal gyri) volume. Furthermore, the ratio of right orbital frontal to right 'archicortical' volume was significantly and positively correlated with level of delusions among male patients. These findings suggest that there may be reciprocal controls on 'archicortical' and 'paleocortical' neurodevelopment among men with schizophrenia, and that larger paleocortical relative to archicortical volumes may be associated with increased delusions.

Brain↗
PubMed1999

[Clinical application of chromosome haplotype analysis and mutation analysis to the diagnosis of Wilson's disease].

OBJECTIVE: To set up the method of gene diagnosis of Wilson's disease (WD) by chromosome haplotype analysis and mutation detection. METHODS: This study selected 3 (CA)n repeat genetic markers,D13S316, D13S133 and D13S314 to construct the chromosome haplotype within 8 Han WD families. PCR-SSCP was used to reconfirm the diagnosis of the siblings of the probands in the families where in the disease-causing mutation had been detected. RESULTS: One asymptomatic WD patient and 5 heterozygotes were detected. CONCLUSION: In the WD families, the analysis of chromosome haplotype helps to make the diagnosis of siblings of the probands;for the WD families in which the disease-causing mutation has been ascertained, mutation analysis can provide direct and definite evidence for diagnosis. The combination of these two methods can provide more evidences for diagnosis.

Genetic Markers↗
PubMed1999

Priming of CD8+ CTL effector cells in mice by immunization with a stress protein-influenza virus nucleoprotein fusion molecule.

Literature is accumulating which suggests the potential for stress proteins to form the basis of a novel vaccine technology. Immunization with mammalian tumor-derived stress proteins and their associated peptides promote anti-tumor immunity. Vaccination with HIV-1 p24 antigen fused to mycobacterial heat shock protein (Hsp) Hsp71 enhances p24-specific immunity, as measured by p24-specific antibody production and in vitro cell proliferation and cytokine induction. An ovalbumin-Hsp71 fusion protein primes ovalbumin-specific CTL activity and resistance to challenge with an ovalbumin-expressing tumor. We have extended these observations by using a mycobacterial Hsp65 fusion molecule to prime CTL specific for a viral antigen. Gene fusion constructs were generated from DNA encoding Mycobacterium bovis strain BCG Hsp65 and individual fragments of influenza virus nucleoprotein (NP) encompassing H-2Kd- and H-2Db-restricted CTL epitopes. The ability of these purified recombinant fusion proteins to prime NP-specific CTL was assessed in mice of appropriate H-2 haplotypes. We observed that adjuvant-free immunization with either fusion protein elicited significant CTL activity when administered at doses of 10-100 micrograms per mouse. An NP fusion protein made with glutathione-S-transferase failed to elicit NP-specific CTL, indicating that the phenomenon requires Hsp65 sequences. A single immunization with the Hsp65-NP fusion protein elicited CTL activity which persisted for a minimum of 4 months post-immunization, at which time it could be boosted by a second immunization. To our knowledge, this is the first report of a member of the Hsp60 family priming for antigen-specific CTL activity when employed as a fusion protein partner.

Animals↗
PubMed1999

The role of lipoprotein processing by signal peptidase II in the Gram-positive eubacterium bacillus subtilis. Signal peptidase II is required for the efficient secretion of alpha-amylase, a non-lipoprotein.

Computer-assisted analyses indicate that Bacillus subtilis contains approximately 300 genes for exported proteins with an amino-terminal signal peptide. About 114 of these are lipoproteins, which are retained in the cytoplasmic membrane. We have investigated the importance of lipoprotein processing by signal peptidase II (SPase II) for cellular homeostasis, using cells lacking SPase II. The results show that lipoprotein processing is important for cell viability at low and high temperatures, suggesting that lipoproteins are essential for growth under these conditions. Although certain lipoproteins are required for the development of genetic competence, sporulation, and germination, these developmental processes were not affected in the absence of SPase II. Cells lacking SPase II accumulated lipid-modified precursor and mature-like forms of PrsA, a folding catalyst for secreted proteins. These forms of PrsA seem to have a reduced activity, as the secretion of alpha-amylase was strongly impaired. Unexpectedly, type I signal peptidases, which process secretory preproteins, were not involved in alternative amino-terminal processing of pre-PrsA in the absence of SPase II. In conclusion, processing of lipoproteins by SPase II in B. subtilis is not strictly required for lipoprotein function, which is surprising as lipoproteins and type II SPases seem to be conserved in all eubacteria.

Amino Acid Sequence↗
PubMed1999

Orbital frontal and amygdala volume reductions in obsessive-compulsive disorder.

BACKGROUND: Functional neuroimaging studies have implicated the frontal lobes and the hippocampus-amygdala complex in the pathophysiology of obsessive-compulsive disorder (OCD). These brain regions have not been well investigated in patients with OCD, however, using magnetic resonance imaging. METHODS: Volumes of the superior frontal gyrus, anterior cingulate gyrus, orbital frontal region, hippocampus, and amygdala were computed from contiguous magnetic resonance images in a sample of 26 patients with OCD and 26 healthy comparison subjects. RESULTS: Patients with OCD had significantly reduced bilateral orbital frontal and amygdala volumes compared with healthy comparison subjects and lacked the normal hemispheric asymmetry of the hippocampus-amygdala complex. Neither brain structure volumes nor asymmetry indices were significantly correlated with total illness duration or length of current OCD episode. CONCLUSIONS: Findings of reduced orbital frontal and amygdala volumes in patients implicate a structural abnormality of these brain regions in the pathophysiology of OCD. Absence of the normal hemispheric asymmetry of the hippocampus-amygdala complex in patients is consistent with an anomalous neurodevelopmental process.

Adult↗
PubMed1999

Human glucosamine-6-phosphate isomerase, a homologue of hamster oscillin, does not appear to be involved in Ca2+ release in mammalian oocytes.

Injections of cytosolic preparations from mammalian sperm into oocytes have been shown to trigger calcium [Ca2+]i oscillations and initiate activation of development. Recently, a protein isolated from hamster sperm has been suggested to be involved in the generation of these oscillations and it was named "oscillin." The human homologue of hamster oscillin is glucosamine 6-phosphate isomerase (GPI, EC no. 5.3.1.10), an enzyme so far described to be involved in hexose phosphate metabolism. To assess the role of GPI on Ca2+ signaling, a human recombinant protein was generated in a prokaryotic system and injected into fura-2-dextran-loaded metaphase II (MII) mouse oocytes. Injection of recombinant GPI failed to induce Ca2+ responses in 12/12 injected MII oocytes despite the fact that the recombinant GPI was active as assessed by an enzymatic assay. Injection of buffer (0/6 oocytes) or fructose-6-phosphate, a product of GPI enzymatic reaction (0/5 oocytes), also failed to initiate Ca2+ responses. Conversely, injections of sperm cytosolic factor induced [Ca2+]i oscillations in all 17/17 oocytes. In addition, injection of recombinant GPI or GPI mRNA failed to induce parthenogenetic activation (0/30 oocytes). Immunofluorescence studies using an anti-GPI polyclonal antibody (GK) resulted in localization of GPI to the sperm's equatorial region. Incubation of the GK antibody with sperm extracts failed to block the [Ca2+]i responses induced by these extracts. Moreover, near complete depletion of GPI from sperm fractions by immunoprecipitation did not impair the ability of these fractions to induce [Ca2+]i oscillations. In summary, our results support the role of a sperm cytosolic component(s) in the generation of [Ca2+]i oscillations during mammalian fertilization, although a protein other than GPI/oscillin is likely to be the active calcium releasing factor.

Aldose-Ketose Isomerases↗
PubMed1999

Preliminary crystallographic studies of human mitochondrial NAD(P)(+)-dependent malic enzyme.

Human mitochondrial NAD(P)(+)-dependent malic enzyme was overexpressed in Escherichia coli and purified by anion-exchange, ATP affinity, and gel filtration chromatography. The protein was crystallized with the hanging-drop vapor diffusion method. Many different crystal forms were observed, five of which were characterized in some detail. A 2.5-A multiple-wavelength anomalous diffraction data set and a 2.1-A native data set were collected using synchrotron radiation on crystals containing selenomethionyl residues. These crystals belong to space group B2, with a = 204.4 A, b = 107.0 A, c = 59.2 A, and gamma = 101.9 degrees. Self-rotation functions demonstrated that the tetramer of this enzyme obeys 222 symmetry.

Crystallization↗