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H Wang

Publications and source records attributed to H Wang.

At least 1,405 records · Page 78Linked to original sources

Alternative gene form discovery and candidate gene selection from gene indexing projects.

Several efforts are under way to partition single-read expressed sequence tag (EST), as well as full-length transcript data, into large-scale gene indices, where transcripts are in common index classes if and only if they share a common progenitor gene. Accurate gene indexing facilitates gene expression studies, as well as inexpensive and early gene sequence discovery through assembly of ESTs that are derived from genes that have not been sequenced by classical methods. We extend, correct, and enhance the information obtained from index groups by splitting index classes into subclasses based on sequence dissimilarity (diversity). Two applications of this are highlighted in this report. First it is shown that our method can ameliorate the damage that artifacts, such as chimerism, inflict on index integrity. Additionally, we demonstrate how the organization imposed by an effective subpartition can greatly increase the sensitivity of gene expression studies by accounting for the existence and tissue- or pathology-specific regulation of novel gene isoforms and polymorphisms. We apply our subpartitioning treatment to the UniGene gene indexing project to measure a marked increase in information quality and abundance (in terms of assembly length and insertion/deletion error) after treatment and demonstrate cases where new levels of information concerning differential expression of alternate gene forms, such as regulated alternative splicing, are discovered. [Tables 2 and 3 can be viewed in their entirety as Online Supplements at http://www.genome.org.]

Alternative Splicing↗

Preliminary study of a metal/a-Se-based portal detector.

A feasibility study has been performed on metal/amorphous selenium detectors for megavoltage portal imaging. The metal plates of the detectors were positioned facing the incident 6 MV and Co-60 photon spectra. The detectors consist of various thicknesses (0.15 mm, 0.30 mm, and 0.50 mm) of amorphous selenium (a-Se) deposited on metal plates of varying thicknesses: aluminum (2.0 mm), copper (1.0 mm and 1.5 mm), stainless steel (0.9 mm), or glass (1.1 mm). The detectors were charged prior to irradiation by corona methods, and the portal images were subsequently digitized after irradiation with a noncontact electrostatic probe. The sensitivity of the detectors to dose, electric field across the a-Se layer, metal plate type and a-Se thickness, was studied. The electrostatic voltage remaining on the a-Se layer was found, both theoretically and experimentally, to exhibit a cubic relationship with respect to dose. An increase in electric field increases the sensitivity (gradient of the a-Se surface voltage vs dose curve) and dynamic range of the resultant image. An increase in a-Se thickness, however, although also increasing the sensitivity, decreases the dynamic range. The metal plate types and thicknesses within the range studied do not have a significant effect on detector sensitivity. Image quality and contrast resolution of the detector were evaluated with a contrast-detail phantom and compared to commercially available film based and electronic portal imaging devices. Image quality of the metal/a-Se detector as a function of dose was studied by discharging the a-Se to various fractions of its initial charge, and as expected, increases with dose due to a decrease in quantum noise. Contrast-detail images obtained by metal/a-Se detectors are superior to those obtained at higher dose levels by other commercial systems.

Biophysical Phenomena↗

Astigmatic single photon emission computed tomography imaging with a displaced center of rotation.

A filtered backprojection algorithm is developed for single photon emission computed tomography (SPECT) imaging with an astigmatic collimator having a displaced center of rotation. The astigmatic collimator has two perpendicular focal lines, one that is parallel to the axis of rotation of the gamma camera and one that is perpendicular to this axis. Using SPECT simulations of projection data from a hot rod phantom and point source arrays, it is found that a lack of incorporation of the mechanical shift in the reconstruction algorithm causes errors and artifacts in reconstructed SPECT images. The collimator and acquisition parameters in the astigmatic reconstruction formula, which include focal lengths, radius of rotation, and mechanical shifts, are often partly unknown and can be determined using the projections of a point source at various projection angles. The accurate determination of these parameters by a least squares fitting technique using projection data from numerically simulated SPECT acquisitions is studied. These studies show that the accuracy of parameter determination is improved as the distance between the point source and the axis of rotation of the gamma camera is increased. The focal length of the focal line perpendicular to the axis of rotation is determined more accurately than the focal length to the focal line parallel to this axis.

Algorithms↗

Antibiotic susceptibility patterns of Streptococcus pneumoniae in china and comparison of MICs by agar dilution and E-test methods.

Beta-lactam resistance by Streptococcus pneumoniae is becoming a significant threat to public health worldwide. However, data concerning antibiotic susceptibility patterns in China have not been published. In this study, a total of 79 clinical isolates and 244 nasopharyngeal isolates of S. pneumoniae were recovered between June and November 1997 in Beijing. The agreement between the MICs (+/-1 log2 dilution) of penicillin and ceftriaxone obtained by the agar dilution and E-test methods for the 79 clinical strains was very good (97.5 and 93.7%, respectively). Of these 79 strains, 9 (11.4%) were intermediate and 2 (2.5%) were resistant to penicillin. Of the 244 nasopharyngeal strains, 32 (13.1%) were intermediate and 3 (1. 2%) were resistant to penicillin. The total of 277 penicillin-susceptible clinical and nasopharyngeal isolates of Streptococcus pneumoniae were 100% susceptible to amoxicillin-clavulanic acid, cefuroxime, ceftriaxone, and cefotaxime. In the 35 penicillin-intermediate and -resistant nasopharyngeal strains, elevated MICs of amoxicillin-clavulanic acid, cefuroxime, ceftriaxone, and cefotaxime were seen for </=4 isolates. Of 244 nasopharyngeal isolates, the overall percentages of tetracycline, erythromycin, chloramphenicol, ofloxacin, and trimethoprim-sulfamethoxazole resistance were 87.6, 74.0, 47.8, 3.7 and 63.3, respectively. Vancomycin and rifampin resistance were not detected. These findings demonstrate that the rate of penicillin-resistant pneumococci is relatively low in China compared to those of other Asian countries. Resistance to non-beta-lactams was much higher than to beta-lactams. The E-test and agar dilution methods appeared to be comparable in identifying resistant strains.

Agar↗

A deficiency in aspartate biosynthesis in Lactococcus lactis subsp. lactis C2 causes slow milk coagulation.

A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc-) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.

Animals↗

Lyme disease Borrelia species in northeastern China resemble those isolated from far eastern Russia and Japan.

Fifty-nine Borrelia burgdorferi sensu lato culture isolates collected from northeastern China were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism (RFLP) analysis and reactivity with monoclonal antibodies (MAbs). Among 59 culture isolates, 30 (50.8%) were Borrelia garinii and 17 (28.8%) were Borrelia afzelii, 2 were mixtures composed of B. garinii with RFLP pattern B and B. garinii with pattern C, and 9 were mixtures composed of B. garinii and B. afzelii. One isolate, ChY13p, produced a unique pattern and was identified as B. garinii based on analyses of 16S rRNA gene sequence, flagellin PCR-RFLP typing, and MAb reactivities. No Borrelia burgdorferi sensu stricto or Borrelia japonica isolates were detected. The results indicate that Lyme disease Borrelia species in northeastern China resemble those of Borrelia isolates from far eastern Russia and Japan.

Base Sequence↗

Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays.

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well (r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States.

Amino Acid Sequence↗

Different classes of proteoglycans contribute to the attachment of Borrelia burgdorferi to cultured endothelial and brain cells.

The Lyme disease spirochete, Borrelia burgdorferi, infects multiple tissues, such as the heart, joint, skin, and nervous system and has been shown to recognize heparan sulfate and dermatan sulfate proteoglycans. In this study, we examined the contribution of different classes of proteoglycans to the attachment of the infectious B. burgdorferi strain N40 to several immortalized cell lines and primary cultured cells, including endothelial cells and brain cells. Bacterial attachment was inhibited by exogenous proteoglycans or by treatment of host cells with inhibitors of proteoglycan synthesis or sulfation, indicating that proteoglycans play a critical role in bacterial binding to diverse cell types. Binding to primary bovine capillary endothelial cells or a human endothelial cell line was also inhibited by digestion with heparinase or heparitinase but not with chondroitinase ABC. In contrast, binding to glial cell-enriched brain cell cultures or to a neuronal cell line was inhibited by all three lyases. Binding of strain N40 to immobilized heparin could be completely inhibited by dermatan sulfate, and conversely, binding to dermatan sulfate could be completely blocked by heparin. As measured by 50% inhibitory dose, heparin was a better inhibitor of binding than dermatan sulfate, regardless of whether the substrate was heparin or dermatan sulfate. These results are consistent with the hypotheses that the species of proteoglycans recognized by B. burgdorferi vary with cell type and that bacterial recognition of different proteoglycans is mediated by the same bacterial molecule(s).

Animals↗

Inhibition of proximal tubular fluid absorption by nitric oxide and atrial natriuretic peptide in rat kidney.

Atrial natriuretic factor (ANF) and nitric oxide (NO) stimulate production of guanosine 3',5'-cyclic monophosphate (cGMP) and are natriuretic. Split-drop micropuncture was performed on anesthetized rats to determine the effects of ANF and the NO donor sodium nitroprusside (SNP) on proximal tubular fluid absorption rate (Jva). Compared with control solutions, SNP (10(-4) M) decreased Jva by 23% when administered luminally and by 35% when added to the peritubular perfusate. Stimulation of fluid uptake by luminal angiotensin II (ANG II; 10(-9) M) was abolished by SNP (10(-4) and 10(-6) M). In proximal tubule suspensions, ANF (10(-6) M) increased cGMP concentration to 143%, whereas SNP (10(-6), 10(-5), 10(-4), 10(-3) M) raised cGMP to 231, 594, 687, and 880%, respectively. S-nitroso-N-acetylpenicillamine (SNAP) also raised cGMP concentrations with similar dose-response relations. These studies demonstrate inhibition by luminal and peritubular NO of basal and ANG II-stimulated proximal fluid absorption in vivo. The ability of SNP to inhibit basal fluid uptake whereas ANF only affected ANG II-stimulated transport may be because of production of higher concentrations of cGMP by SNP.

Absorption↗

Electrophysiological characteristics of the proton-coupled peptide transporter PEPT2 cloned from rat brain.

We have cloned a peptide transporter from rat brain and found it to be identical to rat kidney PEPT2. In the present study we characterize the transport function of the rat brain PEPT2, with special emphasis on electrophysiological properties and interaction with N-acetyl-L-aspartyl-L-glutamate (NAAG). When heterologously expressed in HeLa cells and in SK-N-SH cells, PEPT2 transports several dipeptides but not free amino acids in the presence of a proton gradient. NAAG competes with other peptides for the PEPT2-mediated transport process. When PEPT2 is expressed in Xenopus laevis oocytes, substrate-induced inward currents are detectable with dipeptides of differing charge in the presence of a proton gradient. Proton activation kinetics are similar for differently charged peptides. NAAG is a transportable substrate for PEPT2, as evidenced by NAAG-induced currents. The Hill coefficient for protons for the activation of the transport of differently charged peptides, including NAAG, is 1. Although the peptide-to-proton stoichiometry for negatively charged peptides is 1, the transport nonetheless is associated with transfer of positive charge into the oocyte, as indicated by peptide-induced inward currents.

Amino Acids↗

Effect of feeding diets of varying fatty acid composition on apolipoprotein expression in newborn swine.

The purpose of this study was to determine the effect of chronic (1 wk) feeding of dietary triacylglycerol (TG) of varying fatty acid composition on small intestinal and hepatic apolipoprotein expression, as well as serum lipid and apolipoprotein concentrations, in newborn swine. Two-day-old female swine were fed one of three diets by gavage with the following lipid composition: medium-chain TG (MCT; MCT oil), intermediate-chain saturated TG (ICST; coconut oil), and long-chain polyunsaturated TG (LCPUT; safflower oil) at 753 kJ . kg-1 . day-1 with 51% of energy from fat. After 1 wk, serum lipids and apolipoprotein concentrations were measured, and jejunal apolipoprotein B (apo B) and apo A-I mass and apo B, apo A-I, apo A-IV, and apo C-III synthesis were measured. Liver was processed for determination of apo B and apo A-I mass and apo B, apo A-I, apo C-III, and beta-actin mRNA abundance by slot blot hybridization. Compared with the MCT and LCPUT groups, the ICST group had higher total serum cholesterol, TG, high-density lipoprotein (HDL)-cholesterol, and apo A-I concentrations. There were no differences among the three groups for intestinal apolipoprotein mass or synthesis. In liver, apo A-I mass was highest in the ICST group. Liver apo A-I and apo C-III mRNA abundance was highest in the ICST group. Among all three groups, hepatic apo A-I mass correlated significantly with plasma HDL-cholesterol concentrations, and serum TG concentrations correlated with hepatic apo C-III mRNA abundance. In conclusion, we found that in the newborn piglet, chronic feeding of ICST increases serum total cholesterol, TG, HDL-cholesterol, and apo A-I concentrations and hepatic expression of apo A-I and apo C-III mRNA, compared with feeding of MCT or LCPUT. We speculate that increased hepatic apo A-I expression may contribute to the higher serum HDL and apo A-I concentrations in the ICST animals. Increased hepatic expression of apo C-III with ICST feeding may contribute to the higher serum TG concentrations by apo C-III-mediated inhibition of the catabolism of triacylglycerol-rich lipoproteins.

Animals↗

Molecular characterization of rabbit CPP32 and its function in vascular smooth muscle cell apoptosis.

Vascular remodeling in atherogenesis is marked not only by cellular proliferation and migration but is also impacted by apoptotic cell death. Extensive studies have focused on the signal transduction events leading to apoptosis. CPP32, a member of the caspase/interleukin-1 beta-converting enzyme (ICE) protease family, has emerged as a central player in several reports of apoptosis pathways. Vascular smooth muscle cells (SMC) undergo apoptosis after treatment with various stimuli, including nitric oxide (NO) donors, such as sodium nitroprusside (SNP, 0.1-1 mM). The aim of the present study was to evaluate the role of CPP32 in SNP-induced apoptosis of SMC. We isolated a rabbit CPP32 cDNA by using degenerate primers and polymerase chain reaction technique. The predicted protein encoded by this cDNA contains the conserved sequence (QACRG) necessary for covalent linkage to poly(ADP-ribose) polymerase (PARP) as well as the three amino acids responsible for substrate recognition and catalysis reported in other caspase members. Using a segment of this cDNA as a probe, we found no change of CPP32 mRNA in cultured arterial SMC before and after SNP treatment. We also measured the protease activity of CPP32 against a chromophore p-nitroaniline (pNA)-labeled substrate, DEVD-pNA. Our results showed a dose-dependent increase of CPP32 activity in SMC, with a maximal 10-fold increase after SNP treatment. Addition of a competitive CPP32 inhibitor, DEVD-CHO, produced a 50% reduction in maximal stimulation. Immunoblot analysis illustrated that SNP treatment induced proteolytic cleavage of CPP32 into its enzymatically active subunit p17 as well as the degradation of PARP into a 85-kDa fragment. We further demonstrated that incubation of cultured SMC with DEVD-CHO significantly reduced SNP-induced DNA fragmentation. DNA fragmentation analysis was carried out using several methods including a cell death detection enzyme-linked immunosorbent assay kit, in situ end labeling, and DNA electrophoresis in agarose gel. Our data indicate that CPP32 mRNA is constitutively expressed in rabbit SMC and activation of CPP32 protein has a pivotal role in SNP-induced SMC apoptosis.

Amino Acid Sequence↗

The Drosophila gene Medea demonstrates the requirement for different classes of Smads in dpp signaling.

Signals from transforming growth factor-beta (TGF-beta) ligands are transmitted within the cell by members of the Smad family, which can be grouped into three classes based on sequence similarities. Our previous identification of both class I and II Smads functioning in a single pathway in C. elegans, raised the issue of whether the requirement for Smads derived from different classes is a general feature of TGF-beta signaling. We report here the identification of a new Drosophila class II Smad, Medea, a close homolog of the human tumor-suppressor gene DPC4. Embryos from germline clones of both Medea and Mad (a class I Smad) are ventralized, as are embryos null for the TGF-beta-like ligand decapentaplegic (dpp). Loss of Medea also blocks dpp signaling during later development, suggesting that Medea, like Mad, is universally required for dpp signaling. Furthermore, we show that the necessity for these two closely related, non-redundant Smads, is due to their different signaling properties - upon activation of the Dpp pathway, Mad is required to actively translocate Medea into the nucleus. These results provide a paradigm for, and distinguish between, the requirement for class I and II Smads in Dpp/BMP signaling.

Amino Acid Sequence↗

H2O2 acts on cellular membranes to generate ceramide signaling and initiate apoptosis in tracheobronchial epithelial cells.

Hydrogen peroxide (H2O2) is an inflammatory oxidant which contributes to the pathogenesis of chronic diseases such as lung injury of the respiratory tract, atherosclerosis and cancer. The mechanisms and target sites of this reactive oxidant are mainly unknown. So far there are opposing reports as to whether reactive oxidants inhibit or promote apoptosis. We activated the death pathway in primary tracheobronchial epithelial (TBE) cells with H2O2 (20-200 microM) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation associated with apoptosis. Elevation of ceramide with exogenous ceramide analogs was sufficient for apoptosis induction with the same characteristics and in the same time frame. H2O2 induced rapid sphingomyelin hydrolysis to ceramide, the elevation of which paralleled the induction of apoptosis. Furthermore, H2O2 acted directly on TBE cells membrane preparations devoid of nuclei, stimulating sphingomyelin hydrolysis through a neutral Mg2+ dependent sphingomyelinase (SMase). These data suggest that the formation of ceramide from sphingomyelin in the plasma membrane is a key event in H2O2-induced apoptosis in tracheobronchial epithelial cells.

Animals↗

Identification of lactoferrin complexes in bovine mammary secretions during mammary gland involution.

Part of the antimicrobial activity of lactoferrin resides in its ability to bind to bacteria. The complexing of lactoferrin with other proteins could alter its activity. This study identified the presence of lactoferrin complexes in mammary secretions during mammary gland involution and determined the proportion of free and complexed lactoferrin in mammary secretions. Mammary secretions were collected from Holstein cows on d 7, 14, and 21 of involution. Proteins were fractionated from defatted, filtered mammary secretions by sucrose density gradient ultracentrifugation and by gel filtration chromatography. Proteins contained in separated fractions were identified by SDS-PAGE. The presence of lactoferrin was confirmed by immunoblot analysis. Lactoferrin was present as complexed forms of high molecular mass in mammary secretions at each day of involution. The majority of lactoferrin was present in complexes of higher molecular mass rather than as monomers. A majority of lactoferrin existed in fractions of approximately 250 kDa, although peaks of lactoferrin at 150, 300, and 800 kDa were also found. The presence of lactoferrin complexes may result from interactions with casein or immunoglobulins or from the formation of lactoferrin multimers in the secretions. The interaction of lactoferrin with other proteins in mammary secretions during involution may affect the antimicrobial properties of lactoferrin.

Animals↗

p53 and protein kinase C independent induction of growth arrest and apoptosis by bryostatin 1 in a highly metastatic mammary epithelial cell line: In vitro versus in vivo activity.

We have evaluated the effects of bryostatin 1 on growth of a highly malignant p53-null mouse mammary tumor line, 4T1, and the mechanism by which bryostatin 1 inhibits in vitro growth and in vivo development of tumor and metastases from the orthotopic site. Bryostatin 1 at 20-400 nM concentrations inhibits growth of 4T1 cells by approximately 60% in two-day cultures. Inhibition of growth is associated with an increase in the number of cells undergoing apoptosis with concomitant elevation in the steady state levels of bax protein and drop in bcl-2 levels. The cytotoxic effect of bryostatin 1 on 4T1 cells occurs independently of p53, since there was no evidence of p53-mediated transcriptional activity in 4T1 cells following treatment with bryostatin 1.4T1 cells respond in vivo to bryostatin 1 therapy (75 microg/kg body weight). Intraperitoneal administration of bryostatin 1 inhibits both primary and secondary tumor growth by approximately 50%. However, although bryostatin 1 has a remarkable capacity to slow tumor growth and progression, it is unable to completely eradicate tumor growth and progression due to in vivo development of tumor resistance to bryostatin 1. Levels and cellular distribution of PKCalpha and delta do not correlate with the growth inhibitory effects of bryostatin 1 on 4T1 cells; however, reduction in cytosolic PKCalpha and delta without associated increase in membrane compartment appear to correlate with bryostatin-resistance. Our results suggest that the therapeutic effects of bryostatin 1 in our system do not involve alterations in levels and distribution of PKC but rather a direct upregulation of bax/ bcl-2 ratios that is independent of p53.

Animals↗

p53-independent down-regulation of cyclin D1 and p21Waf1 in the process of immortalization of human esophageal epithelial cells.

Immortalization is considered to be an initial critical step in the process of multistage cell transformation. However, the molecular mechanisms underlying this event are not well understood. Our laboratory previously established the immortalized human esophageal epithelial cell line, HET-1A, by SV40 T-antigen transfection. In the present study, we investigated the role of G1 cyclins and cyclin dependent kinase inhibitors, in the process of immortalization. By using immunoprecipitation and Western blot analysis, sequential changes in the expression of both cyclin D1 and p21Waf1 were detected during the conversion of precrisis esophageal epithelial cells to immortalized HET-1A cells. Reduced expression levels of both cyclin D1 and p21Waf1 were found in early passage and late passage immortalized cells when compared to levels in precrisis cells. In addition, continued subculture of the immortalized cells led to increased expression levels of both cyclin D1 and p21Waf1. No significant changes in the expression of either cyclin E or p53 were observed in early or late passage immortalized cells when compared to precrisis cells. These results suggest that changes in the expression levels of cyclin D1 and p21Waf1, but not cyclin E, may be important for immortalization and continued propagation of human esophageal epithelial cells, and these changes are not dependent on regulation by p53.

Antigens, Polyomavirus Transforming↗