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Biomedical subjects

W Lee

Publications and source records attributed to W Lee.

At least 271 records · Page 15Linked to original sources
PubMed1993

Induced endocytosis in human fibroblasts by electrical fields.

Electroporation creates transient pores through which exogenous molecules can gain access to the cell cytoplasm. However, the electrical events associated with this phenomenon may perturb membrane-dependent events such as endocytosis. To measure the effect of electroporation on endocytosis, suspensions of human gingival fibroblasts were subjected to 5-ms electrical discharges, allowed to recover for variable periods of time, incubated with fluorescent probes, and then analyzed by flow cytometry. Incubation of electroporated fibroblasts with FITC-conjugated bovine serum albumin (BSA) to label moities on cell membranes nonspecifically demonstrated a time-dependent increase of internalized probe for up to 90 min after electroporation. Pretreatment incubation of cells with cytochalasin D abrogated the increased internalization of FITC-BSA due to electroporation. Compared to controls, fluorescence signals due to internalization of surface glycoproteins with FITC-concanavalin A were 43% higher after electroporation and treatment with endoglycosidase F or H to reduce probe associated with surface membrane. Confocal microscopy confirmed intracellular labeling and reduction of membrane-associated probe by the enzyme. Assessment of nonspecific FITC-Con A labeling of cells by pretreatment with alpha-methyl D-mannoside showed that labeling was largely (92%) specific. Compared to controls, electroporation induced a 60% increase of internalization of lucifer yellow, a fluid-phase endocytosis marker. Dual fluorescence labeling of membrane phosopholipids by FITC and TRITC-DHPE demonstrated an increased acidification after electroporation that was time dependent, indicating that electroporation induced more rapid entry of membrane lipid into endosomal compartments. These data demonstrate that the electrical fields used in electroporation of fibroblasts cause an actin-dependent increase in the internalization of all membrane components examined and an increased rate of probe entry in to acidifying compartments.

Actin Cytoskeleton↗
PubMed1993

Characterization of the role of individual protein binding motifs within the hepatitis B virus enhancer I on X promoter activity using linker scanning mutagenesis.

A combination of linker scanning mutagenesis and deletional analyses has been used to determine the role of individual DNA-protein binding sites on expression from the hepatitis B virus (HBV) enhancer I-X promoter (map position (mp) 1042-1354, HBV adw2). Linker scanning mutation of the EF-C site caused a 67.5% drop in X promoter activity in HuH7 cells, but had no effect in HepG2 or HepSK cells. Mutation of the E element resulted in an approximately 50% reduction in X promoter activity in HuH7, HepG2, and HepSK cells. Deletional analysis showed that sequences upstream of the EF-C site (mp 1163) were required for full X promoter activity and implicated the NF-1a site as being sufficient for basal X promoter activity. However, PCR-directed linker scanning mutation of the NF-1a site did not cause a reduction in X promoter activity, indicating that this site was not an essential component of the X promoter. Taken together, these results indicated that multiple, partially redundant protein:DNA interactions in the enhancer I are essential for full X promoter activity. The lack of an essential basal promoter element supports the suggestion that the two separate HBV enhancer elements (enhI and enhII) were created by integration of the X gene into a primordial enhancer element.

Base Sequence↗
PubMed1993

Long-wavelength UVA radiation induces oxidative stress, cytoskeletal damage and hemolysis.

We investigated the ability of the different wavelength regions of UV radiation, UVA (320-400 nm), UVB (290-320 nm) and UVC (200-290 nm), to induce hemolysis. Sheep erythrocytes were exposed to radiation from either a UVA1 (> 340 nm) sunlamp, a UVB sunlamp, or a UVC germicidal lamp. The doses used for the three wavelength regions were approximately equilethal to the survival of L5178Y murine lymphoma cells. Following exposure, negligible hemolysis was observed in the UVB- and UVC-irradiated erythrocytes, whereas a decrease in the relative cell number (RCN), indicative of hemolysis, was observed in the UVA1-exposed samples. The decrease in RCN was dependent on dose (0-1625 kJ/m2), time (0-78 h postirradiation) and cell density (10(6)-10(7) cells/mL).. Hemolysis decreased with increasing concentration of glutathione, hemoglobin or cell number, while the presence of pyruvate drastically enhanced it. Because scanning spectroscopy (200-700 nm) showed that hemoproteins and nicotinamide adenine dinucleotides were oxidized, cytoplasmic oxidative stress was implicated in the lytic mechanism. Further evidence of oxidation was obtained from electron micrographs, which revealed the formation of Heinz bodies near the plasma membrane. The data demonstrate that exposure of erythrocytes to UVA1, but not UVB or UVC, radiation causes oxidation of cytoplasmic components, which results in cytoskeletal damage and hemolysis.

Animals↗
PubMed1993

Morphologic and quantitative study of the efferent vestibular system in the chinchilla: 3-D reconstruction.

An HRP study of the EVN has been performed. Three groups of somas have been identified: Those located in the proximity of the vestibular nuclei, those sandwiched between the facial genu and the IVth ventricle, and those in the RF, surrounding the abducens nucleus. The number of somas is greater in the contralateral brain-stem side. Axons could be followed through the midline, but could not be traced to a labelled soma. A 3-D reconstruction of the EVN within the brain stem is presented.

Animals↗
PubMed1993

Use of FISH to detect chromosomal translocations and deletions. Analysis of chromosome rearrangement in synovial sarcoma cells from paraffin-embedded specimens.

Retrospective cytogenetic analysis was performed on paraffin-embedded cells from five cases of synovial sarcoma to evaluate the frequency of the X;18 translocation characteristic of this tumor. Fluorescent in situ hybridization with DNA probes for the centromeres of chromosomes X and 18 was used with whole chromosome painting probes for X and 18. Translocation was inferred when there were only two X and 18 centromere signals but three painting probe signals of unequal size. On this basis it was possible to identify the t(X;18) in three cases. The fourth case was found to have extra copies of chromosome 18 without translocation, while the fifth case, the only one with a questionable diagnosis, had a normal chromosome pattern with a minor clone showing a translocated 18 but a normal X. Thus this study demonstrates the feasibility and value of using fluorescent in situ hybridization to detect chromosome rearrangements in archival tumor specimens.

Adolescent↗
PubMed1993

Relationship between mitotic delay and the minimum dose rate of X irradiation required to stop cell proliferation.

When cells are subjected to irradiation, their progression through the cell cycle can be arrested. If the arrested cells are subjected to additional damage, their period of arrest is prolonged. Under continuous low-dose-rate irradiation, the cumulative nature of arrest time leads to a geometric increase in the arrest time as a function of the dose rate. Above a certain cell-line-specific dose rate, the arrest duration becomes infinite and cell proliferation ceases. We find that the lowest dose rate (critical dose rate) required to stop cell proliferation during continuous irradiation is the reciprocal of the mitotic delay per gray of high-dose-rate irradiation. The calculated critical dose rates for X rays agree with those measured by Mitchell et al. (Radiat. Res. 79, 537-551, 1979), provided that the critical dose rate is identified with the minimum dose rate which allows no more than one population doubling. This specific identification of the critical dose rate is explained on the basis of cell cycle kinetics. A kinetic mechanism for cell cycle arrest and recovery leads to the critical dose rate as the reciprocal sensitivity for G2 arrest.

Animals↗
PubMed1992

Thromboxane receptor antagonism combined with thromboxane synthase inhibition. 2. Synthesis and biological activity of 8-(benzenesulfonamido)-7-(3-pyridinyl)octaonic acid and related compounds.

A series of arylsulfonamide alkanoic acids substituted with a 3-pyridinyl group along the aliphatic chain were synthesized and tested in vitro for their ability to antagonize thromboxane A2 (TxA2) receptors and inhibit thromboxane synthase. These compounds were found to potently inhibit the U 46619-induced aggregation of human platelets and to also inhibit TxA2 biosynthesis in a human microsomal platelet preparation. However, some members of the series, notably compound 21, were found to display agonist activity on the rabbit aorta TxA2 receptor. This unwanted agonist activity appeared to be related to the presence of a substituent beta to the arylsulfonamido group.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1992

Thromboxane receptor antagonism combined with thromboxane synthase inhibition. 3. Pyridinylalkyl-substituted 8-[(arylsulfonyl)amino]octanoic acids.

A series of 8-[(arylsulfonyl)amino]octanoic acids substituted with a pyridinylalkyl group along the chain were synthesized and tested in vitro for their ability to both antagonize the binding of thromboxane A2 to its receptors and to inhibit the thromboxane synthase enzyme. This series of compounds were found to inhibit the U 46619-induced aggregation of human platelets and the U 46619-induced contraction of dog saphenous vein. The compounds also inhibited TxA2 biosynthesis in a human microsomal platelet preparation. The relative position of the pyridinylalkyl and arylsulfonamide groups had significant effects on the thromboxane receptor antagonist (TxRA) activity and thromboxane synthase inhibitor (TxSI) activity. Compounds with the pyridine ring at the 7- or 8-position of the octanoic acid side chain were weakly active as TxSI but behaved as potent TxRA at the platelet receptor for TxA2. However, these compounds were agonists at the vascular receptor. Substitution of the pyridinylalkyl group at the 2- or 3-position resulted in compounds with potent TxSI activity and weak TxRA activity. The activity profile of the compounds with the pyridinylalkyl substitution at the 4-, 5-, or 6-position was very desirable. Compound 22 with a pyridinylpropyl substituent at the 4-position was found to display extremely potent TxRA and TxSI properties.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1992

Thromboxane receptor antagonism combined with thromboxane synthase inhibition. 4. 8-[[(4-Chlorophenyl)sulfonyl]amino]-4-(3-(3-pyridinyl) propyl)octanoic acid and analogs.

The title compound (10a) and its analogs were synthesized and found to possess two activities, the inhibition of the biosynthesis of thromboxane A2 and antagonism of its receptors. The in vitro and in vivo profile of these compounds as thromboxane receptor antagonists (TxRAs) and thromboxane synthase inhibitors (TxSIs) is described. 10a and its analogs displayed very potent TxRA activity in human washed platelets (IC50 approximately 10(-7)-10(-9) M) and dog saphenous vein (pA2 approximately 9) and also potent TxSI activity (IC50 approximately 10(-9) M). The good bioavailability and the long duration of action of some of these compounds was demonstrated using ex vivo measurement of the TxRA activity upon oral administration to guinea pigs. Compounds 10a, 20, and 33 potently inhibited arachidonic acid induced bronchoconstriction in guinea pigs.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1992

Use of aminotransferase, hepatitis C antibody, and hepatitis C polymerase chain reaction RNA assays to establish the diagnosis of hepatitis C virus infection in a diagnostic virology laboratory.

Clinical and therapeutic decisions for hepatitis C virus (HCV) infection depend on factors that include documentation of past infection as well as identification of those who might benefit from antiviral chemotherapy with systemic interferon. To evaluate the ability of a diagnostic laboratory to accurately identify such patients, we compared results obtained with serum transaminase assays, two HCV antibody assays (enzyme immunoassay [EIA] and immunoblot), and a polymerase chain reaction (PCR)-based assay for HCV RNA using a group of consecutively submitted samples within our university-based diagnostic virology laboratory and sera from a population of random blood donors. One hundred percent of specimens with R values of greater than 3.0 in the HCV EIA were positive in the confirmatory immunoblot. However, 25% of specimens with EIA R values of between 1.0 and 3.0 were not confirmed by either recombinant immunoblot assay (RIBA) or RNA PCR assay (false-positive specimens). A significant correlation (P less than 0.01) between increasing reactivity in the RIBA and positivity in the RNA PCR assay was found. The incidence of HCV viremia, as determined by the RNA PCR assay, was 73% for confirmed seropositive specimens, 33% for seropositive specimens with indeterminate RIBA results, 12% for seronegative specimens obtained from infected patients, and 2.0% for seronegative specimens obtained from uninfected blood donors. In contrast, serum transaminase testing did not correlate with the RNA PCR assay for HCV. Use of the EIA and immunoblot assay followed by RNA PCR testing will identify most patients who are viremic with HCV.

Alanine Transaminase↗
PubMed1992

Maternal respiration and blood gases during aerobic exercise performed at moderate altitude.

We studied whether maternal acid-base status during aerobic exercise performed at moderate altitude is affected by pregnancy. Seven primiparus women were tested at 37 wk gestation and 12 wk postpartum. Subjects were studied at rest, and during two cycle (50 W, 75 W) and two treadmill (67 m.min-1; 2.5% grade, 67 m.min-1; 12% grade) protocols. Exercise bouts lasted 6 min with a 10-min rest between sessions. Minute (VE) and alveolar (VA) ventilation, tidal volume (VT), and ventilatory equivalent for carbon dioxide (VE/VCO2) were significantly (P less than 0.01) greater when exercise was performed during pregnancy. Physiological dead space (VD) was not affected by pregnancy status and did not differ between rest and exercise. Decreases (P less than 0.01) in arterial pH during exercise averaged 0.04 units in both pregnancy and postpartum. Despite similar change in maternal pH, carbon dioxide tension (PaCO2) remained unchanged during exercise at 37 wk gestation but decreased at 12 wk postpartum. Decreases in arterial bicarbonate [HCO3-] associated with exercise were smaller during pregnancy. Our findings indicate that pregnancy did not compromise maternal acid-base status during aerobic exercise.

Acid-Base Equilibrium↗
PubMed1992

Induction of phenotypically altered mammary epithelial cells by neutrons and gamma rays.

These studies have examined alterations in the in vivo growth properties of mammary epithelial cells isolated at 1, 4, and 16 weeks after in vivo irradiation with -137Cs gamma rays or fission-spectrum neutrons. Altered in vitro growth potential was characterized by the proliferation of epithelial foci (EF) from irradiated animals under conditions in which mammary cells from nonexposed animals senesced. These EF were further characterized based on their ability to be subcultured. Both gamma and neutron irradiation resulted in the appearance of cells capable of forming EF. Further, with increased time in situ between irradiation and cell isolation, the frequency of EF which were capable of being subcultured indefinitely (EFs) increased. Reducing the gamma-ray dose rate resulted in fewer EFs while reducing the neutron dose rate resulted in increased frequencies of EFs. These data confirm earlier observations following gamma irradiation and show these cellular changes are also observed following neutron irradiation. In addition, these data indicate that changes in dose rate primarily influence the emergence of immortalized cell populations.

Animals↗
PubMed1991

Quadruple immunosuppression in renal allografts--the Auckland experience.

Recipients of live related and first cadaveric renal allografts were retrospectively reviewed before and after institution of a standardized regimen incorporating greater immunosuppression, (by the addition of antilymphocyte preparations). The two review groups were comparable with 11 live related and 27 first cadaver grafts in each. Despite a small advantage in terms of graft survival and diminished rejection, this was at the expense of septic morbidity. The implications of this are discussed.

Antilymphocyte Serum↗
PubMed1991

The application of epitope mapping in the development of a new serological test for systemic candidosis.

A new serological test for systemic candidosis was developed by raising a rabbit antiserum probe against a specific epitope on Candida albicans, hsp 90. A major fragment at the carboxy terminal end of this immunodominant candidal antigen was epitope mapped by Geysen's method. An epitope, recognised by all infected patients with antibody to the 47 kDa antigen, was synthesized and conjugated to keyhole limpet haemocyanin. A rabbit was successfully immunized against this synthesized peptide epitope and this antiserum was compared, in a dot-immunobinding assay, with unfractionated hyperimmune rabbit antiserum to C. albicans and an affinity-purified rabbit antiserum to the 47 kDa antigen. The epitope-specific antibody probe was more sensitive than the hyperimmune candidal antiserum but less sensitive than the affinity-purified antibody against the 47 kDa antigen, which recognised multiple epitopes. This probe is technically easy to prepare in large amounts and gives no false positives.

Amino Acid Sequence↗
PubMed1991

Regulatory elements mediating transcription of the human Ha-ras gene.

In order to identify transcriptional regulatory elements controlling the expression of the human Ha-ras gene and to quantitatively assess the role of each element, we made mutations of the transcriptional regulatory region, including 5' and internal deletions, linker scanning and replacement mutations, and combinations of these mutations all fused to the bacterial chloramphenicol acetyltransferase gene. The promoter activity of each of these mutants was determined by measuring the transient expression of chloramphenicol acetyltransferase activity after transfection into human epithelial HeLa cells. We found that the most important regulatory region consists of two closely linked but functionally independent elements, the non-consensus GC-II element, CGGGCGGGC, centered at position -153 from the major transcription start site cluster and a new element, CCGGAA, centered at position -161 directly upstream from GC-II. In addition, there are two functional regulatory elements which make minor contributions to the full promoter activity; a double CCAAT NF-I binding site at position -88 and an unidentified upstream element between positions -199 and -252. Aside from GC-II, the GC boxes, of which there are a total of six between positions -185 and +85, make little or no contribution to Ha-ras promoter activity when individual mutations are tested in growing HeLa cells. The three potential AP2 sites and a weak single NF-I binding site make no contribution. The basal promoter region extending to position -75 from the major start site cluster has no independent activity in this TATA-less gene.

Base Sequence↗