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Biomedical subjects

J Wu

Publications and source records attributed to J Wu.

At least 901 records · Page 50Linked to original sources
PubMed1997

A novel methodology for assignment of disulfide bond pairings in proteins.

A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.

Chromatography, High Pressure Liquid↗
PubMed1997

Progression of mouse buthionine sulfoximine cataracts in vitro is inhibited by thiols or ascorbate.

Mouse lens cultures were employed to study the progression of cataracts initiated by injection of buthionine sulfoximine, an inhibitor of glutathione (GSH) biosynthesis. Culture of lenses removed from untreated mice on postnatal day 7, for 48 hr in the presence of 4 mm BSO, resulted in only limited cataractous changes. To enable substantial progression of cataracts in vitro, it was therefore necessary to pretreat the mice with BSO prior to lens culture. A single injection of BSO (4 nmol mg-1 lens), administered on day 7, resulted in >90% depletion of lens GSH within 3 days, but no visible cataractous changes. The clear lenses were incubated for 29+/-1 hr at 37 degrees C in Medium HL-1, supplemented with EGF, insulin and Ca2+, in the presence or absence of BSO, and were scored for cataract development by previously described criteria. In the absence of BSO, only 4 of 10 lenses developed large opacities. However, in the presence of 4 mm BSO, 40 out of 45 experimental lenses developed opacities affecting at least 50% of the lens visual field and were scored as stages 1C-4, depending upon the extent and density of the cataracts. In addition, three lenses had opacities involving 20-50% of the field (stage 1B). By contrast, less than 10% of lenses from untreated mice incubated in the absence of BSO developed opacities. The cataracts developed in 4 mm BSO were accompanied by reduction of lens glutathione levels to <0.010 nmol mg-1 lens. They were almost completely prevented by 1 mm ascorbate, 2 mm GSH, 2 mm GSH monoethyl ester and 2 mm cysteamine. GSH and GSH ester maintained lens glutathione content between 0.1 and 0.2 nmol mg-1 in the presence of BSO, whereas ascorbate did not prevent near-total GSH depletion. The prevention of cataracts by thiols and ascorbate was confirmed by lens Na/K ratios not significantly different from those in control lenses. The above combination of GSH depletion in vivo by a single injection of BSO, followed 3 days later with lens culture in the presence of BSO, may yield a useful system to elucidate and control the biochemical mechanisms involved in oxidative cataract induction by this GSH-depleting agent.

Animals↗
PubMed1997

Triazolam impairs delayed recall but not acquisition of various everyday memory tasks in humans.

A double-blind test battery was administered to 24 human subjects (8 control, 16 drug) to assess the effects of 0.125 mg triazolam (oral) on memory encoding and retention across delay intervals ranging from seconds to 1 week after presentation. Although the drug reduced immediate psychomotor performance, it did not impair recall of previously learned information, nor did it significantly impair encoding of new information. The drug enhanced immediate recall of the location and identity of playing cards, without affecting 4-h delayed recall. The drug treatment impaired correct recall of object names after a delay of 20 min. At 4 h delay, the drug impaired olfactory recognition and free-recall of object names. At both 1 day and 1 week delay, the drug impaired recall of biographical information and correct identification of picture-photographer pair associations. The drug also impaired the daily improvement of the drug group as compared with the control group in a geometric puzzle solving task. The time course of these memory impairments compares well with the known effects of triazolam on long-term potentiation (LTP), a candidate biological mechanism underlying telencephalic memory formation and expression.

Adolescent↗
PubMed1997

Detection of Babesia bovis using a DIG-labeled DNA probe.

A plasmid DNA containing the inserted Babesia bovis cDNA clone designated c51A was used to prepare a DNA probe for B. bovis. The purified 0.6 kb specific DNA fragment was labeled by DIG DNA labeling. After denaturation, the probe was hybridised with the blotted target DNA extracted from bovine red blood cells infected with B. bovis or other protozoa or bovine red blood cells plus extra white blood cells. It was found that the probe produced from purified 0.6 kb DNA fragment could detect sample of B. bovis DNA equivalent to 0.015 microliter of 10% whole infected blood. Compared to the same DNA fragment labeled with photobiotin, this new probe is more sensitive giving a darker hybridization signal, a lighter hybridization background and without any non-specific reactions. These results indicated that this DIG-labeled B. bovis C5lA probe could provide a sensitive and specific method to diagnose clinically suspected B. bovis infections and distinguish B. bovis DNA from other haemoprotozoan infections.

Affinity Labels↗
PubMed1997

Urine levels of aluminum after drinking tea.

A microwave-assisted acid digestion procedure coupled with a graphite furnace atomic absorption method has been applied in the determination of aluminum (Al) in urine to verify the correlation of free forms of Al in tea infusions and urinary excretion of Al. Significant urinary Al excretion has been found in 24-h urine of four volunteers after tea drinking. However, the difference in amount of Al excretion in urine between the consumption of Oolong (black tea) and Long-Jin (green tea), each of them with unique Al contents and species, was not significant. These findings indicated that the high levels of free Al species in tea infusions did not result in significant change in urinary excretion of the metal, possibly owing to the transformation by ligands present in food and the gastrointestinal tract (GIT). However, it could not be assumed that there was no big difference in absorption of the metal in the human body if fractions of consumed Al retained in the body or excreted by bile or feces were considered.

Adult↗
PubMed1997

Characterization of proinflammatory cytokine production and CD14 expression by murine alveolar macrophage cell lines.

Alveolar macrophages, which play a central role in lung defense, produce cytokines that help orchestrate local inflammatory responses. In sepsis and other pathological conditions, bacterial lipopolysaccharide endotoxin can induce alveolar macrophages (AM) to release proinflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1, and interleukin-6. Studying the mechanisms that control alveolar macrophage cytokine production may lead to better therapies for conditions involving inflammatory lung injury. We and others have noted significant differences between alveolar macrophages and peritoneal macrophages, but large numbers of human or murine alveolar macrophages are rarely available for detailed mechanistic studies. We have obtained three murine alveolar macrophage cell lines (AMJ2C8, AMJ2C11, and AMJ2C20) and have begun to characterize their cytokine responses to proinflammatory stimuli. We measured the effects of endotoxin, interferon gamma, and the combination of the two on production of tumor necrosis factor, interleukin-1 beta, and interleukin-6 in each line. We also studied the expression of the endotoxin receptor CD14 by these cells, and investigated the effect of serum on their endotoxin responsiveness. We show here that all three of the cell lines responded in a manner comparable to that of primary murine alveolar macrophages. Observed variations between these lines may reflect the documented heterogeneity seen in populations of primary alveolar macrophages. These cell lines should expand the repertoire of tools available to investigators studying regulation of murine alveolar macrophage responses.

Animals↗
PubMed1997

Effects of bath resistance on action potentials in the squid giant axon: myocardial implications.

This study presents a simplified version of the quasi-one-dimensional theory (Wu, J., E. A. Johnson, and J. M. Kootsey. 1996. A quasi-one-dimensional theory for anisotropic propagation of excitation in cardiac muscle. Biophys. J. 71:2427-2439) with two components of the extracellular current, along and perpendicular to the axis, and a simulation and its experimental confirmation for the giant axon of the squid. By extending the one-dimensional core conductor cable equations, this theory predicts, as confirmed by the experiment, that the shapes of the intracellular and the extracellular action potentials are related to the resistance of the bath. Such a result was previously only expected by the field theories. The correlation between the shapes of the intracellular and the extracellular potentials of the giant axon of the squid resembles that observed during the anisotropic propagation of excitation in cardiac muscle. Therefore, this study not only develops a quasi-one-dimensional theory for a squid axon, but also provides one possible factor contributing to the anisotropic propagation of action potentials in cardiac muscle.

Action Potentials↗
PubMed1997

Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap.

Purified smooth muscle myosin in the in vitro motility assay propels actin filaments at 1/10 the velocity, yet produces 3-4 times more force than skeletal muscle myosin. At the level of a single myosin molecule, these differences in force and actin filament velocity may be reflected in the size and duration of single motion and force-generating events, or in the kinetics of the cross-bridge cycle. Specifically, an increase in either unitary force or duty cycle may explain the enhanced force-generating capacity of smooth muscle myosin. Similarly, an increase in attached time or decrease in unitary displacement may explain the reduced actin filament velocity of smooth muscle myosin. To discriminate between these possibilities, we used a laser trap to measure unitary forces and displacements from single smooth and skeletal muscle myosin molecules. We analyzed our data using mean-variance analysis, which does not rely on scoring individual events by eye, and emphasizes periods in the data with constant properties. Both myosins demonstrated multiple but similar event populations with discrete peaks at approximately +11 and -11 nm in displacement, and 1.5 and 3.5 pN in force. Mean attached times for smooth muscle myosin were longer than for skeletal-muscle myosin. These results explain much of the difference in actin filament velocity between these myosins, and suggest that an increased duty cycle is responsible for the enhanced force-generating capacity of smooth over skeletal-muscle myosin.

Actins↗
PubMed1997

Genesis of MEG signals in a mammalian CNS structure.

Neuromagnetic signals of guinea pig hippocampal slices were characterized and compared with the extracellular field potential to elucidate the genesis of magnetoencephalographic signals in a mammalian CNS structure. The spatial distribution of magnetic evoked field (MEF) directed normal to bath surface was similar for transverse CA1, longitudinal CA1 and longitudinal CA3 slices in the presence of 0.1 mM picrotoxin (PTX) which blocks inhibitory synaptic transmission. Their MEFs were produced by currents along the longitudinal axis of the pyramidal cells. Comparisons of the MEF with the laminar potential profile revealed that the MEF was generated by intracellular longitudinal currents. The dipolar component of the intracellular currents was the dominant factor generating the MEF even at a distance of 2 mm from the slice. The MEF from a slice in Ringer's solution without PTX became similar in temporal waveform with time to the MEF in the presence of PTX, indicating the predominance of excitatory connections in generating the MEF and the existence of highly synchronous populations activities across the slice even in PTX-free Ringer's solution. The presence of such highly synchronous population activities underlying the MEFs was verified directly with field potentials recorded across the slice. A systematic variation of the stimulation site revealed a characteristic waveform for each site. The variation of the with stimulation site suggested the contribution of many factors, both synaptic and voltage-sensitive conductances, to the overall waveform of the MEF.

Action Potentials↗
PubMed1997

The antiproliferative effects of calcitonin gene-related peptide in different passages of cultured vascular smooth muscle cells.

Previously our laboratory showed that calcitonin gene-related peptide (CGRP) is released from perivascular nerves exposed to endotoxin or inflammatory mediators bradykinin and prostaglandins. CGRP contributes significantly to the vasodilation of inflammation and septic shock. Another potential action of CGRP is inhibition of vascular smooth muscle cell (VSMC) growth, which could serve to counterbalance the stimulatory effects of IL-1 and TNF on VSMC proliferation. VSMCs from rabbit and rat aorta (in the second and fifth passages) were plated at 100,000 cells/well in 24-well trays in 10% fetal bovine serum (FBS) for 24 h, incubated for an additional 24 h without FBS, and then exposed to 2.5% FBS for 24 h in the presence or absence of CGRP. 3H-thymidine incorporation was used to measure DNA synthesis and proliferation. CGRP caused significant inhibition of 3H-thymidine incorporation, which correlated with elevations of cAMP in both rat and rabbit aortic VSMCs. Interestingly, the responses of both the elevation of cAMP and the inhibition of DNA synthesis became larger in VSMCs with an increasing number of passages. The data suggest that the CGRP, released during vascular inflammation, may serve to inhibit the proliferation of VSMCs, thus limiting the growth of atheromatous lesions.

Animals↗
PubMed1997

Effects of vitamins E, C and catalase on bromobenzene- and hydrogen peroxide-induced intracellular oxidation and DNA single-strand breakage in Hep G2 cells.

BACKGROUND/AIMS: Water-soluble vitamin E (Trolox C), ascorbic acid and catalase were shown in our previous study to protect isolated rat hepatocytes against bromobenzene-induced toxicity. METHODS: In order to study the mechanisms of this protection and the pathogenesis of bromobenzene-induced hepatocellular injury, a fluorometric assay for the investigation of intracellular oxidation, indicated by conversion of dichlorofluorescein diacetate to dichlorofluorescein, was used. Single-strand DNA breakage was also evaluated in Hep G2 cells by a radio-labelling method. RESULTS: Bromobenzene (2.4 and 4.8 mM) induced a significant increase in dichlorofluorescein fluorescence intensity compared to the controls. Trolox C, ascorbic acid or catalase significantly inhibited bromobenzene-induced enhancement of fluorescence intensity (p<0.05-0.001), as well as reduced auto-intracellular oxidation in untreated Hep G2 cells. Hydrogen peroxide (H2O2) evoked a dose-dependent increase in dichlorofluorescein fluorescence intensity in Hep G2 cells, and the effect was completely blocked by Trolox C (2.0 mM) and catalase (4800 unit/ml). Bromobenzene caused significant single-strand DNA breakage in Hep G2 cells during 2 h suspension incubation and 24 h primary incubation. H2O2 (400 microM) led to marked single-strand DNA breakage in 20 min, and the effect was attenuated by Trolox C. CONCLUSIONS: Metabolism of bromobenzene in Hep G2 cells induces production of H2O2, indicated by enhancement of dichlorofluorescein fluorescence intensity, or other free radicals, which leads to single-strand DNA breakage in the cells. Vitamins E and C and catalase display strong intracellular antioxidative effects. Vitamin E could partially inhibit H2O2-induced single-strand DNA breakage in the cells.

Antioxidants↗
PubMed1997

Measurement of velocity and attenuation of shear waves in bovine compact bone using ultrasonic spectroscopy.

The ultrasonic spectroscopy (broadband pulse) technique was applied to simultaneously measure phase velocity and attenuation coefficient of shear waves in bovine compact bone at frequencies ranging from 4.0-10.0 MHz. It was found that the ratio of attenuation coefficient of shear waves to that of longitudinal waves at a particular frequency for bovine compact bone was smaller than that of other polymer synthesizing materials. The anisotropy of phase velocity and attenuation coefficient of compact bone was also experimentally examined.

Animals↗
PubMed1997

Nitric oxide synthase-expressing neurons are area-specifically distributed within the cerebral cortex of the rat.

Neuronal nitric oxide synthase produces nitric oxide, a radical involved in neurotransmission as well as in cytotoxicity during stroke and neurodegenerative diseases. In the adult Wistar rat neuronal nitric oxide synthase-positive neurons are inhomogenously distributed along defined cortical areas, with highest densities (18 cells/mm2) in cingular area 1, piriform cortex, frontal motor area Fr 2 and in the medial visual association area Oc 2MM. A medium packing density of neuronal nitric oxide synthase neurons (10/mm2) characterizes primary sensory areas, whereas retrosplenial cortices contain lowest cell numbers (3-5/mm2). The data suggest that functions of certain cortical areas are more dependent on intracortically produced nitric oxide than others, and that cortical injury may cause more severe nitric oxide related cytotoxicity in areas with higher numbers of neuronal nitric oxide synthase-positive neurons.

Animals↗
PubMed1997

Evidence for involvement of the neuronal isoform of nitric oxide synthase during induction of long-term potentiation and long-term depression in the rat dentate gyrus in vitro.

The possible role of nitric oxide in the induction of long-term potentiation and long-term depression of field excitatory postsynaptic potentials in the dentate gyrus of the hippocampal slice has been investigated, in the rat, using two novel nitric oxide synthase inhibitors, 1-(2-trifluoromethylphenyl)imidazole, which is selective for the neuronal isoform in vitro, and 3-bromo-7-nitro-indazole. Long-term potentiation was induced by a series of high-frequency trains, and long-term depression was induced by prolonged low-frequency stimulation at 1 Hz. The induction of long-term potentiation was inhibited by both 1-(2-trifluoromethylphenyl)imidazole and 3-bromo-7-nitro-indazole at concentrations which did not alter the amplitude of the test excitatory postsynaptic potential. The inhibitory effect of 1-(2-trifluoromethylphenyl)imidazole on the induction of long-term potentiation was prevented by pretreatment with L-arginine, the substrate amino acid used by nitric oxide synthase for nitric oxide production. The induction of long-term depression was inhibited by both 3-bromo-7-nitro-indazole and 1-(2-trifluoromethylphenyl)imidazole at concentrations which did not affect the test excitatory postsynaptic potential. The inhibitory effect of 1-(2-trifluoromethylphenyl)imidazole was prevented by pretreatment with L-arginine. The present experiments provide strong support for the involvement of the neuronal isoform of nitric oxide synthase in the induction of long-term potentation and long-term depression.

Animals↗
PubMed1997

Activation of fibroblast growth factor 8 gene expression in human embryonal carcinoma cells.

To study the role of fibroblast growth factor 8 (FGF-8) in human development and malignancies, we have isolated and characterized its gene. The gene spans 6.0 kbp and is comprised of five exons. Using reverse transcription-polymerase chain reaction, we were able to show that FGF-8 is expressed in two of the seven human mammary carcinoma cell lines tested and in only one of nine breast tumors. In contrast, both of the two normal breast tissues tested express FGF-8. FGF-8 was previously shown to be present in adult testis and ovary. Surprisingly, only one of the seven testis carcinomas and one of 12 ovary carcinomas express FGF-8, whereas all three kidney carcinomas tested express FGF-8. We further showed that fetal brain and lung express FGF-8, whereas fetal intestine and liver do not. Finally, we showed that a teratocarcinoma cell line, Tera-2, can be induced to express FGF-8 mRNA by fetal bovine serum.

Adult↗