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Biomedical subjects

W Hu

Publications and source records attributed to W Hu.

At least 109 records · Page 6Linked to original sources

Sensitivity-enhanced MQ-HCN-CCH-TOCSY and MQ-HCN-CCH-COSY pulse schemes for (13)C/(15)N labeled RNA oligonucleotides.

Sensitivity enhanced multiple-quantum 3D HCN-CCH-TOCSY and HCN-CCH-COSY experiments are presented for the ribose resonance assignment of (13)C/(15)N-labeled RNA sample. The experiments make use of the chemical shift dispersion of N1/N9 of pyrimidine/purine to distinguish the ribose spin systems. They provide a complementary approach for the assignment of ribose resonance to the currently used HCCH-COSY and HCCH-TOCSY type experiments in which either (13)C or (1)H is utilized to separate the different ribose spin systems. The pulse schemes have been demonstrated on a 23-mer (13)C/(15)N-labeled RNA aptamer complexed with neomycin and tested on a 32-mer RNA complexed with a 23-residue peptide.

Carbon Isotopes↗

Analysis of inorganic anions by electrostatic ion chromatography using zwitterionic/cationic mixed micelles as the stationary phase.

The inability to separate fluoride, phosphate and sulfate by electrostatic ion chromatography (EIC) was overcome by using an ODS silica column coated with mixed zwitterionic-cationic surfactants as the stationary phase. The best results were obtained using the zwitterionic surfactant, 3-(N,N-dimethylmyristylammonium)-propanesulfonate (C19H41NO3S), and the cationic surfactant, myristyltrimethylammonium, CH3(CH2)13N+(CH3)3, in a 10:1 molar ratio in the column coating solution. With a dilute solution of sodium tetraborate as the eluent the model analyte anions were completely separated in the following elution order: F, HPO42-, SO42-, Cl-, NO2-, Br-, NO3-. The very early elution of phosphate and sulfate is most unusual and is unique to this system. Detection limits better than 1.1 x 10(-4) mM and linear calibration plots up to 7.0 mM were obtained with a suppressed conductivity system.

Journal Article↗

Use of zwitterionic micelles in the eluent: a new approach for ion chromatographic (IC) analysis of ions in biological fluids with direct sample injection.

The problem of column performance degradation due to irreversible binding of proteins encountered in ion chromatographic (IC) analysis of ions in protein-containing samples was overcome by using zwitterionic micelles (e.g., Zwittergent-3-14) as a portion of the eluent. A zwitterionic micellar eluent showed high ability for solubilization of proteins, and, hence, the protein-containing samples could be analyzed without need for deproteinization. On the other hand, the zwitterionic micelle was insensitive to conductivity but interacted with the analyte ions, due mainly to its unique configuration of charges (namely, the zwitterionic micelle containing both positively and negatively charged groups but carrying no net charge). Using a zwitterionic micellar eluent, the analyte ions could be detected selectively and sensitively, and moreover, the selectivity for the analyte ions was unique. A conventional anion-exchange column conditioned with a Zwittergent-3-14 micellar eluent was applied for the analysis of real biological samples (serum and urine) with direct sample injection. The results of the successful detection of inorganic anions (Cl-, SO4(2-), NO2-, Br-, and NO3-) have demonstrated the usefulness of this new IC approach for the analysis of biological samples.

Animals↗

Biochemical responses of the mycorrhizae in Pinus massoniana to combined effects of Al, Ca and low pH.

Biochemical responses of Pinus massoniana, with and without the inoculation mycorrhizal fungus Pisolithus tinctorius at the root, to artificial acid rain (pH 2.0) and various Ca/Al ratios were investigated. Some enzymes associated with the nutritive metabolism, such as acid phosphatase, alkaline phosphatase, nitrate reductase, mannitol dehydrogenase and trehalase, in the roots, stems and leaves of plant were obviously inhibited by the artificial acid rain and Al. After treatment with pH 2.0 + Ca/Al (0/1 or 1/10) artificial acid rain, the protein content in the organs was decreased. However, the activities of superoxide dismutase (SOD) and peroxidase (POD) and glutathione (GSH) concentrations were induced. It demonstrated that acid rain and Al could induce oxygen radicals in plant. Compared with the treatments with lower pH or Al, respectively, the combination of lower pH and Al concentration was more toxic to P. massoniana. Al toxicity could be ameliorated by the addition of Ca and the amelioration was the most when the ratio was 1/1 among the various Ca/Al ratio. Infection with mycorrhizal fungus P. tinctorius at the root of P. massoniana increased the ability of the plant to resist the toxicity of artificial acid rain and Al stress.

Acid Phosphatase↗

Expression of the cyclin-dependent kinase inhibitor p27 in keratoacanthoma.

BACKGROUND: Keratoacanthomas are characterized by initial rapid enlargement followed by clinical regression. A series of cyclin and cyclin-dependent kinase complexes regulate cell cycle progression. p27(kip) inhibits a variety of cyclin-cyclin-dependent kinase complexes in vitro and may act to hold eukaryotic cells in a quiescent state (G0). OBJECTIVE: We examined expanding and regressing keratoacanthomas for expression of p27(kip). METHODS: An immunohistochemical method was used to visualize and count p27(kip)-labeled cells in 5 expanding and 15 regressing keratoacanthomas. RESULTS: In normal epidermis p27(kip) was found overlying the nuclei of suprabasilar keratinocytes. In expanding keratoacanthoma there was little expression of p27(kip) in nuclei of atypical keratinocytes composing the tumor (1.25 +/- 2.1 labeled cells per high-power field); in regressing keratoacanthoma the nuclei of most suprabasilar keratinocytes in atypical tumor aggregates contained p27(kip) (55.1 +/- 28.6 labeled cells per high-power field). The difference was significant at P values of less than.001. CONCLUSION: The identification of p27(kip) in regressing keratoacanthoma but not in expanding keratoacanthoma suggests that p27(kip) may be playing a role in promoting regression of keratoacanthoma and is a potential target for pharmacologic intervention.

Cell Cycle Proteins↗

Sensitivity enhanced NMR spectroscopy by quenching scalar coupling mediated relaxation: application to the direct observation of hydrogen bonds in 13C/15N-labeled proteins.

In this paper, we demonstrate that the sensitivity of triple-resonance NMR experiments can be enhanced significantly through quenching scalar coupling mediated relaxation by using composite-pulse decoupling (CPD) or.an adiabatic decoupling sequence on aliphatic, in particular alpha-carbons in 13C/15N-labeled proteins. The CPD-HNCO experiment renders 50% sensitivity enhancement over the conventional CT-HNCO experiment performed on a 12 kDa FK506 binding protein, when a total of 266 ms of amide nitrogen-carbonyl carbon defocusing and refocusing periods is employed. This is a typical time period for the direct detection of hydrogen bonds in proteins via trans-hydrogen bond 3hJ(NC') couplings. The experimental data fit theoretical analysis well. The significant enhancement in sensitivity makes the experiment more applicable to larger-sized proteins without resorting to perdeuteration.

Carbon Isotopes↗

Detection of very weak side chain-main chain hydrogen bonding interactions in medium-size 13C/15N-labeled proteins by sensitivity-enhanced NMR spectroscopy.

We describe the direct observation of very weak side chain-main chain hydrogen bonding interactions in medium-size 13C/15N-labeled proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation. between the hydrogen acceptor side chain carboxylate carbon 13CO2delta of glutamate 54 and the hydrogen donor backbone amide 15N of methionine 49 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond 3hJ(NCO2delta) coupling by employing a novel sensitivity-enhanced HNCO-type experiment, CPD-HNCO. The 3hJ(NCO2delta) coupling constant appears to be even smaller than the average value of backbone 3hJ(NC') couplings, consistent with more extensive local dynamics in protein side chains.

Carbon Isotopes↗

NMR detection of side chain-side chain hydrogen bonding interactions in 13C/15N-labeled proteins.

We describe the direct observation of side chain-side chain hydrogen bonding interactions in proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the guanidinium nitrogen 15Nepsilon of arginine 71, which serves as the hydrogen donor, and the acceptor carboxylate carbon 13CO2gamma of aspartate 100 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond 3h JNepsilonCO2gamma coupling by employing a novel HNCO-type experiment, soft CPD-HNCO. The 3h JNepsilonCO2gamma coupling constant appears to be even smaller than the average value of backbone 3h JNC' couplings, consistent with more extensive local dynamics in protein side chains. The identification of trans-hydrogen bond J-couplings between protein side chains should provide useful markers for monitoring hydrogen bonding interactions that contribute to the stability of protein folds, to alignments within enzyme active sites and to recognition events at macromolecular interfaces.

Animals↗

Increased expression of fascin, motility associated protein, in cell cultures derived from ovarian cancer and in borderline and carcinomatous ovarian tumors.

Fascin bundles actin microfilaments within dynamic cellular structures such as microspikes, stress fibers and membrane ruffles. Fascin overexpression induces membrane protrusions and increased cell motility, and is highly expressed in various transformed cells, and in specialized normal cells including neuronal, endothelial and dendritic cells. In breast cancer, fascin expression correlates with high-grade tumors. To investigate whether fascin might be a predictor factor for ovarian cancer progression, eighteen cell cultures derived from ovarian cancer, and thirty four archival paraffin-embedded material of normal versus borderline and carcinomatous ovaries were stained by immunocytochemistry and immunohistochemistry with fascin Mab 55K-2. Overall expression of the fascin protein was found in 50% (9/18) of cell cultures derived from original samples of ovarian tumors. Expression of fascin protein was found in 67% (6/9) of cell cultures derived from patients diagnosed with stage IV disease, and 33% (3/9) of cell cultures from patients diagnosed with stage II/III. There was no clear relationship between fascin expression and histologic types, tumor grade, or DNA ploidy. However, 75% of cell cultures, which developed into a xenograft after intraperitoneal inoculation, showed fascin expression, while 86% of non-tumorigenic cell cultures did not show fascin expression. Expression of fascin in these established ovarian tumor cell cultures was significantly associated with the ability for these cells to grow intraperitoneally (P < 0.05). Furthermore, fascin was never expressed in normal epithelial ovarian tissues, but was present in all pathologic ovaries. Both diffuse and focal patterns were observed in borderline ovarian tumors (67% and 33%), advanced primary ovarian cancer (67% and 33%) and metastatic ovarian cancer (89% and 11%). Therefore, our data suggest that fascin could serve as a prognostic factor for abnormal ovarian epithelial pathology and could be a novel target for the treatment of ovarian cancer.

Carrier Proteins↗

Determination of inorganic anions in biological fluids with direct sample injection by electrostatic ion chromatography using zwitterionic micelles in both stationary and mobile phases.

A new ion chromatographic (IC) system, which uses zwitterionic (e.g., Zwittergent 3-14) micelles as both stationary and mobile phases, highly useful for the analysis of inorganic anions in biological samples, was developed. The zwitterionic micellar stationary phase (which is obtained by immobilizing the zwitterionic surfactant on surfaces of the reversed-phase ODS) showed high ability to confine the elution bands of the large amount of SO4(2-) and Cl- to narrow zones. As a result, a base-line separation of NO2-, Br- and NO3- from SO4(2-) and Cl- is always achieved. The zwitterionic micellar mobile phase, (which is obtained by dissolving the zwitterionic surfactant with a suppressive electrolytic solution, e.g., aqueous NaHCO3 solution), on the other hand, showed high ability for rapid elution of proteins. The separation column is therefore always being cleaned up even after the protein-containing sample is directly injected. The zwitterionic micelles are also insensitive to conductivity detection, therefore either the suppressed or the non-suppressed conductivity detection method is applicable for detection of the analyte ions. Urine and serum were chosen as the model real samples and were analysed with direct sample injection; the results of successful determination of a number of inorganic anions (SO4(2-), Cl-, NO2-, Br- and NO3-) in both samples have demonstrated the usefulness of this new IC system.

Animals↗

Ion chromatographic analysis of hydronium ion using ODS modified with dodecylsulfate as the stationary phase.

The hydronium ion (H3O+), the most essential species to every field of chemistry, was successfully separated from other cations. This major goal was achieved by a HPLC approach in which a reversed-phase ODS which had been initially modified with dodecylsulfate was used as the stationary phase and an aqueous KNO3 solution was used as the mobile phase. Of all tested monovalent cations (analyte ions), the hydronium ion (H3O+) showed the highest affinity towards the stationary phase, followed by NH4+, Na+, and then Li+. The hydronium ion, after the separation, was directly detected using conductivity and the detection signal (conductivity) was in linear proportion (linear range 7.8 x 10(-4)-0.45 M; injection volume, 100 microL) to the concentration of the acid species of the tested sample. This proposed HPLC method was applicable to separate/detect H3O+ from both strong and weak acidic samples.

Journal Article↗

Helicobacter pylori sero-prevalence in asthma.

Asthma is a very common respiratory disease which is characterized by the presence of cytokine-mediated airway inflammation leading to smooth muscle contraction, oedema and progressive airway damage in some cases. In light of our recent finding of an increased sero-prevalence of Helicobacter pylori in bronchiectasis, we have determined serum levels of H. pylori-specific IgG in asthmatic and control subjects. Altogether 90 consecutive asthmatic [mean age +/- SD 42.6+/-16 years and 52 female (F)] and 97 healthy control subjects (mean age +/- SD 43.2+/-13.3 years and 51 female (F); P = 0.78 and 0.39 respectively) were recruited prospectively. H. pylori sero-prevalence was not significantly different between asthmatic and control subjects (P>0.05). Serum H. pylori IgG levels did not correlate with FEV1 % predicted, FVC % predicted or duration of asthma (P>0.05). Similar to the results of previously published sero-epidemiological studies, there was a weak correlation between serum H. pylori IgG with increasing age (r = 0.43, P = 0.004). Despite the sero-epidemiological association of H. pylori infection with many inflammatory conditions, our data showed no such association for middle age asthmatic patients with mild intermittent asthma in our locality.

Adult↗

Myeloablative chemotherapy for recurrent aggressive oligodendroglioma.

The objective of this study was to ascertain the duration of tumor control and the toxicities of dose-intense myeloablative chemotherapy for patients with recurrent oligodendrogliomas. Patients with previously irradiated oligodendrogliomas, either pure or mixed, that were contrast enhancing, measurable, and behaving aggressively at recurrence were eligible for this study. Only complete responders or major partial responders (75 % reduction in tumor size) to induction chemotherapy--either intensive-dose procarbazine, lomustine, and vincristine or cisplatin plus etoposide-could receive high-dose thiotepa (300 mg/m2/day for 3 days) followed by hematopoietic reconstitution using either bone marrow or peripheral blood stem cells. Thirty-eight patients began induction chemotherapy and 20 (10 men, 10 women; median age 46 years; median Karnofsky score 80) received high-dose thiotepa. For the high-dose group, the median event-free, progression-free, and overall survival times from recurrence were 17, 20, and 49 months, respectively. Tumor control in excess of 2 years was observed in 6 patients (30%). Four patients (20%) are alive and tumor free 27 to 77 months (median, 42 months) from the start of induction therapy; however, fatal treatment-related toxicities also occurred in 4 patients (20%). Three patients died as a result of a progressive encephalopathy which, in 2 instances, was accompanied by a wasting syndrome; 1 patient died as a consequence of an intracerebral (intratumoral) hemorrhage. Fatal toxicities occurred in patients with pretreatment Karnofsky scores of 60 or 70. High-dose thiotepa to consolidate response was a disappointing treatment strategy for patients with recurrent aggressive oligodendroglial neoplasms, although several patients had durable responses. Moreover, as prescribed, high-dose thiotepa had significant toxic effects in previously irradiated patients, especially those with poorer performance status.

Adult↗

Identification and analysis of bacterial protein secretion inhibitors utilizing a SecA-LacZ reporter fusion system.

Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds for Staphylococcus aureus strain MN8 were found to be < or =128 microg/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.

Adenosine Triphosphatases↗

Expression of a functional antizearalenone single-chain Fv antibody in transgenic Arabidopsis plants.

The efficacy of cloning a recombinant mycotoxin antibody in plants was tested using Arabidopsis as a model. An antizearalenone single-chain Fv (scFv) DNA fragment was first cloned in the newly constructed phage display vector (pEY.5) and then recloned in the plant transformation vector pKYLX71::35S(2). After transformation, constructs of antizearalenone scFv were introduced into immature Arabidopsis seeds via Agrobacterium tumefaciens mediation by vacuum infiltration. Only plants transformed with the construct containing a PR-1b signal peptide sequence produced transgenic offspring. The antizearalenone scFv "plantibody" from these transgenic plants bound zearalenone with a high affinity (50% inhibitory concentration, 11.2 ng/ml) that was comparable to that of bacterially produced scFv antibody and the parent monoclonal antibody (MAb). By electron microscopic immunogold labeling, the presence of antizearalenone scFv was detected mainly in the cytoplasm and only occasionally outside the cell. Like bacterially produced scFv antibody, antizearalenone scFv plantibody exhibited greater sensitivity to methanol destabilization than did the parent MAb. The sensitivity of antizearalenone scFv plantibody to acidic disassociation was similar to the sensitivities of bacterially produced scFv antibody and MAb. Expression of specific plantibodies in crops might be useful for neutralizing mycotoxins in animal feeds and for reducing mycotoxin-associated plant diseases.

Antibody Formation↗

Brain tissue sodium is a ticking clock telling time after arterial occlusion in rat focal cerebral ischemia.

BACKGROUND AND PURPOSE: Many patients with acute stroke are excluded from receiving thrombolysis agents within the necessary time limit (3 or 6 hours from stroke onset) because they or their family members are unable provide the time of stroke onset. Brain tissue sodium concentration ([Na(+)]) increases gradually and incessantly during the initial hours of experimental focal cerebral ischemia but only in severely damaged brain regions. We propose that this steady increase in [Na(+)] can be used to estimate the time after arterial occlusion in the rat middle cerebral artery occlusion model of ischemic stroke. METHODS: Sixteen anesthetized Sprague-Dawley rats underwent permanent middle cerebral artery occlusion combined with bilateral common artery occlusion. After 100 to 450 minutes, diffusion-weighted MRI was used to generate apparent diffusion coefficient (ADC) maps, cerebral blood flow (CBF) was determined with (14)C-iodoantipyrine (in a subset of 7 animals), and the brain was frozen. Autoradiographic CBF sections and punch samples for Na(+) analysis were obtained from the brain at the same level of the MR image. Severely at risk regions were identified with an ADC of <520 microm(2)/s and, in the subset, with both ADC of <520 microm(2)/s and CBF of <40 mL. 100 g(-1). min(-1). RESULTS: Both CBF and the ADC dropped quickly and remained stable in the initial hours after ischemic onset. Linear regression revealed strong linearity between [Na(+)] and time after onset, with a slope of 0.95 or 1.00 (mEq/kg DW)/min, with both ADC and ADC-plus-CBF criteria, respectively. The 95% CIs at 180 and 360 minutes were between 41 and 52 minutes. CONCLUSIONS: The time after ischemic onset can be estimated with this 2-step process. First, ADC and CBF are used to identify severely endangered regions. Second, the [Na(+)] in these regions is used to estimate time after onset. The favorable 95% CIs at the time limits for thrombolytic therapy and the availability of measurements of ADC, CBF, and [Na(+)] in humans through the use of MRI suggest that this time-estimation scheme could be used to assess the appropriateness of thrombolysis for patients who do not know when the stroke occurred.

Animals↗

Activation of fibrinolysis by SMTP-7 and -8, novel staplabin analogs with a pseudosymmetric structure.

Two novel staplabin analogs, SMTP-7 and -8, have been isolated from cultures of Stachybotrys microspora IFO 30018. Spectroscopic analyses showed that the SMTP-7 molecule consisted of two identical staplabin core structures and ornithine which bridges the two partial structures. In the SMTP-8 molecule, the bridging unit was lysine. At concentrations of 80 approximately 150 microM, the two compounds caused 2- to 12-fold increase in urokinase-catalyzed plasminogen activation, fibrin binding of plasminogen, and urokinase- and plasminogen-mediated fibrinolysis. These activities of SMTP-7 and -8 were two to ten times higher than those of staplabin and previously isolated SMTPs, which exerted such effects at concentrations ranging from 150 to 800 microM.

Benzopyrans↗