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The hydroxysteroid dehydrogenases: beta-HSDH, 20 beta-HSDH, and 3 alpha-HSDH, were immobilized on CNBr-activated Sepharose. The effect of various immobilization conditions on the activity recovery and stability were examined. The presence of cofactor during the immobilization reaction increased the activity recovery (40--60% of the total) and also led to materials highly stable in the presence of organic solvents. For example, beta-HSDH maintained 60% of its original activity two months after continuous use in the water--ethyl-acetate system. Kinetic experiments showed that the increase of the apparent Km values is poor and demonstrated that the organic solvent behaves as a weak inhibitor (ki greater than 0.2M) for the substrate. The immobilized enzymes lyophilized in the presence of sucrose had full activity restored even after several months storage at room temperature. Immobilized hydroxysteroid dehydrogenases were shown to be suitable for preparative transformation of steroids in water--organic solvent systems.

Two methods utilizing ion-exchange high-performance liquid chromatography have been developed for the separation of choline- and ethanolamine-containing metabolic precursors for phosphatidylcholine and phosphatidylethanolamine. Both methods employ an analytical anion-exchange column supplemented with a cation-exchange column; the latter is required only to separate betaine from choline. Complete separation of choline- and ethanolamine-labeled metabolites from extracts of Chinese hamster ovary cells can be achieved in 20 min.

The denaturation by urea and renaturation of 20 beta-hydroxysteroid dehydrogenase, a tetrameric enzyme consisting of four identical subunits, were followed by high-performance size exclusion chromatography to detect intermediates in the processes. During the denaturation process no intermediate form (structured monomers or dimers) between the tetramer and the denatured monomer was observed. During the renaturation process, carried out either with or without NADH, high molecular weight aggregates, native tetramers, and low molecular weight intermediates were evidenced and quantified. The contemporaneous measurement of recovery of activity unambiguously demonstrated that the tetrameric structure is essential for enzymatic activity.

Our study sought to determine whether, where and how the consonance or dissonance of musical intervals might be reflected in the electrical activity (depth EEG) of the human brain. Presenting an adequate musical test program to a patient with depth electrodes implanted within the planum temporale (near Heschl's gyrus) and the hippocampal formations of both sides, we found that the reactions shown by the depth EEG of the limbic system correspond in precise and distinctly differing patterns to the consonances and dissonances presented. The main findings are: the EEG of the left hippocampus reflects the consonance-dissonance dichotomy; the activity of the left hippocampus but not the right is affected by a previous sequence of dissonances in responding to a single consonance; the EEG activity of the right Heschl's gyrus (the left Heschl's gyrus was not analyzed), but neither the left nor the right hippocampus, responded differently to a given succession of intervals and its pitch inversion. These results may help to understand the different emotional responses which usually arise in response to consonant or dissonant musical intervals.

We investigated the denaturation of tetrameric 20 beta-hydroxysteroid dehydrogenase (20R)-17 beta,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) to find out whether intermediate states are formed during the process. The denaturation process was studied in the presence and absence of stabilizers, both specific, such as NADH, and non-specific, such as the salting-out anion phosphate. Changes in enzymatic activity, intrinsic protein fluorescence and far-ultraviolet circular dichroism were monitored. When NADH was present, denaturation of the enzyme by urea was a one-step transition between the native and the completely denatured state. In dilute phosphate, and even more so in concentrated phosphate, the existence of intermediate states with different stability is evidenced by the noncoincidence of the transition curves that probe for different functional and conformational aspects of the enzyme. Therefore, for 20 beta-hydroxysteroid dehydrogenase the formation of intermediates can be prevented by adding NADH, or enhanced by adding concentrated phosphate.

Use of L-asparaginase by the extracorporeal route in the therapy of acute lymphoblastic leukemia (ALL) has been proposed. Results, however, are not so satisfactory as i.v. administration of this enzyme, because the levels of L-asparagine do not fall for a sufficient length of time due to the antagonistic action of the L-asparagine-synthetase. To avoid the L-asparagine rebound we have utilized, by extracorporeal route, L-glutaminase together with L-asparaginase, in order to reduce L-asparagine and L-glutamine levels. We have therefore performed a series of experiments in vitro and in vivo either using L-asparaginase alone or together with L-glutaminase. Results show that, contrary to what happens when L-asparaginase is used alone, L-asparagine levels decrease and remain low even after 24 hours from the treatment, when L-glutaminase is added to the system. Thus a lowering of L-glutamine levels, which seems to play an important role in the therapy of ALL, is obtained.

Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

We evaluated systemic and central hemodynamics in 10 lean hypertensives and in 10 obese hypertensives (WHO stage I-II) after 8 weeks treatment regimen with a serotoninergic antagonist such as ketanserin. At the beginning and at the end of the study, body weight, BMI, blood pressure and heart rate were measured and a first pass radionuclide angiocardiography was performed to determine cardiac output, cardiac index and ejection fraction of left ventricle. Total peripheral resistances were also calculated. In both hypertensive groups ketanserin significantly reduced diastolic (p less than 0.05) and mean (p less than 0.005) blood pressure. No significant change in systolic blood pressure, cardiac output, cardiac index and ejection fraction of left ventricle has been observed in lean and obese hypertensives. Total peripheral resistance values significantly (p less than 0.05) decreased in lean hypertensives; in obese hypertensives total peripheral resistance moderately reduced. Our results indicated that monotherapy with ketanserin is effective in treating mild to moderate hypertension both in lean and in obese hypertensives and did not interfere with left ventricular performance. At last the effectiveness of ketanserin treatment appears moderately higher in lean than in obese hypertensives.

The chemical and physical properties of the high-molecular-weight glycoprotein (SO20, w = 8S; Ve=Vo on Sephadex G-200) with gastric antisecretory activity extracted from the urine of pregnant women were studied. Gel filtration in the presence of sodium dodecyl sulphate and sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis indicated subunit mol.wts. of 16 000 +/- 1500 and 13 000 +/- 1000 respectively. Reaggregation of the subunits and partial recovery of the biological activity were observed on removal of the detergent. The partial C-terminal sequence was found to be Phe-Tyr-Leu-Val-OH, whereas glycine appears to be the N-terminal amino acid. The carbohydrate composition was examined; all galactosamine was found to be O-glycosidically linked to the polypeptide chain.

To study the sonographic (US) evolution of hepatocellular carcinoma, 53 tumors in 45 untreated patients were observed regularly with real-time US for a period of 6 to 56 months. At the beginning, 25 tumors were hypoechoic, 18 isoechoic, 4 hyperechoic, and 6 had mixed hypo/hyper echo patterns. At the follow-up, 7 initially hypoechoic tumors had changed to hyperechoic or to mixed echo patterns; 8 hypoechoic tumors had become isoechoic; 9 of the 25 initially hypoechoic neoplastic lesions had maintained the same echo density. Ten of the 15 initially isoechoic tumors had changed to mixed echo patterns, and 5 had remained unchanged. Three initially isoechoic lesions and a hypoechoic one had turned into diffuse patterns; 2 initially hyperechoic neoplastic lesions had remained unchanged; 1 had switched into hypoechoic, and 1 changed to mixed echo pattern; 4 out of 6 tumors with echo pattern had remained unchanged, 1 had become hyperechoic and 1 hypoechoic. The current study has proven various tumors less than or equal to 3 cm phi to be isoechoic, and most tumors greater than 3 cm phi to have mixed hypo/hyper echo patterns. The echogenicity of small hepatocellular carcinomas increases with the tumor growth, and remains unchanged when they do not increase in size.