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Biomedical subjects

H M Wang

Publications and source records attributed to H M Wang.

At least 73 records · Page 4Linked to original sources
PubMed1993

Angiotensin II regulates parathyroid hormone-related protein expression in cultured rat aortic smooth muscle cells through transcriptional and post-transcriptional mechanisms.

Parathyroid hormone-related protein (PTHrP), a tumor product responsible for malignancy-associated hypercalcemia, is also produced in many normal tissues, including vascular smooth muscle cells (SMC). As PTHrP exhibits vasodilatory properties, we postulated that other vasoactive agents may control PTHrP gene expression in SMC. Addition of angiotensin II to serum-deprived SMC resulted in a marked induction of PTHrP mRNA by 2 h, with a peak (6-10-fold) at 4-6 h. Angiotensin II effects on PTHrP gene expression were inhibited by saralasin, an angiotensin II receptor antagonist, and blocked by actinomycin D and cycloheximide, suggesting a requirement for gene transcription and protein synthesis. Nuclear run-off assays revealed a 3-fold increase in PTHrP gene transcription 1 h after angiotensin II treatment. Angiotensin II also prolonged PTHrP mRNA half-life by 2-3-fold. Angiotensin-induced PTHrP mRNA is partially dependent on cyclooxygenase products and protein kinase C activation. Other vasoconstrictor substances, including serotonin and bradykinin, also stimulated PTHrP expression, whereas the vasodilator atrial natriuretic peptide did not. Addition of recombinant PTHrP-(1-141) significantly inhibited angiotensin II-induced SMC DNA synthesis. PTHrP expression is increased by angiotensin II through transcriptional and post-transcriptional mechanisms. In addition, PTHrP modulates the effect of angiotensin II on SMC proliferation. This suggests that PTHrP acts locally in SMC, possibly to oppose the vasoactive and/or growth-promoting effects of vasoconstrictor agents such as angiotensin II.

Angiotensin II↗
PubMed1993

Transplantation of cultured human retinal pigment epithelium into rabbit subretina.

Transplantation of normal retinal pigment epithelium (RPE) into a diseased eye holds promise for treatment of several blinding disorders. Previous studies have involved immunosuppression and implantation of freshly isolated cells. We report here the successful transplantation of cultured human RPE cells into rabbits that were not immunosuppressed. A modified pars plana transvitreal technique was used for RPE transplantation. The cultured RPE cells, loaded with carbon as a marker, were transplanted into the denuded Bruch's membrane of albino rabbits. The animals were followed for from 1 week to 3 months. On histologic examination at 2 months, no infiltrating lymphocytes were found in the vitreous cavity or choroid, even though Bruch's membrane was damaged. At about 3 months there were some macrophages in the subretina of transplanted eyes, indicating that an immunoreaction does occur eventually. Electron microscopy of the transplanted RPE showed apical-basal polarity and gap junctions. Restored function was attested to by the presence of phagosomes and phagocytosed outer segments in the transplanted cells. Our findings suggest that there is a weak, delayed immunoreaction to human RPE cells transplanted beneath the retina of the rabbit; however, functional recovery of the transplanted cells occurs before this immune response develops.

Adult↗
PubMed1993

Allotransplantation of rabbit retinal pigment epithelial cells double-labelled with 5-bromodeoxyuridine (BrdU) and natural pigment.

To facilitate identification of transplanted retinal pigment epithelial (RPE) cells, we sought to double-label the cells with 5-bromodeoxyuridine (BrdU) and with natural pigment. The BrdU is not lost during cell division but does require immunohistochemical methods for visualization; the pigment, on the other hand, allows immediate, obvious identification, but is gradually lost with cell division. Together they provide a convenient, long-term double label. Herein we report the successful allotransplantation of double-labelled RPE cells onto Bruch's membrane of albino rabbits. The labelled cells were localized by anti-BrdU antibody and the avidin biotin-alkaline phosphatase complex (ABC-AP) method, and by visible inclusions of pigment. Using this double-label method, allotransplanted RPE cells could be readily and reliably identified on the recipient Bruch's membrane eight months after transplantation. The cells had distinct basal and apical morphology, and were in close contact with the photoreceptor outer segments of the host. This successful allotransplantation raises the possibility that the subretinal space of the rabbit might enjoy some degree of immunologic privilege.

Animals↗
PubMed1993

Synthetic and cytotoxic studies of alpha-methylene-gamma-butyrolactone bearing pyrimidines.

A total of ten pyrimidine alpha-methylene-gamma-butyrolactones were synthesized as potential antitumor agents on the basis of their possible action as Michael acceptors for DNA/RNA and cellular enzymes. The synthesis of these heterocycles involved a convenient Reformatsky-type reaction of pyrimidinyl ketones with ethyl alpha-(bromomethyl)acrylate. The preliminary in vitro cytotoxic assay indicated that these synthetic compounds were essentially active against the growth of KB, Hep-2, HeLa and Colo-205 cells. Among them, 5'-biphenyl-5'-(uracil-1-ylmethyl)-2'-oxo-3'-methylenetetrahydr ofuran (5f) demonstrated to be the most potent antileukemic agent.

4-Butyrolactone↗
PubMed1992

Monoclonal antibodies specific for low-affinity interleukin-3 (IL-3) binding protein AIC2A: evidence that AIC2A is a component of a high-affinity IL-3 receptor.

Mouse interleukin-3 (IL-3) binds to its receptor with high and low affinities. Using anti-Aic2 antibody, two distinct cDNAs (AIC2A and AIC2B) were isolated. The AIC2A gene encodes a protein of 120 Kd that binds IL-3 with low affinity, whereas the AIC2B gene encodes a protein that is 91% identical to AIC2A at the amino acid level, but which does not bind IL-3. To study the structure of the functional high-affinity IL-3 receptor (IL-3R), we generated specific monoclonal antibodies against the AIC2A protein. We produced a soluble AIC2A protein by inserting a termination codon at the beginning of the transmembrane domain of the AIC2A cDNA. Soluble AIC2A protein expressed in COS7 cells was purified to homogeneity and three anti-AIC2A monoclonal antibody-producing hybridomas (3D1, 3D4, and 9D3) were obtained from a rat immunized with the purified soluble AIC2A protein. The antibodies were specific for the AIC2A protein and did not bind to the AIC2B protein. Using chimeric receptors between AIC2A and AIC2B, recognition sites of the antibodies were mapped. The antibodies immunoprecipitated a 120-Kd protein from IL-3-dependent PT18 cells. The N-terminal sequence of the 120-Kd protein was consistent with the predicted processing site of the signal sequence of the AIC2A protein. Staining of IL-3-dependent and IL-3-independent cell lines with the 9D3 antibody were consistent with the IL-3 binding. The 9D3 antibody inhibited both the high-affinity IL-3 binding and the low-affinity binding, as well as IL-3-dependent proliferation. These results indicate that the AIC2A protein is a binding component of a high-affinity IL-3R.

Amino Acid Sequence↗
PubMed1992

Search for the heparin antithrombin III-binding site precursor.

The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins.

Animals↗
PubMed1992

Structure of mouse interleukin 3 (IL-3) binding protein (AIC2A). Amino acid residues critical for IL-3 binding.

Despite the high degree of sequence homology between two mouse proteins AIC2A and AIC2B (91% at the amino acid level), only the AIC2A protein binds interleukin 3 (IL-3). Soluble AIC2A protein bound IL-3 with affinity similar to the membrane-bound AIC2A protein, indicating that binding of IL-3 to AIC2A was mediated by the external domain alone. The extracellular domain of the AIC2A protein has two repeats of the common motif shared by members of the cytokine receptor family. Neither one of these repeats alone bound IL-3. Hybrids of AIC2A and AIC2B revealed that the first domain of the cytokine receptor motif could be replaced with the AIC2B sequence without an affinity change, suggesting the importance of the second domain. By changing individual amino acid residues of AIC2A in the second domain which differ from those of AIC2B, we identified several amino acid residues critical for IL-3 binding. All these residues are located at the putative hinge region within the second domain.

Amino Acid Sequence↗
PubMed1992

Retinal pigment epithelium implantation in the rabbit: technique and morphology.

Dysfunction of the retinal pigment epithelium (RPE) is a major component of many degenerative disorders of the retina. We have developed a modified pars plana technique for subretinal implantation of homologous, cultured, carbon-loaded RPE cells in the rabbit. The natural history and morphologic behavior of the implanted RPE cells for up to 6 weeks after transplantation were observed in successful procedures.

Animals↗
PubMed1992

In vitro stimulation of retinal pigment epithelium proliferation by taurine.

Taurine is an amino acid that is essential for retinal integrity and function. Although it has been suggested that the ratio of melatonin to taurine in the interphotoreceptor matrix may regulate the phagocytosis of outer segments by retinal pigment epithelial (RPE) cells, the effect of taurine on the RPE has not been studied. Using cultured RPE cells, we found that in vitro taurine specifically stimulated proliferation of human and rabbit RPE, but had only minimal effect on cultured scleral fibroblasts. The RPE proliferation was due to more cells entering into S-phase and thus an increase in DNA synthesis, was not dependent upon cell density, and was most pronounced in the presence of a low concentration of fetal bovine serum.

Animals↗
PubMed1992

Measurement of free radicals from smoke inhalation and oxygen exposure by spin trapping and ESR spectroscopy.

Research in smoke inhalation has established that free radicals are produced from gases released during combustion and these species impair lung function. Using spin traps and their adducts in an animal model free radicals were measured. Various hyperbaric oxygen regimens were tested in an attempt to attenuate pulmonary damage caused by free radical reactions. Our data demonstrated that persistent oxygen- and carbon-centered free radicals are detectable in intravascular fluids after smoke inhalation. The smoke inhalation model showed however, clearing of spin trap adducts one hour after smoke exposure. Other researchers have found that when 100% oxygen is given at 1 atmosphere absolute (ATA) for 1 h, free radicals were not detectable. However, oxygen given at 2.5 ATA does produce detectable free radicals. With continued exposure at this pressure, the levels of free radicals increase for up to 60 min. This study suggests that the level of free radical induced oxygen toxicity may be a function of oxygen pressure and duration of oxygen exposure.

Animals↗
PubMed1992

[Autonomic nerve preserving operation for rectal carcinoma: preliminary postoperative urinary function of 15 cases].

The autonomic nerve preserving operation was performed on 15 patients with rectal carcinoma (9 men and 6 women), from August 1990 to February 1991. The average age was 57.2 years ranging from 28 to 70 years. Low anterior resection was performed on 6 patients having middle rectal carcinoma. Seven Colonic anastomosis and two abdominoperineal resections were performed for lower rectal carcinoma. The average operation time was 4 hours and 22 minutes. The average blood loss was 1560 cc. Urinary functions were evaluated pre- and post-operatively. None of the patients needed re-catheterization postoperatively. Urodynamic studies showed no significant differences. We recommend this operation for rectal cancer because it prevented the postoperative autonomic dysfunction without affecting the curability.

Adult↗
PubMed1991

Determination of the pKa of glucuronic acid and the carboxy groups of heparin by 13C-nuclear-magnetic-resonance spectroscopy.

As part of our continuing studies on heparin, the present paper uses 13C-n.m.r. spectroscopy to examine the acidity of heparin's uronic acid carboxylate groups. Heparin contains three different uronic acids. In porcine mucosal heparin these account for approx. 91, 7 and 2 mol% of the total uronic acid residues. These are alpha-L-idopyranosyluronic acid 2-sulphate, beta-D-glucopyranosyluronic acid and alpha-L-idopyranosyluronic acid. The pKa values of their carboxylate groups were determined as 3.13 (using heparin), 2.79 (using heparin) and 3.0 (predicted by using model compounds) respectively. 18C-n.m.r. spectroscopy, performed at various pH values, provided a convenient method of simultaneously determining the pKa of multiple carboxylate groups, of similar acidity, within heparin D-Glucopyranosyluronic acid and heparin-derived di-, tetra- and hexa-saccharides were used as model compounds to determine pKa values of the different carboxy groups. The results suggested that molecular size had an effect on pKa. Unambiguous assignment of carboxy carbon resonances were accomplished through the use of two-dimensional n.m.r. spectroscopy. Finally, application of this method to the simplest model compound, D-glucopyranosyluronic acid, permitted the determination of the pKa of both its alpha- and beta-anomers.

Chemical Phenomena↗
PubMed1991

Synthesis of extracellular matrix by macrophage-modulated retinal pigment epithelium.

In proliferative vitreoretinopathy, macrophages and retinal pigment epithelial cells are associated with microfibrillar matrix proteins in the vitreous cavity, but the contribution of this extracellular matrix to the pathophysiology is not known. We used radiolabeling techniques on cultured human retinal pigment epithelial cells to correlate the secretion of extracellular matrix proteins with macrophage-induced modulation of cell proliferation and morphologic features. Retinal pigment epithelial cells incubated in a macrophage-conditioned medium assumed fibrocytelike morphologic characteristics, grew faster, and exhibited a decreased cellular release of fibrillar and nonfibrillar matrix components. However, due to a simultaneous greater increase in cell numbers in these modulated cultures, the total production of fibrillar and nonfibrillar matrix components by the culture population was increased.

Cell Division↗
PubMed1991

Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure delta UA2X(1----4)-D-GlcNY6X (where delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R.J. Linhardt, Anal. Biochem. 195, 68-73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1----4)-D-GlcNAc and delta UA(1----4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.

Carbohydrate Sequence↗
PubMed1991

Adaptive changes in visual cell transduction protein levels: effect of light.

Long-term environmental light-mediated changes in visual cell transduction proteins were studied to assess the influence of rearing environment on their levels and their potential effects on intense light-induced retinal damage. The levels of rhodopsin, S-antigen and the alpha subunit of transducin were measured in whole eye detergent extracts, retinal homogenates or rod outer segments isolated from rats reared in weak cyclic light or darkness, and following a change in rearing environment. Rats changed from weak cyclic light to darkness had 22% more rhodopsin per eye than cyclic-light rats after 12-14 days in the new environment. Western trans-blot analysis of retinal proteins from these dark-maintained animals contained 65% higher levels of immunologically detectable alpha transducin; S-antigen levels were approximately 45% lower than in cyclic-light rats. In rats changed from the dark environment to weak cyclic light, rhodopsin levels decreased by 18% during a comparable period; retinal alpha transducin was 35% lower, S-antigen was 30% higher. At various times after the change in rearing environment, some rats were exposed to intense visible light to determine their susceptibility to retinal damage. Two weeks after an 8-hr exposure, cyclic-light reared rats had rhodopsin levels only 10% lower than control (2.1 nmol per eye). However, rhodopsin was 75% lower when cyclic-light rats were maintained in darkness for 2 weeks before intense light. For animals originally reared in darkness, rhodopsin was 78% lower following 8 hr of intense light, whereas only 30% rhodopsin loss occurred in dark-reared rats after previous maintenance for 2 weeks in weak cyclic-light.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMed1991

High salt mimics effects of light pulses on circadian pacemaker in cultured chick pineal cells.

Chick pineal cells in static culture display a persistent photosensitive circadian rhythm of melatonin production and release. Pulses of white light or darkness, in otherwise constant red light, induce phase shifts in subsequent cycles whose magnitude and direction depend on the phase at which the pusle is given. Such "phase-dependent phase shifts" are mediated by effects on the underlying pacemaker. We reported previously that inhibiting the Na+-K+-adenosinetriphosphatase with ouabain evokes phase shifts with the same phase dependence as those induced by pulses of darkness. Two of the consequences of inhibiting the Na+ pump are membrane depolarization and cell swelling. To test the relevance of these effects and to distinguish between them, we exposed chick pineal cells to pulses of high concentrations of added KCl, which should cause both membrane depolarization and cell shrinkage. We also compared the effects of added NaCl, sodium gluconate, and mannitol, which should cause cell shrinkage without comparable depolarization. Each of these hypertonic solutions induced phase shifts in the melatonin rhythm with the same phase dependence as those caused by pulses of light rather than those caused by ouabain or darkness. These results argue strongly against a dominant role for changes in membrane potential in phase shifting the melatonin rhythm. Rather, they suggest that cell swelling may mediate the darklike effects of ouabain on the circadian pacemaker in chick pineal cells and indicate that cell shrinkage mimics the effects of light.

Animals↗