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V C Yang

Publications and source records attributed to V C Yang.

At least 73 records · Page 4Linked to original sources
PubMed1991

Versatile, non-clotting-based heparin assay requiring no instrumentation.

A non-clotting-based heparin assay requiring no instrumentation has been developed by attaching protamine (a heparin antidote) to a porous filter paper strip and monitoring the subsequent capillary migration of heparin sample through the paper. The area of paper to which heparin adsorbed, which is proportional to the concentration of heparin in the sample, is made visible by spraying Methylene Blue NNX solution onto the paper strip when the sample reservoir has emptied. The dye interacts with heparin to cause a metachromatic shift of the dye's absorption maximum from blue to purple. Heparin concentrations in the samples are estimated according to the length of the purple region on the paper strip. Heparin in plasma (2 to 35 int. units/mL) can be differentiated in approximately 6 min. The plasma heparin values derived by the new method agree reasonably well with those obtained by conventional assays (r greater than 0.99, n = 48).

Extracorporeal Circulation↗
PubMed1990

Preparation of diethylaminoethyl hollow fibres for high-flow liquid chromatography.

A novel anion-exchange column for fast-flow liquid chromatography was developed. The column was prepared by immobilizing diethylaminoethyl groups on to the inner walls of regenerated cellulose hollow fibres. A detailed investigation was conducted of the parameters affecting diethylaminoethyl immobilization such as the NaOH concentration, the reaction temperature, the diethylaminoethyl concentration and the reaction span. Based upon the findings, a protocol to optimize the preparation of the diethylaminoethyl hollow fibres was established. The diethylaminoethyl hollow fibres prepared adsorbed 186 +/- 30 mg of albumin/g fibres.

Adsorption↗
PubMed1990

Late effects in mouse heart after 60Co gamma-irradiation of the brain: an ultrastructural study.

A single dose of 8 or 20 Gy 60Co gamma-rays was given to C3H male mice at 4 months of age. Degenerative changes in the cardiac muscle due to brain irradiation were observed first at 6 months after irradiation, and became progressively more severe at 12-24 months. The changes seen at the ultrastructural level included myofibrillolysis, the presence of lysosomal-like bodies and interstitial fibrosis. Ultrastructural changes in the control cardiac muscle throughout the experimental period were monitored and only minor aging changes were noted. The coronary arteries of control mice began to show a slight amount of smooth muscle degeneration and fibrosis 1 year into the experiment. At 18 months the lesions became more severe, and at 24 months there was relatively less distinction between the control and the 20 Gy treated group. Degenerative changes in the coronary arteries were noticed at 6 months after irradiation, and became progressively more severe at later times (12-24 months). The major changes included smooth muscle degeneration with fibrosis and the accumulation of debris and extracellular matrix. At 18 months the medial smooth muscle showed severe damage, with accumulations of matrix material and debris. There was additional fibrosis in the adventitial layer. There were few additional changes at 24 months after 20 Gy irradiation. Quantitative analyses indicated that the average fractional volumes of degenerated smooth muscle cells were 13, 27 and 39% in the unirradiated group at 12, 18 and 24 months, respectively, and 13 and 29% in the sham-irradiated group at 12 and 18 months into the experiment, respectively. These percentages were 12, 32 and 49% (P less than 0.05) after 8 Gy irradiation, and 19% (P less than 0.05), 46% (P less than 0.01), and 42% after 20 Gy irradiation, respectively.

Animals↗
PubMed1989

A facile colorimetric protamine titration method.

On the basis of the reversible, competitive binding of protamine and azure A dye to heparin, a facile, colorimetric protamine titration method was developed. The method uses azure A dye as the titration indicator and has thus replaced the time-consuming clotting assay in the traditional protamine titration method with a rapid colorimetric assay. It offers the same accuracy in estimating the titration end point as the traditional titration method but allows the processing time to be significantly shortened. With the use of a premade diagnostic kit containing a fixed amount of azure A dye and various amounts of protamine, the titration end point can be determined in less than 5 minutes. This new colorimetric protamine titration method should provide clinicians with an easy and reliable means to accurately estimate the protamine dose required for heparin neutralization. It should also assist medical laboratories in preparing plasma samples that are free of heparin interference for routine coagulation tests. In a reverse manner, the colorimetric assay can also be used with a heparin titration procedure to quickly assess the heparin dose required for protamine reversal. For patients who have received overdoses of protamine, the availability of a facile heparin titration method may offer the prospect of exercising a heparin "back titration."

Azure Stains↗
PubMed1988

Contrast in levels of metabolic enzymes in human and mouse ova.

A methodology is described for analyzing single human ova for 8 or 9 different metabolic enzymes, or 4 or 5 enzymes plus as many metabolites. This overcomes an obstacle to the study of human ovum metabolism: the severe limitation of usable material. Results obtained with this methodology, applied to discarded specimens from an in vitro fertilization program, indicate that in spite of imperfections these ova can provide a valid picture of the metabolic characteristics of normal human ova. Data are presented for 17 enzymes from 8 metabolic pathways in human and mouse ova. Relative to size, 10 of the enzymes were substantially higher in human than mouse ova. Most dramatically so were 2 enzymes of fatty acid metabolism (10-fold and 15-fold), hexokinase (9-fold), and aspartate aminotransferase (19-fold). This suggests that major species differences in metabolism are present. The validity of the human data, in spite of restriction to discarded material, is supported by (1) consistency of results among most of the ova, 2] concordance between average levels with those of rare specimens that were discarded because sperm were not available, and (3) the presence of adenosine triphosphate (ATP) concentrations similar to those of normal mouse ova. Surprisingly, both human and mouse ova contain phosphocreatine at levels nearly equal of those of ATP.

Acetyl-CoA C-Acetyltransferase↗
PubMed1988

Structural changes in the mouse heart one year after brain exposure to 60Co gamma ray.

This study was conducted to investigate the sequential structural changes in the hearts of C3H male mice 1 to 12 months after brain irradiation. A single brain dose of 8 or 20 60Co gamma Gy was given to the animals at 4 months of age. Degenerative changes in the heart occurred, firstly at 6 months after irradiation, and became progressively more severe at 12 months. The cardiac muscle showed areas of focal myofibrillolysis, myofibrillar degeneration with loss of entire myofibrils, the presence of lysosomal-like bodies, and interstitial fibrosis. Coronary artery degeneration was also found at 12 months after irradiation; the major changes included smooth muscle degeneration with fibrosis, and the accumulation of debris and extracellular matrix. Quantitative analysis indicated that the degeneration of the arterial smooth muscle after 20 Gy irradiation (18.9%) was significantly higher than that of the unirradiated control (13.2%), and shammed control (13.3%) groups, p less than 0.05.

Animals↗
PubMed1987

Extracorporeal enzymatic heparin removal: use in a sheep dialysis model.

Extracorporeal medical devices such as the hemodialyzer rely on systemic heparinization to prevent thrombus formation. Heparin, however, can lead to serious hemorrhagic complications. A blood filter containing immobilized heparinase, a heparin specific enzyme, was used to degrade heparin into small fragments which have significantly less anticoagulant activity than the parent compound. The heparinase filter was tested in the extracorporeal circuit during the hemodialysis of adult sheep. At a blood flow of 200 ml/min, the clearance of heparin varied from 50 to 70 ml/min (N = 16) depending on the amount of immobilized heparinase in the filter. Hemolysis was insignificant as measured by the animals' red cell counts, hematocrit, total hemoglobin and a plasma-free hemoglobin value of 89 +/- 33 mg/dl (N = 16) (less than 1% of the total hemoglobin). The white cell counts dropped to 47 +/- 7% (N = 16) of the initial value at 20 minutes and rebounded to 72 +/- 10% (N = 16) after one hour. The platelet counts decreased to 55 +/- 8% (N = 16) of the initial value after one hour. No change in heparin clearance was observed when reactors were used repeatedly in adult sheep over a 10 week period. The red cell counts, white cell counts, platelet counts, total hemoglobin and hematocrit did not change after 10 weeks of exposure to the device. These results suggest that with further study, heparinase may be useful in removing heparin used to anticoagulate blood in extracorporeal circuits.

Animals↗
PubMed1987

A histological study on the cataractogenic effects of heavy charged particles.

The morphology of the mouse lens was studied by light and electron microscopy between 4 and 30 months after graded single doses of photons or charged particle exposure. Four-month-old mice were given upper body or head only doses of 220 kVp x-rays or to 40Ar (570 MeV/amu), 40Ar(500 MeV/amu), 20Ne(470 MeV/amu) and 12C (400 MeV/amu) particles. The lenses examined in this pilot study were largely from mice dedicated to other designed experiments; nor all radiation conditions are represented at each sample time. At 4 months after 0.05, 0.3 or 0.9 Gy 20Ne radiation, the micronuclei appeared in the epithelial cells at the bow region. The anterior fibers showed swollen cytoplasm containing amphorous materials after 0.3 or 0.9 Gy 20Ne exposure. The posterior fibers were relatively normal. The results at 15 months after irradiation indicated heavy charged particles were more effective than x-rays for inducing morphological changes; however there were no qualitative differences in damage produced by heavy charged particles and x-rays. Characteristic changes after irradiation included abnormal bow patterns, posterior and anterior migration of nucleated cells in the cortex, swelling of fibers and the development of swirls of epithelium at the anterior pole. The results also indicated that the degree of degenerative changes in lens fibers increases with increasing radiation dose and sampling time. The radiation-induced injury to lens fibers also showed a relationship to linear energy transfer (LET). The qualitative observations at 24 months indicated that low energy 40Ar (600 KeV/micron) is less effective than high energy 40Ar particles (100 KeV/micron)1 and radiation-induced injury in the lens fibers shows an LET-dependence when the ionizing density is less than 100 KeV/micron. At 30 months after heavy charged particle irradiation, the changes in control lens were the swollen fibers, while in the 40Ar-irradiated animals, aberrant fibers with abnormal nuclei, amorphous materials and debris were observed.

Animals↗
PubMed1986

Removal of the anticoagulant activities of the low molecular weight heparin fractions and fragments with flavobacterial heparinase.

Recently, the development of low molecular weight heparin fractions and fragments (LMHF) as potential antithrombotic agents has gained increased attention. However, the lack of antagonists to neutralize the anticoagulant effects of these drugs may seriously exclude them from possible uses in extracorporeal therapy. This is mainly because of the concern that the high dosage of the drugs employed in extracorporeal therapy could lead to serious bleeding risks. Our earlier work has demonstrated that immobilized heparinase can remove polydisperse heparin both in vitro and in vivo. To examine whether such a system may be used as a novel approach to neutralize the anticoagulant effects of LMHF, different LMHF were tested using heparinase. In vitro data showed that both the APTT and anti-FXa activities of the LMHF including Kabi 2165, PK 10169, Cy 216 and CY 222 were nearly completely eliminated by heparinase in less than 20 min. This study suggests that an immobilized heparinase system may be an useful element for the acceptance of the LMHF for their use in extracorporeal therapy.

Anticoagulants↗
PubMed1985

Biophysical properties of cytidine diphosphate diacylglycerol in solution.

The physical properties of CDP diacylglycerol derived from egg phosphatidylcholine are very different from those of the common glycerophospholipids, such as phosphatidylcholine. Gently dispersed in buffer (5 mM phosphate, 0.15 M NaCl, pH 7.4), the liponucleotide initially forms an opalescent suspension of spherical vesicles, up to 50 micron in diameter, which appear to be unilamellar. These large vesicles are unstable and, independently of initial concentration, unstirred suspensions are no longer turbid after being incubated for about 1 h at room temperature. The passage of samples through Sepharose and Sephadex at increasing time intervals after the first hour reveals a continuing but slow diminution in size until, at about two days, a final peak is obtained which remains invariant for longer times. Chromatography of these ultimate stable micelles on Sephadex G-200 gives a Stokes radius of 4.2 nm. Their sedimentation coefficient extrapolated to zero concentration is 6.1 S. These numbers, combined with a partial specific volume of 0.835 ml X g-1, give an anhydrous mass of 155 000 Da and an aggregation number of 158. Although the data suggest the particles to be spherical, other compact forms cannot be excluded. Proton NMR at 220 MHz shows time-dependent spectral changes which are consistent with the slow structural transformation observed by gel-filtration chromatography, and indicate that the sugar and cytosine groups in the ultimate micelles apparently are motionally restricted. The critical micelle concentration is near 6 microM, but micelle-free molecule equilibration requires at least 7 days at a total concentration of 89 microM. Sonication considerably decreases the time required for the vesicle-micelle transformation and the micelle-free molecule equilibration. Some implications for enzymology are discussed.

Biophysical Phenomena↗
PubMed1985

pH-dependent binding analysis, a new and rapid method for isoelectric point estimation.

Based upon the pH-dependent binding affinity of amphoteric molecules for an ion exchanger, and by taking advantage of batch procedures, a facile method was developed for estimating isoelectric points of these molecules. The new method allows pI measurements to be accomplished within 1 h. Moreover, any possible protein-ampholyte interaction or artifact formation, as may be introduced from the presence of carrier ampholytes when conventional focusing methods are employed, is eliminated by the method. In addition, because of the short processing time, isoelectric points of proteins can be measured at any desired temperature without much risk of protein denaturation. Seven proteins with well-defined isoelectric points were examined by the method. The measured pI values were within a range of 0.2 pH unit or less of the reported values. The precision of pI measurements by the method can be even further improved with the employment of a narrower pH gradient. Since the isoelectric point is an important parameter which governs much of the art of separating proteins, the advent of a simple and rapid method for its measurement would be of use for selecting the proper strategy for protein isolation and purification.

Binding Sites↗
PubMed1985

Purification and characterization of heparinase from Flavobacterium heparinum.

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.

Amino Acids↗
PubMed1982

Physical properties of arabinofuranosylcytosine diphosphate diacylglycerol, an antitumor liponucleotide.

Dispersed from a dry film into buffer (5 mM phosphate, 0.15 M NaCl, pH 7.4), the liponucleotide 1-beta-D-arabinofuranosylcytosine 5'-diphosphate L-1,2-diacylglycerol (ara-CDPdiacylglycerol) spontaneously forms vesicles which are several microns in diameter and probably unilamellar. Their average size immediately begins to decrease, and after 2 h none can be seen in the light microscope. During 1-2 days in unstirred solutions at 25 degrees C, the vesicles are transformed to spherical or nearly spherical micelles having an apparent partial specific volume of 0.835 ml . g-1, a maximum possible aggregation number of about 150, and an anhydrous radius of about 37 A. The critical micelle concentration (CMC) is about 10 microM in buffer and 20 microM in distilled water, but micelle-monomer equilibration requires at least 1 week at a total concentration of 66 microM. This exceedingly slow equilibration is unique among reported detergents. The standard enthalpy and entropy of micellization are - 13 kJ . mol-1 and 87 J . mol-1 . K-1, respectively. These values are within the range reported for other detergents. Sonication accelerates the vesicle-micelle transformation to 30 min.

Chemical Phenomena↗
PubMedDate not supplied

Large scale preparation and characterization of mucopolysaccharase contamination free heparinase.

By a combination of hydroxylapatite chromatography and negative adsorption on QAE-Sephadex at pH 8.3, heparinase (E.C.4.2.2.7) can be successfully isolated from all the other mucopolysaccharase contaminants present in Flavobacterium heparinum. Hydroxylapatite isolates heparinase primarily from chondroitinases, hyaluronidase, and most glycuronidases. QAE-Sephadex chromatography at pH 8.3 further separates heparinase from heparitinases, sulfatases, and the remaining glycuronidases. The heparinase preparation thus obtained contains no statistically significant levels of other contaminating mucopolysaccharases except for heparitinases that are present at an apparent maximum level of 3.4%. Owing to the presence of a crossreaction of heparinase on heparitin sulfate at conditions employed for the assay of heparitinase, the heparitinase level of 3.4% could be misleading because of the action of heparinase on heparitin sulfate. Characterization of this heparinase preparation shows that the enzyme has an optimum salt concentration of 0.08M NaCl, an optimum pH of 6.5, an activation energy of 5 kcal/mol, and a Km of 7.95 X 10(-6) M. These parameters are almost identical to those displayed by a homogeneous heparinase preparation. The method described here is suitable for scale-up purposes using batch chromatographic procedures.

Chromatography, Gel↗
PubMedDate not supplied

An investigation of heparinase immobilization.

A systematic investigation of the parameters that affect the efficiency of immobilizing heparinase onto cyanogen bromide activated crosslinked 8% agarose beads was conducted. Two experimental measures, the "fraction bound" and the "fraction retained," were used to monitor the coupling efficiency. The fraction bound is the portion of the total initial enzyme that is bound to the agarose gel. The fraction retained is the fraction of bound enzyme that is active. The product of the two measures indicates the coupling efficiency. The activity of the immobilized heparinase was measured under conditions free of both internal and external mass transfer limitations, and thus, the fraction retained represents the true immobilized enzyme activity. Increasing the degree of activation of the beads results in an increase in the fraction bound, the fraction retained, and consequently, the coupling efficiency. As the ratio of enzyme solution to gel volume increases from 1.5 to 2.2, the fraction bound remains constant but the fraction retained decreases (heparinase concentration; 0.15 mg/mL and degree of activation; 9.5 mumol of cyanate esters/g of gel). At volume ratios greater than 2.2, both the fraction bound and the fraction retained decline continuously. Changing the heparinase concentration in the coupling solution changes the coupling efficiency in a manner similar to that of the volume ratio change. When heparin is added during the coupling process, the fraction bound declines as the heparin concentration increases, whereas the fraction retained increases up to a heparin concentration of 12 mg/mL and decreases thereafter. When arginine, lysine, and glycine are used to block the unreacted cyanate ester groups after the coupling process, the immobilized heparinase shows different pH optima of 6.5, 6.9, and 7.2, respectively. Based upon these findings, a protocol to optimize heparinase immobilization is developed.

Cross-Linking Reagents↗