PubMed Health⌕ Search

Biomedical subjects

C T Wang

Publications and source records attributed to C T Wang.

At least 91 records · Page 5Linked to original sources
PubMed1988

Changes in both calcium pool size and morphology of human platelets incubated in various concentrations of calcium ion. Calcium-specific bleb formation on platelet-membrane surface.

In this study, the response of gel-filtered human platelets to extracellular Ca2+ at Ca2+ concentrations [Ca2+]o of 1-10 mM was investigated. The distribution of Ca2+ among various pools was studied using: (1) quin2, to estimate the cytosolic free Ca2+ concentration ([Ca2+]i); and (2) 45CaCl2 plus EGTA, to quantitate the sizes of the EGTA-releasable, EGTA-nonreleasable and surface-bound Ca2+ pools. The morphological changes were revealed by scanning electron-microscopy (scanning EM), and the effect on thrombin-stimulated aggregation was examined using an aggregometer. Platelets continuously sequestered Ca2+ into both EGTA-releasable and EGTA-nonreleasable pools to maintain a low [Ca2+]i level. The rate of sequestration to the EGTA-releasable pool was independent of [Ca2+]o, while that of the EGTA-nonreleasable pool exhibited first-order kinetics. The cell morphology changed gradually from discoid to the tadpole-like type, and finally to irregular forms. This morphological change correlated with the gradual increase in [Ca2+]i. The EGTA-nonreleasable pool saturated at about 3000 pmol/10(8) cells. This saturation resulted in a drastic increase in the EGTA-releasable pool size, and the cell was lysed concomitantly. The maximum safety capacity of the EGTA-releasable pool was estimated to be 1100 pmol/10(8) cells. The contribution of the cellular compartments to these two pool sizes is extensively discussed. The surface-bound pool size also increased continuously. When two different capacities were reached, i.e., 160 and 600 pmol/10(8) cells, the binding rate increased above the initial rate by 7- and 11-fold, respectively. Hence, the surface-binding capacity might be a critical factor which alters the membrane structure and exposes more binding sites. The cell surface appeared to have blebs, after the binding size had reached more than 600 pmol/10(8) cells. Bleb formation resulted in the inhibition of platelet function. Divalent cations, such as Mg2+, Sr2+ and Ba2+ did not cause bleb formation, which could mean that this formation is a Ca2+-specific phenomenon.

Barium↗
PubMed1988

Applications of BOP reagent in solid phase synthesis. Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]-GRF(1-29)-NH2.

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219-1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22-29) and (13-29) fragments of [Ala15]-GRF(1-29)-NH2. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala15]-GRF(1-29)-NH2 was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1-29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (less than 10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.

Growth Hormone-Releasing Hormone↗
PubMed1988

Synthesis, biological activity and conformational analysis of cyclic GRF analogs.

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.

Animals↗
PubMed1987

Different susceptibilities of platelet phospholipids to various phospholipases and modifications induced by thrombin. Possible evidence of rearrangement of lipid domains.

On the membrane surface of the human platelet, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were hydrolyzed to different extents by the snake venom phospholipases A2 of varying pI values. The susceptibility of platelet phospholipids to basic phospholipase A2 of Naja nigricollis (pI 10.6) has been reported (Wang et al. (1986) Biochim. Biophys. Acta 856, 244-258). The susceptibilities of platelet phospholipids to acidic phospholipase A2 of Naja naja atra (pI 5.2) and to neutral phospholipase A2 of Hemachatus haemachatus (pI 7.3) were investigated in this study. In gel-filtered platelets, acidic phospholipase A2 hydrolyzed 35% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 18% PC and 3% PE. In thrombin-induced shape-changed platelets, acidic phospholipase A2 hydrolyzed 20% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 15% PC and 6% PE. In thrombin-activated platelets, acidic phospholipase A2 hydrolyzed 25% PC and 7% PE, while neutral phospholipase A2 hydrolyzed 25% PC and 10% PE. Sequential lipid hydrolysis experiments showed that basic phospholipase A2 of Naja nigricollis could hydrolyze the remaining PC and PE in the membrane previously treated with the neutral enzyme. The results may mean that: the PC and the PE domains exist on the platelet membrane surface; and the lipid domains on the membrane surface of resting platelets are rearranged by thrombin.

Blood Platelets↗
PubMed1986

Estimation of the phospholipid distribution in the human platelet plasma membrane based on the effect of phospholipase A2 from Naja nigricollis.

Human platelets in three physiological states were prepared. These states were the gel-filtered, the thrombin-induced shape-changed, and the thrombin-activated platelets. The phospholipid distributions in these three types of membrane were probed by using the basic phospholipase A2 of Naja nigricollis. This enzyme could penetrate through these membranes to hydrolyze all of their accessible phospholipids and to cause cell lysis. The hydrolytic time-courses displayed three phases. The state of platelet in each lipid hydrolytic phase was examined by: (1) measuring the leakage of lactate dehydrogenase; (2) analyzing the morphology by both scanning and transmission electron microscopy (scanning EM and transmission EM); and (3) estimating the hydrolysis of the [32P]phosphate-labeled platelets. The existence of these three hydrolytic phases may signify that the phospholipase A2 sequentially hydrolyzed its substrates in the membrane outer leaflet, in the inner one, and in the cytosol. The content and the distribution of each phospholipid class in the plasma membranes of the resting and of the shape-changed platelets were similar. These membrane surfaces consisted mainly of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Phosphatidylserine (PS) was not exposed on the surface of the shape-changed platelet. The content of each lipid class in the activated platelet membrane was 10% more than that in the resting platelet. PS was found on the activated platelet cell surface. This implies that PS is exposed only during platelet secretion.

Animals↗
PubMed1985

Analysis of different forms of recombinant human leukocyte interferons and synthetic fragments by high-performance liquid chromatography.

Analytical high-performance liquid chromatography (HPLC) conditions are reported for the evaluation of recombinant human interferon monomers: recombinant human leukocyte interferon A (rIFN-alpha A), rIFN-alpha D, and the hybrid rIFN-alpha A1-62/D64-166. The two monomeric forms of rIFN-alpha A (slow-migrating monomer and fast-migrating monomer) were also resolved by HPLC. Conditions are reported for the HPLC separation of oligomers (dimers and trimers) of rIFN-alpha A. The synthesis and analytical HPLC of the carboxy-terminus fragment, corresponding to IFN-alpha D (140-166), and a series of analogues comprising the IFN-alpha A (105-125) region is reported. The syntheses were accomplished by the solid-phase peptide synthesis procedure and the products were purified by preparative HPLC.

Amino Acid Sequence↗
PubMed1985

Synthesis of thymosin alpha 1 by fragment condensation using tert.-butyl side chain protection.

A novel synthesis of thymosin alpha 1 by classical methods using seven tert.-butyl side chain protected fragments is described. Optimum conditions were found for the final DCC/HOBt coupling of the two key intermediates; decapeptide and octadecapeptide. Thymosin alpha 1 was purified by two stages of preparative HPLC (partial purification with C8 and final purification with C18 reverse phase silica gel) to give a 30% overall yield for the final four stages of synthesis (including catalytic hydrogenation of octadecapeptide, coupling, deprotection and purification). The product was shown to be homogeneous by thin-layer and paper high voltage electrophoresis, isoelectric focusing analysis, thin-layer chromatography and high performance liquid chromatography. Amino acid analysis, optical rotation, 1H-n.m.r. spectroscopy, FAB mass spectroscopy and peptide mapping after tryptic digestion confirmed the structure of thymosin alpha 1. Three minor stereoisomer contaminants were isolated by HPLC and characterized as [D-Lys14]-thymosin alpha 1, [D-Lys17]-thymosin alpha 1 and [D-Ala3]-thymosin alpha 1 resulting from racemization at Lys14, Lys17 and Ala3 during the coupling of the fragments. A final contaminant, isolated by HPLC, was characterized as N alpha-isobutyloxycarbonyl-thymosin alpha 1 (15-28), which results from "wrong way opening" of an activated mixed anhydride.

Amino Acid Sequence↗
PubMed1983

The determination of the separate Ca2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction.

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47-72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.

Animals↗
PubMed1981

31P-NMR studies of oriented multilayers formed from isolated sarcoplasmic reticulum and reconstituted sarcoplasmic reticulum.

31P-NMR spectra were obtained from oriented multilayer preparations of normal sarcoplasmic reticulum and reconstituted sarcoplasmic reticulum with lipid to protein ratios varying between 41 : 1 and 110 : 1. The dependence of the 31P-NMr spectra on the alignment of the membranes with respect to the magnetic field was used to draw two conclusions about the motion of the phospholipid molecules that contribute to the observed spectra. First, the phosphate group and the two adjacent methylene groups are able to rapidly rotate (i.e., tau R much less than 10-5 S) around the normal to the plane of the membrane. Second, the restricted internal motion of the phosphate group and the glycerol CH2OP group is very similar to that found in liposomes formed from sarcoplasmic reticulum phospholipids. Calibration experiments showed that all (100 +/- 7%) of the phospholipid molecules in the membrane can be accounted for in the observed spectra. Thus, essentially all the phospholipid molecules in the sarcoplasmic reticulum and the reconstituted sarcoplasmic reticulum membranes have the same motion in the polar headgroup region as found in model bilayer membranes. Since a large fraction of the phospholipid molecules (between one-quarter and one-half, depending on ;the lipid to protein ratio) are immediately surrounding the calcium-pump protein, we conclude that the calcium-pump protein does not perturb the motion of these 'boundary-layer' lipids.

Animals↗
PubMed1981

The effect of thrombin on the organization of human platelet membrane glycosphingolipids. The sphingosine composition of platelet glycolipids and ceramides.

Surface-exposed glycolipids in human platelets were investigated with nonpenetrating membrane probes, NaB3H4/galactose oxidase and NaB3H4/periodate. Trihexosylceramide, globoside, and hematoside were exposed in resting platelets. The incubation of platelets with thrombin resulted in a 50% reduction in the labeling of both trihexosylceramide and globoside and a 100% increase in labeling of hematoside on the platelet surface. The quantitation of glycolipids in platelets incubated with thrombin revealed no changes in the amounts of trihexosylceramide and globoside but there was an increase in hematoside. These experiments indicate that there is a specific rearrangement of glycolipids on the surface of thrombin-treated platelets. The study also showed that both ceramide A and B are present in platelets. The former contained 3.36 nmol of sphingosine and the latter 0.12 nmol of sphingosine/10(9) platelets. The sphingosine composition of ceramide B, glucosylceramide, and the minor platelet gangliosides was similar and they were composed of two-thirds erythrosphingosine (18:1) and one-third threosphingosine (18:1). The composition of lactosylceramide, trihexosylceramide, globoside, and hematoside was related to ceramide B and they contained about 60% erythrosphingosine (18:1), 25% threosphingosine (18:1), and 10% dihydrosphingosine (18:0). Ceramide A differed in that it was composed of 56% erythrosphingosine (18:1), 34.7% dihydrosphingosine (18:0), 4.3% threosphingosine (18:1), 2.7% erythrosphingosine (16:1), and 2.3% erythrosphingosine (17:1). The sphingosine composition of sphingomyelin resembled that of ceramide A but did not contain threosphingosine (18:1). The possibility that platelet ceramide B is involved n glycolipid metabolism and ceramide A in sphingomyelin metabolism is considered.

Blood Platelets↗
PubMed1981

Functional characteristics of reconstituted sarcoplasmic reticulum membranes as a function of the lipid-to-protein ratio.

The ATP-induced Ca2+ accumulation efficiency and rates of Ca2+ uptake of the reconstituted sarcoplasmic reticulum (RSR) model membrane system were measured over an extended range of lipid-to-protein (L/P) molar ratios and were compared to those of isolated light sarcoplasmic reticulum (LSR). Highly purified sarcoplasmic reticulum (SR), dissociated in the presence of deoxycholate, was reconstituted for several L/P ratios, according to the same procedure, forming closed membranes vesicles composed of greater than 95% Ca2+ pump protein and SR lipids which were capable of ATP-induced Ca2+ accumulation in the absence of oxalate or other Ca2+ precipitating agents. This suggests that dissociation of SR and reconstitution to form RSR does not significantly affect the ability of the Ca2+ pump protein incorporated into the SR lipid bilayer to establish Ca2+ gradients. Electron micrographs of fixed and stained dispersions of RSR revealed a structural organization of the membrane that was dependent upon the L/P molar ratio. RSR with L/P greater than 88 were composed of closed vesicles whose membranes stained asymmetrically, similar to that observed for LSR. Closed vesicles of RSR with L/P less than 88 were composed of membrane that stained symmetrically. In addition, reconstituted SR preparations with well-defined L/P molar ratios greater than 88 possess a functional behavior similar to that of LSR (in the absence of oxalate, energy efficiencies are 60-70% and apparent initial uptake rates are 80% that of isolated LSR controls); RSR preparations with L/P less than 88 are characterized by significantly depressed values of the energy efficiencies and apparent initial uptake rates especially at low L/P ratios. Thus, we are the first to report a reconstituted SR model membrane system capable of attaining rates of Ca2+ uptake comparable to isolated LSR controls at comparable L/P ratios in the absence of oxalate or other Ca2+ precipitating agents.

Animals↗
PubMed1981

Comparison of the profile structures of isolated and reconstituted sarcoplasmic reticulum membranes.

The profile structures of functional reconstituted sarcoplasmic reticulum (RSR) membranes were investigated as a function of the lipid/protein (L/P) ratio via x-ray diffraction studies of hydrated oriented multilayers of these membranes to a resolution of 10-15 A, and neutron diffraction studies on these multilayers to lower resolutions. Our results at this stage of investigation indicate that reconstitution of SR with variable amounts of Ca2+ pump protein for L/P ratios greater than 88 results in closed membraneous vesicles in which the Ca2+ pump protein is distributed asymmetrically in the membrane profile; a majority of the protein density is contained primarily in the extravesicular half of the membrane profile whereas a relatively lesser portion of the protein spans the hydrocarbon core of the RSR membranes. These RSR membranes are functionally similar and resemble isolated light sarcoplasmic reticulum in both profile structure and function at a comparable L/P ratio. Reconstitution with greater amounts of Ca2+ pump protein (e. g. L/P approximately 50-60) resulted in substantially less functional membranes with a dramatically thicker profile structure.

Animals↗