Biomedical subjects

E Wang

Publications and source records attributed to E Wang.

At least 127 records · Page 7Linked to original sources

PubMed2000

Structure and expression of a membrane component of the inhibin receptor system.

The purification and cloning of a membrane-anchored proteoglycan with affinity for inhibin A are described. Bovine pituitary membranes were isolated, and membrane-anchored proteins were solubilized and used as an enriched source of inhibin binding protein. The extract was passed over an inhibin A affinity column, and a protein, designated p120, was identified as an inhibin-binding moiety. A partial amino acid sequence was determined for the protein, which matched two human complementary DNAs (cDNAs) in the database. The full-length cDNA predicts a 1336-amino acid glycoprotein. Full-length p120-encoding cDNAs were isolated from human testis RNA and cloned into expression vectors. Two p120 messenger RNA transcripts of 4.6 kb and 2 kb are detected in rat pituitary by RNA blot analysis. Similar analysis of rat testis RNA revealed transcripts of identical molecular mass, albeit at lower abundance. To determine the cellular localization of p120 in pituitary and testis, an antibody directed against the predicted extracellular domain of the protein was generated and used in an immunohistochemical analysis of thin tissue sections. p120 immunostaining is coincident with FSHbeta immunopositive gonadotrope cells in rat pituitary. p120 staining is intense in the testicular Leydig cells, which bind iodinated inhibin but not iodinated activin. In summary, an inhibin-binding protein has been isolated that is produced in tissues that are targets of inhibin action.

Activin Receptors↗

PubMed2000

Optical aberrations and objective choice in multicolor confocal microscopy.

Refinements in design have simplified confocal microscopy to the extent that it has become a standard research tool in cell biology. However, as confocal microscopes have become more powerful, they have also become more demanding of their optical components. In fact, optical aberrations that cause subtle defects in image quality in wide-field microscopy can have devastating effects in confocal microscopy. Unfortunately, the exacting optical requirements of confocal microscopy are often hidden by the optical system that guarantees a sharp image, even when the microscope is performing poorly. Optics manufacturers provide a wide range of microscope objectives, each designed for specific applications. This report demonstrates how the trade-offs involved in objective design can affect confocal microscopy.

Animals↗

PubMed2000

Blood flow distribution in sorbent beds: analysis of a new sorbent device for hemoperfusion.

A new polymer-based sorbent cartridge has been recently developed for enhancing middle molecule removal during hemodialysis. The cartridge (Betasorb, Renaltech, New York, USA) has been designed to be placed in series with the dialyzer in the blood circuit. It is therefore important to evaluate the distribution of flow into the blood compartment of the device in order to assess if the surface of the sorbent is utilized to the best. For this purpose, a special imaging technique was utilized. Cartridges were analyzed during a simulated in vitro circulation at 250 and 350 ml/min of blood flow and 25% and 40% hematocrit. Cartridges were placed in vertical position and a cross longitudinal section 1 cm thick was analyzed in sequence by a helical scanner. Dye was injected into the arterial inlet and the progressive distribution was evaluated by sequential densitometrical measures carried out automatically by the machine. The sequential images analyzed by the scanner demonstrated excellent distribution of the flow in the blood compartment with minimal difference between the central and the peripheral regions of the compartment. In particular the following flow velocity pattern could be observed under the different experimental conditions tested. We may conclude that the cartridge design is adequate and no channelling effects could be detected in the blood compartment. The flow distribution is slightly affected by changes in flow rate and hematocrit showing an optimal utilization of the available surface for molecule adsorption.

Adsorption↗

PubMed2000

A clinical investigation on tong xin luo capsule in treatment of coronary heart disease with silent myocardial ischemia.

36 cases of coronary heart disease with silent myocardial ischemia (SMI) were treated sequentially with Tong Xin Luo (TXL) capsule and isosorbide dinitrate in random order. The results showed that both drugs were effective in decreasing episodes of SMI and shortening the duration of asymptomatic myocardial ischemia. TXL, however, was found with a better action and was superior to isosorbide dinitrate. It was also found that TXL could improve diastolic function of the left ventricle as well.

Adult↗

PubMed2000

Predictive value of access blood flow and stenosis in detection of graft failure.

AIMS: Low access flow and the diagnosis of high degrees of venous stenosis have been recommended as indications for prophylactic angioplasty. However, recent studies have shown that prophylactic angioplasty for > 50% stenosis did not prolong graft patency, and that a single flow measurement may not accurately predict graft failure. In this study we compared the value of monthly measurement of access flow and of the maximal degree of stenosis in the detection of graft failure over a three-month period. METHODS: Thirty-nine hemodialysis patients with polytetrafluoroethylene (PTFE) grafts were evaluated by Doppler ultrasound at monthly intervals for three months. Graft failures were defined as thrombosis, or surgical and angioplastic revisions required because of the presence of access recirculation, and patients with graft failure were followed within the subsequent one-month periods of observation. RESULTS: Twelve graft failures occurred during the three-month period of observation. The risk for subsequent graft failure significantly increased at flows < 300 ml/min. Nine (20%) graft failures occurred with stenoses of 30 to 50%, and three (13%) with stenoses of> 50%. The grafts that failed in the second and the third study months had a 25.8% (380 +/- 62 vs. 287 +/- 190 ml/min, p < 0.05) and a 36.5% (393 +/- 142 vs. 226 +/- 41 ml/min, p < 0.05) decrease in access flow, respectively. There was no significant change in access flow for the grafts patent throughout the study (911 +/- 333, 794 +/- 302, and 919 +/383 ml/min, p = ns). No significant increases in maximal stenosis were found for the grafts that failed in the second month (44 +/- 6.1 vs. 48 +/- 15%, p = ns) and the third month (48 +/- 9 vs. 51 +/- 16%, p = ns). There were no significant changes in the maximum stenosis for the grafts patent throughout the three-month study periods (37 +/- 15,43 +/- 11, and 44 +/- 15%, p = ns). CONCLUSIONS: Access flow is a more sensitive predictor of graft failure than stenosis. Examination of trend in decline of access flow is a more powerful indicator to detect graft dysfunction than an individual single flow value.

Aged↗

PubMed1999

A single base substitution in the variable pocket of yeast tRNA(Arg) eliminates species-specific aminoacylation.

Early biochemical data showed that aminoacyl-tRNA synthetases often displayed species-specific recognition of tRNA. We compared the ability of purified Saccharomyces cerevisiae and Escherichia coli arginyl-tRNA synthetases to aminoacylate native and transcribed yeast tRNA(Arg) as well as E. coli tRNA(Arg). The kinetic data revealed that yeast ArgRS could charge E. coli tRNA(Arg), but at a lower efficiency than it charged either the transcribed or native yeast tRNA(Arg). E. coli ArgRS can acylate only its cognate E. coli tRNA. Strikingly, a single base change from C to A at position 20 in yeast tRNA(3)(Arg) altered the species specificity. The transcript of yeast tRNA(3)(Arg)CA20 mutant was aminoacylated by E. coli ArgRS with a 10(6) increase in k(cat)/K(m) over that for aminoacylation of yeast tRNA(3)(Arg) transcript. This indicates that A20 is not only an important identity of E. coli tRNA(Arg), but is also the key to altering species-specific aminoacylation of yeast tRNA(Arg).

Amino Acyl-tRNA Synthetases↗

PubMed1999

Functional analysis of antigen-specific T lymphocytes by serial measurement of gene expression in peripheral blood mononuclear cells and tumor specimens.

The cloning of cancer Ags recognized by T cells has provided potentially new tools to enhance immunity against metastatic cancer. The biological monitoring of effective immunization has, however, remained a dilemma. We describe here a sensitive molecular quantitation methodology that allows analysis of in vivo immune response to vaccination. Metastatic melanoma patients were immunized with a synthetically modified peptide epitope (209-2M) from the melanoma self-Ag gp100. Using serial gene expression analysis, we report functional evidence of vaccine-induced CTL reactivity in fresh cells obtained directly from the peripheral blood of postimmunized patients. Further, we demonstrate in vivo localization of vaccine-induced immune response within the tumor microenvironment. The results of these molecular assays provide direct evidence that peptide immunization in humans can result in tumor-specific CTL that localize to metastatic sites.

Biopsy, Needle↗

PubMed1999

Increased vaccine-specific T cell frequency after peptide-based vaccination correlates with increased susceptibility to in vitro stimulation but does not lead to tumor regression.

Although in vitro sensitization assays have shown increased melanoma Ag (MA)-specific CTL reactivity after vaccination with MA peptides, clinical responses have been uncommon. This paradox questions whether data obtained from the in vitro stimulation and expansion of T cells lead to an overestimation of the immune response to vaccines. Using HLA/peptide tetramer (tHLA), we enumerated MA-specific T cell precursor frequency (TCPF) directly in PBMC from 23 melanoma patients vaccinated with gp100:209-217(210M) (g209-2M) peptide. Vaccine-specific TCPF was higher in postvaccination PBMC from seven of seven patients treated with peptide alone and four of five patients treated with peptide plus IL-12 (range of postvaccination TCPF, 0.2-2.4% and 0.2-2.5%, respectively). The increased TCPF correlated with enhanced susceptibility to in vitro stimulation with the relevant epitope. Paradoxically, no increase in postvaccination TCPF was observed in most patients who had been concomitantly treated with IL-2 (1 of 11 patients; range of postvaccination TCPF, 0.02-1.0%), a combination associated with enhanced rates of tumor regression. The lack of increase in TCPF seen in these patients corresponded to inability to elicit expansion of vaccine-specific T cells in culture. This study shows that a peptide-based vaccine can effectively generate a quantifiable T cell-specific immune response in the PBMC of cancer patients, though such a response does not associate with a clinically evident regression of metastatic melanoma.

CD8-Positive T-Lymphocytes↗

PubMed1999

Optimized lymphocyte protein extraction performed simultaneously with DNA and RNA isolation: application to the study of factors affecting DNA, RNA, and protein recovery from lymphocytes of the oldest individuals.

We describe an optimized procedure for protein extraction performed simultaneously with that of DNA and RNA from a single tissue sample that is, unlike the original protocol, suitable for quantitative studies. This optimized protocol is particularly well adapted to studies where gene regulation at DNA, RNA, and protein levels must be examined simultaneously, and when the amount of starting biological material is limited. We applied this procedure to the study of factors affecting both qualitatively and quantitatively the extraction of DNA, RNA, and proteins from lymphocytes of very old individuals, since we observed variability in the recovery of these molecular species with advanced age. Therefore, we investigated the combined effects of age and time delay between blood collection and lymphocyte isolation on the recovery of DNA, RNA, and proteins simultaneously extracted from Danish nonagenarians and centenarians versus younger adult samples. Our results suggest that neither RNA nor DNA nor protein contents of lymphocytes are altered with aging. However, the quantity of RNA and protein recovery is affected by a 24-h delay in blood processing. This effect is more pronounced in the oldest, particularly for RNA, and may affect data interpretation of age-dependent gene expression studies.

3T3 Cells↗

PubMed1999

Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.

Acylation↗

PubMed1999

Discrimination of tRNALeu isoacceptors by the insertion mutant of Escherichia coli leucyl-tRNA synthetase.

A variant (LeuRS-A) of Escherichia coli leucyl-tRNA synthetase (LeuRS) carrying a 40-residue duplication in its connective peptide 1 (CP1) has a 3-fold lower specificity for than for, whereas wild-type LeuRS has the same specificity for these two isoacceptors. The replacement of the acceptor stem of with yields a chimeric tRNA(Leu) for which wild-type LeuRS has the same specificity as it does for the two normal isoacceptors mentioned, but for which LeuRS-A has a reduced specificity similar to that for, indicating a difference between these two acceptor stems. LeuRS-A is slightly less stable than the native enzyme. Wild-type LeuRS and LeuRS-A have almost same K(d) value for their interaction with as determined by fluorescence quenching. No difference was detected between these two proteins by CD and fluorescence spectroscopy. These results show that LeuRS-A can discriminate between the two isoacceptors of tRNA(Leu).

Acylation↗

PubMed1999

Intrachromosomal triplication of 2q11.2-q21 in a severely malformed infant: case report and review of triplications and their possible mechanism.

A female fetus with brain malformations, multicystic kidneys, absence of the right thumb, and a posterior cleft of palate was delivered at 32 weeks of gestation. Cytogenetic studies including FISH showed a novel intrachromosomal triplication of the proximal long arm of chromosome 2 (q11.2-q21), resulting in tetrasomy for this segment. The middle repeat was inverted. At least 11 patients with intrachromosomal triplications have been reported, mostly involving chromosome 15q. The mechanism involved in formation of these rearrangements is compatible with U-type exchange events among three chromatids.

Abnormalities, Multiple↗

PubMed1999

Separation of promethazine and thioridazine using capillary electrophoresis with end-column amperometric detection.

Promethazine and thioridazine were separated and detected by capillary electrophoresis with end-column amperometric detection. The influence of pH value on oxidation potential, the peak current and the resolution were studied and the following conditions was selected: 0.03 M Na2HPO4 and 0.015 M citric acid at pH 3.0, detection potential at 1.10 V. The detection limits of these two substances were in the range of 10(-8) mol/l. The linear range spanned two to three orders of magnitude. This method was applied to the detection of promethazine and thioridazine spiked in urine.

Antipsychotic Agents↗

PubMed1999

Toxin injury-dependent switched expression between EF-1 alpha and its sister, S1, in rat skeletal muscle.

Elongation factor-1 alpha, (EF-1 alpha), a translation factor involved in peptide chain elongation, is found ubiquitously in all cells. Previously, we identified a highly homologous EF-1 alpha sister gene, S1, whose transcript is found in only three tissues: brain, heart, and muscle, where the tissue-specific expression of S1 is caused by its exclusive presence in cells such as neurons and myocytes. Using sequence-specific synthetic peptides, we have recently produced polyclonal antibodies that can distinguish the protein product of EF-1 alpha from that of its sister, S1. Results of Western blotting show that these two proteins appear in S1-positive muscle tissue in inverse relationship, i.e., when S1 protein is in abundance, EF-1 alpha protein is in contrast in low quantity, and vice versa. During early embryonic stages, EF-1 alpha is the predominant protein species, whereas S1 is hardly detectable. This high EF-1 alpha versus low S1 protein presence undergoes a switch in that by postnatal day 14, EF-1 alpha is scarce whereas S1 is abundant; thus, there is a development-dependent shift of EF-1 alpha/S1 ratio from high to low, and the low EF-1 alpha/S1 ratio is maintained in adulthood. In this report, we describe the reversal of the EF-1 alpha/S1 ratio from low to high during muscle injury (experimentally induced by Marcaine injection), and a return to the original low ratio once the injury is repaired by regeneration. In this injury condition, EF-1 alpha is rapidly upregulated immediately after the Marcaine treatment, possibly reflecting an injury-dependent response of regeneration. The increase of EF-1 alpha corresponds with a decrease of S1 protein presence, thus resulting in a change of EF-1 alpha/S1 ratio from low to high. However, the high EF-1 alpha/S1 ratio eventually reverts to low, when regeneration-associated proliferation ceases, and fully differentiated myotubes are reestablished in the injured cells. This result shows that: (1) a high EF-1 alpha/S1 ratio is an early molecular diagnostic marker for injury-elicited regeneration; and (2) when injury repair is accomplished, there is a reversion to the low EF-1 alpha/S1 ratio, reflecting the restoration of the muscle fiber to the preinjury functional status. Results presented here not only show that a high EF-1 alpha/S1 ratio is a molecular marker for injured muscle, but also reveal the underpinning translational regulation in muscle during injury.

Actins↗

PubMed1999

Signaling pathway for apoptosis: a racetrack for life or death.

Apoptosis, or programmed cell death, is a gene-directed mechanism activated as a suicidal event to get rid of excess, damaged, or infected cells. The recent astounding pace of research in this area has expanded our horizon of understanding that this mechanism is regulated largely by pro- and anti-apoptosis factors acting for or against the final death event. The driving force behind these factors, either pro-apoptosis or pro-survival, is largely determined by signal transduction pathways, starting with the initiation of a death signal at the plasma membrane, and following through a complex cytoplasmic network before reaching the end point of cell demise. Enmeshed in this intricate cytoplasmic network are many checkpoints, where complexes of pro- and anti-apoptosis factors function to facilitate or deter the death signals. The culmination of the balancing act between these two camps of factors at these signal transduction checkpoints may then result in the final decision to die or to live. Thus, the eventual death of a cell may require successful passage through all the checkpoints, a mechanism Nature has provided as a safeguard to prevent erroneous triggering of death. With the advent of a new biotechnology revolution at the dawn of the new millenium, we look forward to an exciting era when we can gain fuller understanding of the operation of all these checkpoints. Ultimately, this gain will pave the way to control the apoptosis event at the checkpoints, and to support the organism's functionality as long as possible. J. Cell. Biochem. Suppls. 32/33:95-102, 1999.

Animals↗

PubMed1999

Alteration in sphingolipid metabolism: bioassays for fumonisin- and ISP-I-like activity in tissues, cells and other matrices.

The first discovered naturally occurring inhibitor of de novo sphingolipid biosynthesis was fumonisin B1. There are now 11 identified fungal inhibitors of ceramide synthase or 'fumonisin B1-like' compounds. With the exception of the australifungins, all other fungal ceramide synthase inhibitors are structurally sphingoid-like. There are several recently discovered fungal inhibitors of another enzyme in the de novo sphingolipid biosynthesis pathway: serine palmitoyltransferase (SPT). One of the SPT inhibitors is named ISP-I. While ceramide synthase inhibitors are toxic to animals, plants and fungi, the SPT inhibitors are not known to cause animal or plant disease, but are potent inhibitors of fungal growth. Very little is known about their toxicity in animals. There are at least 24 fungal SPT inhibitors produced by a variety of fungi. Given that the fungal inhibitors of sphingolipid biosynthesis are chemically and biologically diverse, two bioassays have been developed to screen for fumonisin-like or ISP-I-like activity in naturally contaminated products or fungal culture materials. These bioassays are based on the changes in free sphingoid base concentration that occur when the ceramide synthase or SPT are inhibited. The bioassays have the advantage that they are functionally rather than chemically specific and thus will detect ceramide synthase and SPT inhibitors regardless of their chemical structure.

Acyltransferases↗

PubMed1999

Differential regulation of inducible nitric oxide synthase gene expression by ethanol in the human intestinal epithelial cell line DLD-1.

We have examined the regulation of inducible nitric oxide synthase (iNOS) gene expression by ethanol in monolayers of DLD-1 cells, an epithelial cell line derived from human intestinal adenocarcinoma. Optimum induction of iNOS mRNA in these cells was obtained with IFN-gamma and IL-1beta treatment, while further addition of TNF-alpha did not have significant effect. In a set of experiments to study ethanol effects, DLD-1 monolayers were pretreated with ethanol for 24 h and were then treated with IFN-gamma + IL-1beta for an additional 24 h. Cells pretreated with ethanol showed decreased iNOS mRNA levels, indicating that ethanol may inhibit cytokine-induced iNOS transcription or affect mRNA destabilization. The suppression was ethanol-dose dependent with an IC50 of 50 mM. In another set of experiments to study ethanol effects, DLD-1 monolayers were pretreated with 66 mM ethanol for 24 h. These cells showed significant upregulation of IL-1beta mRNA and protein as detected in the supernatants. Aliquoted supernatants from these cells (i.e., conditioned media) were added to naive DLD-1 monolayers together with IFN-gamma. Conditioned medium from ethanol-treated cells increased the IFN-gamma-induced iNOS mRNA of naive cells by threefold. Two different effects of ethanol are now reported: (a) ethanol inhibits IFN-gamma + IL-1beta-induced iNOS mRNA of the same DLD-1 cells and (b) ethanol induces cellular paracrine signals by releasing IL-1beta into the medium, which in combination with IFN-gamma increases iNOS mRNA levels of the recipient naive DLD-1 cells. Because IFN-gamma and IL-1beta are produced by intestinal immune cells, these findings may have implications for differential in vivo regulation of epithelial iNOS genes by ethanol, depending on the inflammatory and immune status of the host.

Cell Line↗

PubMed1999

High-level expression and single-step purification of leucyl-tRNA synthetase from Escherichia coli.

A T7 promoter-based His6-tagging vector has been constructed that directs the synthesis in Escherichia coli of fusion proteins containing a stretch of six histidine residues at the N terminus. The vector allows overproduction and single-step purification of His6-fusion protein by immobilized metal (Ni2+) chelate affinity chromatography. The gene encoding leucyl-tRNA synthetase (leuS) was cloned into this vector and expressed in high level. The specific activity of the synthetase in the crude extract of E. coli JM109(DE3) transformant containing the His6-tagging vector with the gene leuS was approximately 110 times that of JM109(DE3) (the host strain without the vector). The overproduced His6-fusion leucyl-tRNA synthetase can be purified to homogeneity under native conditions within 2 h by one-step affinity chromatography with an overall yield of 55%. The His6-tag at the N terminus of leucyl-tRNA synthetase did not affect its aminoacylation activity or the secondary structure.

Chromatography, Affinity↗