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↗

PubMedDate not supplied

Mitochondrial perturbation attenuates bile acid-induced cytotoxicity.

Hydrophobic bile acids such as deoxycholate (DOC) are known to damage liver cells during cholestasis and promote colon cancer. Cellular stresses induced by bile acids, which include mitochondrial and endoplasmic reticulum (ER) stresses, can result in apoptosis. We found that inhibition of mitochondrial complexes I-V with rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, myxothiazol or oligomycin strongly protected against DOC-induced apoptosis of HCT-116 cells. To understand the mechanism of this protection, we explored the ability of these specific inhibitors to reduce DOC-induced mitochondrial and ER stresses. Different inhibitors markedly reduced DOC-induction of mitochondrial condensation, the DOC-induced decrease in mitochondrial membrane potential and the DOC-induced dilatation of the ER (evidence of ER stress). A dramatic induction of nucleolar segregation by antimycin A and myxothiazol, two distinct complex III inhibitors, was also observed. These findings strongly implicate mitochondrial crosstalk with apoptotic signaling pathways and mitochondrial-nucleolar crosstalk in the development of apoptosis resistance in the colon.

Antimycin A↗

PubMedDate not supplied

The mutagenicity of bile acids using a fluctuation test.

The mutagenicity of bile acids was detected by a fluctuation test using Salmonella typhimurium TA100 and TA98 as tester strains. Cholic acid, chenodeoxycholic acid, deoxycholic acid and ursodeoxycholic acid were mutagenic in this test while lithocholic acid was not. The mutagenicity of the bile acids on a molar basis was roughly one-fourth that of methyl methanesulfonate, a moderately potent mutagen. Epidemiological studies have shown a high correlation between levels of bile acids excreted and colon cancer. However, no evidence has previously been reported showing that bile acids are mutagenic. Our results suggest that bile acids may be important in the etiology of colon cancer.

Bile Acids and Salts↗

PubMedDate not supplied

'Big Bang' emergence of the combinatorial immune system.

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Animals↗

PubMedDate not supplied

Concerning dose efficiency in image intensified fluoroscopy.

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Fluoroscopy↗

PubMedDate not supplied

An analysis of dose efficiency in image intensified fluoroscopic examinations.

In this paper we have developed expressions for assessing radiographic systems where grids, air gaps, or both are used. The application of these expressions to film-screen detectors gives predictions in good agreement with data that are found in the literature. Using this analytical tool, image intensified fluoroscopy has been studied. We have shown that under certain circumstances with air gaps there are potential gains in the efficient use of dose in fluoroscopy. The air gaps are of particular value where relatively thin body parts or small lesions are being examined using small focal spot generators. An independent gain control on the image intensifier would be useful for achieving the greatest flexibility when using magnification in fluoroscopy.

Fluoroscopy↗

PubMedDate not supplied

A detailed experimental and theoretical comparison of the angular and energy dependencies of grid transmission.

Grid transmission characteristics have been predicted, with good agreement demonstrated between experiment and theory. Differences between the present work and an earlier theoretical analysis have been noted. It is shown that comparison between experiment and theory can lead to a sensitive method for determining grid parameters. Generally, the derived grid parameters deviate somewhat from manufacturer supplied data. Use of the manufacturer's nominal parameters can lead to large errors in predicting grid transmission.

Radiography↗

PubMedDate not supplied

On the significance of very small angle scattered radiation to radiographic imaging at low energies.

We have studied the angular distribution of scattered radiation at low energies for angles between 2 degrees and 15 degrees from the outward normal to the exit surface of several phantoms, with 1 degree resolution. A cryogenically cooled germanium detector was used to measure the spectra of the scattered radiation. The differential scattering fluences, or numbers of photons per unit solid angle per unit surface area, exhibit distinct peaks at angles in the vicinity of 5 degrees, with the angular position being only slightly energy dependent but very material dependent. The scattered spectra show large changes as a function of angle, in some cases actually becoming harder than the exit unscattered beam. The significance of this behavior relative to the imaging of targets in mammography examinations is discussed.

Humans↗

PubMedDate not supplied

Experimental and theoretical energy and angular dependencies of scattered radiation in the mammography energy range.

Predicted scatter characteristics from a Monte Carlo code have been compared to an experiment for a 4.0-cm-thick acrylic resin (Plexiglas) phantom and 27.3-keV radiation. The results show generally good agreement, except that in the scattered spectra the predictions appear to underestimate the proportion of photons in the low-energy tail of the scattered radiation. The angular dependency of the energy-integrated scattered radiation fluence is in excellent agreement with the predictions.

Mammography↗