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Biomedical subjects

M Wu

Publications and source records attributed to M Wu.

At least 271 records · Page 15Linked to original sources
PubMed1998

Determination of intraocular lens tilt and decentration using simple and rapid method.

PURPOSE: To determine the tilt and decentration of implanted intraocular lens (IOL) in vivo one year postoperatively. METHODS: A retrospective study was performed on 67 eyes by the method introduced by Guyton using the third and fourth Purkinje imagesm through undilated pupil. RESULTS: The average decentration of implanted IOL was 0.68 mm, and the average tilt was 6.03 degrees. The result was similar to the histopathological study in postmortem eyes and the measurement in vivo. Linear-regression analysis showed there was no relationship between the tilt and decentration and the residual astigmatism, axial length, IOL power, etc. CONCLUSION: The result is similar to the other methods. The method using the third and fourth Purkinje images is simple and easy to perform. It can be used for determining the IOL position through undilated pupil and does not require special apparatus.

Adult↗
PubMed1998

Recovery cycles of neurons in the inferior colliculus, the pontine nuclei and the auditory cortex of the big brown bat, Eptesicus fuscus.

This study examines the recovery cycles of neurons in the inferior colliculus (IC), the pontine nuclei (PN) and the auditory cortex (AC) of the big brown bat, Eptesicus fuscus, using a pair of identical pulses at different interpulse intervals. Although the recovery cycle varies among neurons, on average, AC neurons have the longest recovery cycle and PN neurons have the shortest one. The recovery cycle of IC neurons is slightly longer than PN neurons. Neurons tend to have longer recovery cycles when determined with long pulse duration than short one. The minimum threshold of these neurons to the second pulse increases with decreasing interpulse interval. The difference in the recovery cycles of these neurons supports previous findings that IC and PN neurons can follow sound pulses at higher rates than AC neurons do.

Acoustic Stimulation↗
PubMed1998

[Construction and application of retroviral vector carrying green fluorescent protein].

OBJECTIVE: To construct retroviral vector carrying rapidly selective marker. METHODS: The recombination retroviral vector GCGFPPXSN was constructed by cloning the green fluorescent protein (GFP) cDNA into the retroviral vector containing putative internal ribosome entry sites GCXPXSN and transferred in ecotropic packaging cell line PE501 by electroporation method. The supernatants of the PE501GCGFPPXSN were used to infect the amphotropic packaging cell line PA317. The G418 resistant clones were selected in 4 weeks and were detectable by fluorescence microscopy or by fluorescence-activated cell sorting(FACS). RESULTS: A recombination retroviral vector GCGFPPXSN carrying rapidly selective marker GFP was constructed. GFP expression in packaging cell line PA317-GCGFPPXSN transferred by GCGFPPXSN was detected by fluorescence microscopy of FACS. PA317-GCGFPPXSN grew to a titer of 1.2 x 10(5) cpu/ml. By cocultivating retroviral vector producing cells and T lymphocytes, expression of GFP was observed in T lymphocytes 2 days after the end of the cocultivation. T lymphocytes expressing GFP were separated. CONCLUSION: The mammalian cell can be efficient gene transfected by retroviral vector carrying GFP. The use of GFP for cell marking represents an important advantage over conventional strategies which typically involve the use of neomycin resistance. GFP, in fact, allows a rapid in vitro selection of transduced cell by FACS. The selection requires only two-day culture with this retroviral vector, compared with 10-14 day culture with a classical retroviral vector.

Cell Line↗
PubMed1997

Proteolysis of the carboxyl-terminal GPI signal independent of GPI modification as a mechanism for selective protein secretion.

Variable amounts of soluble forms of a variety of glycosyl-phosphatidylinositol (GPI)-anchored proteins occur extracellularly, but the molecular mechanisms governing their release are not entirely clear. When the GPI-anchored folate receptor (FR) type beta was expressed transiently in human 293 fibroblasts, there was a roughly equal distribution of [3H]folic acid binding protein between the cell surface and the medium after 24 h over a wide range of expression levels of FR-beta. The difference in apparent molecular masses between the soluble FR-beta and the PI-PLC-treated membrane protein indicated that the former was not released from the membrane by the action of phospholipase. Brefeldin A inhibited the release of soluble FR-beta from both the transfected 293 cells and stable recombinant CHO (CHO-FR-beta) cells while pre-existing levels of cell surface FR were unaltered suggesting the absence of a precursor-product relationship between the membrane-associated FR-beta and the soluble protein in the medium. [35S]Cysteine pulse-chase analysis was consistent with this finding. Interchanging of carboxyl-terminal peptides between FR-beta and FR-alpha revealed that the nature of the processed signal for GPI modification was responsible for the quantitative membrane anchoring of FR-alpha and the production of soluble FR-beta. When total cell lysates were analyzed by Western blot, a diffuse band of apparent 41 kDa and three additional sharp bands of apparent 35, 33, and 29.3 kDa were seen. The 41 kDa band was identified as the PI-PLC sensitive cell surface receptor. Several mutant constructs of FR-beta, in which the carboxyl-terminal signal for GPI modification was either disrupted or deleted only gave the three lower bands. The three sharp bands from the wild-type and the mutant forms of FR-beta were identified as nonglycosylated (29.3 kDa) or glycosylated polypeptides in which the carboxyl-terminal peptide was at least partially proteolyzed without GPI modification. All of the mutations in the GPI signal resulted in the recovery of [3H]folic acid binding protein in the media which, similar to the wild-type FR recovered from the media, were converted to the 29.3 kDa band by N-glycanase. The results from this study indicate that a carboxyl-terminal peptide in FR-beta is efficiently proteolyzed intracellularly by a pathway that is independent of GPI signal recognition resulting in proper protein folding and secretion. Such carboxyl-terminal sequences could represent a simple adaptation for proteins whose physiologic functions reside both at the cell surface and in extracellular fluids, allowing their selective and tissue-specific release.

Amino Acid Sequence↗
PubMed1997

Clustering of GPI-anchored folate receptor independent of both cross-linking and association with caveolin.

The distribution of the glycosyl-phosphatidylinositol (GPI)-anchored folate receptor (FR) in a diffuse pattern vs. functional clusters associated with caveolae has been debated. The equivocal nature of direct localization studies is due to possible experimental artifacts such as cross-linking of the protein by the antibody probes prior to fixation and alternatively the use of a disruptive fixation method. Such studies have also been complicated by the use of cells that vastly overexpress FR. In this study a monovalent probe, i.e., a biotinylated folate affinity analogue was used to covalently label FR. Cells expressing moderate levels of FR, i.e., JAR epithelial cells expressing FR-alpha and recombinant CHO fibroblasts expressing FR-beta, were used. The affinity label and either caveolin or antigenic sites on FR were localized by electron microscopy using colloidal gold conjugated antibody probes post-embedding in the relatively permeable LR White resin. The method avoided both receptor cross-linking and early fixation steps and also enabled the use of transport permissive conditions while labeling FR at the cell surface. The results indicate that in steady-state FR is not significantly colocalized with caveolin. However, the receptor molecules occur predominantly in clusters, independent of cross-linking, providing a physical basis for the observed kinetics of receptor internalization and recycling during folate transport. Evidence is also presented to suggest that early mild fixation will disrupt the clustering of FR.

Animals↗
PubMed1997

Mutations of O6-methylguanine-DNA methyltransferase gene in esophageal cancer tissues from Northern China.

Epidemiologic studies have implicated the involvement of environmental factors in the etiology of esophageal cancer (EC). Our previous data have indicated that EC patients and their blood relatives show genomic instability and are deficient in repair of DNA damage induced by N-nitroso-compounds and related genotoxic agents. Thus, exposure to high levels of N-alkylnitrosamines, which are known animal carcinogens and which induce alkyl adducts in DNA, may be causally linked to EC. Among the alkyl adducts, O6-alkylguanine was shown to be the critical one related to carcinogenic lesions; its repair varies widely in a tissue-specific fashion. O6-methylguanine-DNA methyltransferase (MGMT) is responsible for its repair. Hence, inactivating mutations in this protein would impair the efficiency of the repair process. To screen for possible mutations in the MGMT polypeptide in EC patients, we analyzed a highly conserved region of the MGMT gene in 40 EC tissues and lymphocytes of 6 high-risk EC family members from high EC areas of Northern China by polymerase chain reaction single-strand conformation polymorphism and by direct sequencing of PCR products. Ten base substitutions (point mutations) within 8 codons of 7 EC samples were identified. However, no germline mutation was found in the high EC families studied so far. Concurrent study in 30 pairs of fresh EC tissues and their adjacent normal mucosa by Southern blot and Western blot analyses showed deletion of the MGMT gene in 2 samples. Thus, the high frequency of mutations (7/40) and deletions (2/30) of the MGMT gene may be partially responsible for the overall high mutation rate observed in EC tissues.

Base Sequence↗
PubMed1997

Validation of a high-performance liquid chromatographic assay method for pharmacokinetic evaluation of busulfan.

The development and validation of a high-performance liquid chromatographic (HPLC) assay for determination of busulfan concentrations in human plasma for pharmacokinetic studies is described. Plasma samples containing busulfan and 1,6-bis(methanesulfonyloxy)hexane, and internal standard, were prepared by derivatization with sodium diethyldithiocarbamate (DDTC) followed by addition of methanol and extraction with ethyl acetate. The extract was dried under nitrogen and the samples reconstituted with 100 microl of methanol prior to HPLC determination. Chromatography was accomplished using a Waters NovaPak octadecylsilyl (ODS) (150 x 3.9 mm I.D.) analytical column, NovaPak ODS guard column, and mobile phase of methanol-water (80:20, v/v) at a flow-rate of 0.8 ml/min with UV detection at 251 nm. The limit of detection was 0.0200 microg/ml (signal-to-noise ratio of 6) with a limit of quantitation (LOQ) of 0.0600 microg/ml for busulfan in plasma. Calibration curves were linear from 0.0600 to 3.00 microg/ml in plasma (500 microl) using a 1/y weighting scheme. Precision of the assay, as represented by C.V. of the observed peak area ratio values, ranged from 4.41 to 13.5% (13.5% at LOQ). No day-to-day variability was observed in predicted concentration values and the bias was low for all concentrations evaluated (bias: 0 to 4.76%; LOQ: 2.91%). The mean derivatization and extraction yield observed for busulfan in plasma at 0.200, 1.20 and 2.00 microg/ml was 98.5% (range 93.4 to 107%). Plasma samples containing potential busulfan metabolites and co-administered drugs, which may be present in clinical samples, provided no response indicating this assay procedure is selective for busulfan. This method was used to analyze plasma concentrations following administration of a 1 mg/kg oral busulfan dose.

Busulfan↗
PubMed1997

Solution structure of (rGGCAGGCC)2 by two-dimensional NMR and the iterative relaxation matrix approach.

The three-dimensional solution structure of the RNA self-complementary duplex [formula in text] was derived from two-dimensional NMR and the iterative relaxation matrix approach. Each GA mismatch forms two hydrogen bonds: A-NH6 to G-O6 and A-N1 to G-NH1 (imino). This GA structure differs from the sheared tandem GA structure in [formula in text] which also has two hydrogen bonds: A-N7 to G-NH2 and A-NH6 to G-N3 [SantaLucia, J., Jr & Turner, D. H. (1993) Biochemistry 32, 12612-12623], although the only difference between the two sequences is the order of the two GA mismatches. Inspection of three-dimensional structures indicates that substituting [formula in text] for [formula in text] makes GA mismatches unable to have stable sheared conformations. This may explain why the [formula in text] motif is rarely observed in nature, whereas [formula in text] common.

Crystallography, X-Ray↗
PubMed1997

Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway.

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on beta-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that beta-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with beta-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32-cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous beta-catenin. Steady-state levels and nuclear accumulation of beta-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased beta-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of beta-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in beta-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in beta-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.

Animals↗
PubMed1997

Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs.

The dorsal-ventral axis in frog embryos is specified during the first cell cycle, when the cortex rotates relative to the cytoplasmic core along parallel microtubules associated with the core. Cytoplasmic transfer experiments suggest that dorsal determinants are transported 90 degrees from the vegetal pole to the dorsal equator, even though the cortex rotates only 30 degrees. Here we show that, during rotation, small endogenous organelles are rapidly propelled along the subcortical microtubules toward the future dorsal side and that fluorescent carboxylated beads injected into the vegetal pole are transported at least 60 degrees toward the equator. We also show that deuterium oxide, which broadens the zone of dorsalization even though it reduces the extent of rotation and is known to randomize the microtubules, also randomizes the direction of organelle transport. Moreover, beta-catenin, a component of the Wnt signaling pathway that possesses dorsalizing activity in Xenopus, colocalizes with subcortical microtubules at the dorsal side of the egg at the end of rotation. We propose that cortical rotation functions to align subcortical microtubules, which then mediate the transport of dorsal determinants toward their plus ends on one side of the egg.

Animals↗
PubMed1997

Inherited somatic mosaicism caused by an intracisternal A particle insertion in the mouse tyrosinase gene.

A recessive, fully penetrant mutation (c(m1OR)) at the mouse albino locus that results in coat-color mottling has been characterized at the molecular level. Restriction mapping and DNA sequencing analyses provide evidence that mutants carry a 5.4-kb intracisternal A particle (IAP) element insertion upstream of the tyrosinase (Tyr) promoter. Northern blot analysis and reverse transcription-PCR results show that the tyrosinase gene is expressed at much lower levels in mutant than in wild-type mice. The mutant Tyr gene still retains the tissue-specific expression pattern, and the Tyr transcript is not initiated from the IAP long terminal repeat promoter. We propose that the IAP insertion isolates the promoter of the tyrosinase gene from upstream cis-acting regulatory elements, leading to a substantially decreased level of Tyr gene expression in mutants.

Animals↗
PubMed1997

Cymbidium mosaic potexvirus RNA: complete nucleotide sequence and phylogenetic analysis.

The complete nucleotide sequence of the genomic RNA of cymbidium mosaic potexvirus (CymMV) was determined to be 6227 nucleotides in length, excluding the poly (A) tail at the 3' terminus. Similar to other potexviruses, its genome organisation is comprised of five major open reading frames (ORFs 1 to 5), encoding a Mr 160 KDa putative RNA-dependent RNA polymerase (RdRp); a Mr 26KDa/13KDa/10KDa triple-gene-block (TGB) and a Mr 24 KDa coat protein. The CymMV encoded proteins shared a high degree of homology to their corresponding proteins of other members of the potexvirus group. The nucleotide sequence of the 5' noncoding region (NCR) of CymMV and all other potexviruses initiates with GAAAA. CymMV possesses the shortest 5' NCR among all potexviruses. Based on phylogenetic comparisons of RdRp and coat protein, CymMV shares a close relationship to PAMV, NMV, WClMV and SMYEaV. This is believed to be the first record of the complete nucleotide sequence of CymMV.

Amino Acid Sequence↗
PubMed1997

Mapping the distribution of outer hair cell voltage-dependent conductances by electrical amputation.

The mammalian outer hair cell (OHC) functions not only as sensory receptor, but also as mechanical effector; this unique union is believed to enhance our ability to discriminate among acoustic frequencies, especially in the kilohertz range. An electrical technique designed to isolate restricted portions of the plasma membrane was used to map the distribution of voltage-dependent conductances along the cylindrical extent of the cell. We show that three voltage-dependent currents, outward K, I(K,n), and I(Ca) are localized to the basal, synaptic pole of the OHC. Previously we showed that the lateral membrane of the OHC harbors a dense population of voltage sensor-motor elements responsible for OHC motility. This segregation of membrane molecules may have important implications for auditory function. The distribution of OHC conductances will influence the cable properties of the cell, thereby potentially controlling the voltage magnitudes experienced by the motility voltage sensors in the lateral membrane, and thus the output of the "cochlear amplifier."

Acoustic Stimulation↗
PubMed1997

Incidence of premature birth and neonatal respiratory disease in infants of HIV-positive mothers. The Pediatric Pulmonary and Cardiovascular Complications of Vertically Transmitted Human Immunodeficiency Virus Infection Study Group.

OBJECTIVE: We sought to determine the prematurity rate in infants of HIV-positive mothers and to characterize the incidence and severity of neonatal respiratory disease in this population. STUDY DESIGN: From 1990 to 1994, 600 live-born infants of HIV-infected mothers were enrolled prenatally (73%) or postnatally (27%) from five U.S. centers. Logistic regression was used to determine the association of HIV status in the infant with prematurity (< or = 37 weeks), low birth weight (< or = 2.5 kg), and very low birth weight (< or = 1.5 kg) rates. The incidence of respiratory distress syndrome (RDS), bronchopulmonary dysplasia, meconium aspiration syndrome, and neonatal pneumonia was compared with anticipated rates for gestational age and birth weight. RESULTS: Very high rates of prematurity (19%), low birth weight (18.3%), and very low birth weight (3.3%) were found in the infants of HIV-positive mothers; and HIV infection in the infant was associated with younger gestational age. The overall incidence of RDS was 3% (17/600), which coincided with the anticipated rate, after adjusting for prematurity and birth weight. Only five infants (all < or = 1.5 kg) had bronchopulmonary dysplasia, and none required assisted ventilation beyond 14 days. Three term infants had mild meconium aspiration syndrome, and there were no cases of documented neonatal pneumonia. CONCLUSION: Infants born to HIV-positive mothers exhibited high prematurity and low birth weight rates, and the odds of prematurity were higher in infants who were infected with HIV. Despite the high incidence of prematurity and perinatal risk of this population, incidence and severity of neonatal respiratory disease were not higher than would be expected from available neonatal data in populations not exposed to HIV.

Adult↗
PubMed1997

Are immunoconjugates useful for therapy with autoimmune diseases?

Immunoconjugates or immunotoxins (ITs) are targeting molecules which consist of a monoclonal antibody together with a toxin--thereby they can selectively kill target cells in a highly efficient manner. The use of ITs as a drug targeting approach is one of the most attractive research fields for tumor therapy; however, the study of ITs for the treatment of autoimmune diseases has been given little attention until recently. It has been shown that ITs could help alleviate the symptoms of myasthenia gravis and rheumatoid arthritis in animal models. In the last 3 yr ITs have been used in clinical trials (phase I and phase II) for the treatment of various autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus and psoriasis. This article reviews the main progress on the application of ITs for the therapies of autoimmune diseases. The preliminary results suggest the future may hold some promise, but side effects, in addition to there being no convincing efficacy, remain unresolved.

Animals↗
PubMed1997

Immunological screening of a genomic M. bovis BCG library expressed in M. smegmatis and identification of the M. bovis BCG analog of ClpB.

This work describes the screening of a M. bovis BCG cosmid library in M. smegmatis with a hyperimmune rabbit anti-BCG serum. Cross-reactive antibodies interfere with the detection of BCG specific antigens in M. smegmatis culture filtrates. We, therefore, screened parallel western blots with serum adsorbed with a M. smegmatis cell lysate and unadsorbed serum. Comparison of the western blots allowed distinction between BCG specific and cross-reactive M. smegmatis antigens. Thirty-one cosmids expressed BCG specific antigens. One of them, a hitherto undescribed 100 kDa antigen was subcloned, sequenced and expressed in E. coli. It shows a high degree of homology to ClpB, a member of the Clp family of proteases and was immunologically reactive with the rabbit hyperimmune serum against M. bovis BCG. A positive signal was also obtained with sera of patients with tuberculosis. This antigen is a previously unrecognized target of the human immune response to mycobacteria.

Animals↗