Clinical changes in "true" hobnail hemangioma during menstruation.

Hobnail hemangioma (HH) was known in the past as targetoid hemosiderotic hemangioma (THH). We present a case that meets the criteria of HH in a 34-year-old woman. The lesion presented as a reddish papule on her left hip. It changed periodically during menstruation and the changes included. The biopsy showed the characteristics of a typical HH with moderate iron deposits. Immunohistochemical staining failed to demonstrate either estrogen or progesterone receptors (Fig. 1, Ref. 15). Full Text (Free, PDF) www.bmj.sk.

beta-Galactosidase immobilization for milk lactose hydrolysis: a simple experimental and modelling study of batch and continuous reactors.

The experiment described in this paper introduces students to the practical use of an enzyme (beta-galactosidase, or lactase) acting on a natural substrate. The enzyme is immobilized onto a cheap support, and the immobilized derivative is used in a packed-bed reactor for continuous milk lactose hydrolysis. The results are compared to those obtained for discontinuous batch reactors with soluble enzyme. A mathematical model of the two types of reactors is run, and its results are compared with the experimental data obtained.

Determination of L-carnitine by flow injection analysis with NADH fluorescence detection.

A flow injection analysis method for determining L-carnitine is reported. The system uses the enzyme L-carnitine dehydrogenase covalently immobilized to Eupergit C. The NADH produced by the action of the enzyme, which is proportional to the L-carnitine concentration, is quantified using fluorescence detection. The system response was rapid and had a wide range of linearity. At a flow rate of 0.2 ml/min, a detection limit of 1 microM (20 pmol) was obtained for L-carnitine, peak areas were linear up to 100 microM, and samples could be injected every 4 min. The method performed well as a routine assay, showing high sensitivity (54,000 AU/microM), a precision of 0.96%, and the ability to carry out 144 consecutive assays with an RSD of 1.47% (good stability). Comparisons were made with other known methods for L-carnitine determination. Presence of D-carnitine had no effect on L-carnitine assay. The analysis was valid for determining L-carnitine concentrations in commercial pharmaceutical preparations.

Retention and regeneration of native NAD(H) in noncharged ultrafiltration membrane reactors: application to L-lactate and gluconate production.

NAD(H) was retained in a noncharged ultrafiltration membrane reactor for the simultaneous and continuous production of L-lactate and gluconate with coenzyme regeneration. Polyethyleneimine (PEI), a 50-kDa cationic polymer, achieved coenzyme retentions above 0.8 for PEI/NAD(H) molar ratios higher than 5. The ionic strength of the inlet medium caused a decrease of NAD(H) retention that can be counterbalanced by an initial addition of 1% bovine serum albumin (BSA). Continuous reactor performance in the presence of PEI and BSA showed that NAD(H), glucose dehydrogenase, and lactate dehydrogenase were retained by 10-kDa ultrafiltration membranes; L-lactate and gluconate were produced at conversions higher than 95%. PEI enhanced the thermal stability of the enzymes used and increased the catalytic efficiency of glucose dehydrogenase, while no effect was found on the kinetic parameters of lactate dehydrogenase. A model that implements the kinetic equations of the two enzymes describes the reactor behavior satisfactorily. In brief, the use of PEI to retain NAD(H) is a new interesting approach to be widely applied in continuous synthesis with the large number of known dehydrogenases.

Influence of polyhydroxylic cosolvents on papain thermostability.

Papain thermostability was studied, and non-first-order deactivation kinetics were observed. The results obtained were analyzed by a two-step series-type deactivation model involving the native and active enzyme, an active intermediate enzyme state, and a final inactive state, with excellent agreement. The influence of different polyhydroxylic cosolvents (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of papain at 60 degrees C was also studied. Analysis of the results by the assayed model showed that the main protective effect of cosolvents was observed in the second step of the deactivation profile. The results obtained were analyzed as a function of both the thermodynamic parameters and a protective effect, defined as the ratio of papain half-lives (with and without cosolvents) for the second deactivation step, showing in both cases an important stabilizing effect of these cosolvents on the enzyme. The overall protective effect of cosolvents was also related simultaneously to their concentration and their water activity-depressing power.

Glycylglycylphenylalaninamide synthesis catalysed by papain in a medium containing polyols.

The influence of several polyols (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on both the thermostability and tripeptide(Gly-Gly-PheNH2)-synthesis capability of papain was studied at 60 degrees C. The results obtained from the thermostability studies on papain showed that polyols increased the half-life time of the esterase activity of the enzyme proportionally to their molecular size and concentration, except for ethylene glycol. The presence of polyols, as water-activity-depressing agents, also enhanced the enzyme activity for Gly-Gly-PheNH2 synthesis in a way which was directly proportional to the molecular size of the polyol molecule and its water-activity-depressing power. A linear relationship between the increase in the synthetic/hydrolytic activity ratio and the overall concentration of hydroxy groups in the reaction media was obtained, indicating that these latter groups are mainly responsible for the modification of the catalytic behaviour of the enzyme, as a result of a change in their microenvironment.

Kinetic and operational study of a cross-flow reactor with immobilized pectolytic enzymes.

The kinetics and operational behavior of a nylon membrane derivative with immobilized pectolytic enzymes in a cross-flow reactor were analyzed. A high dependence on the recycling flow rate was observed. A design equation of the system has been proposed by taking into account both the shear stress deactivation and the control of the external diffusional limitations. Integration of the resulting design equation allowed us to study the effect of different operational parameters on substrate conversion. The catalytic efficiency of the immobilized derivative in a cross-flow reactor showed the highest pectin hydrolysis capability when it was compared with two different configurations of packed-bed reactors.

[Active antihelminitic alkaloids: active in vitro against Leishmanic Tropica the protozoa involved in leishmaniasis]. / Activité antiparasitaire d'alcaloïdes bisbenzylisoquinoleiques. I: Activité in vitro sur des promastigotes de trois souches de Leishmania.

Leishmaniasis caused by protozoan Leishmania ssp., is an endemic parasitic disease in Central and South America. The chemotherapeutic agents against Leishmania ssp. (pentavalent antimony compounds, pentamidine and amphothericine B) are toxic and expensive products. Basing on the Bolivian folk medicine, we tried to find new active principles. Fourteen isoquinoline alkaloids, especially bisbenzylisoquinoline alkaloids extracted from Annonaceae, Berberidaceae, Hernandiaceae and Menispermaceae, demonstrate highly effective activity against this protozoan. Among them gyrocarpine, daphnandrine and obaberine seem to be of particular interest. The therapeutic effect was studied by biological assays on culture forms in vitro three strains of Leishmania, L. donovani, L. braziliensis (cutaneous and mucocutaneous leishmaniasis), L. mexicana amazonensis (cutaneous) and L. donovani (visceral leishmaniasis).

[ Active trypanocidal alkaloids, bisbenzylisoquinoles ii. active in vitro against trypanosoma cruzi the agent responsible for trypanosomiasis, south america]. / Activité antiparasitaire d'alcaloïdes bisbenzylisoquinoléiques. II. Activité in vitro sur des epimastigotes de trois souches typifiees de Trypanosoma cruzi.

Chagas disease caused by the protozoan Trypanosoma cruzi is an endemic parasitic disease in Central and South America. The chemotherapeutic agents against Trypanosoma cruzi (imidazol compounds, lampit and benznidazol) are not very convenient products. Since it is known that protozoan Trypanosoma are close to Leishmania we studied the action of 14 bisbenzylisoquinoline alkaloids, in vitro, on three strains of T. cruzi (Tulahuen, C8C11, and 1979 CL1). As in the case of Leishmania, gyrocarpine, daphnandrine and obaberine showed an interesting activity and we judge them to be worthy of in vivo assays.

Evaluation of the effectiveness factor along immobilized enzyme fixed-bed reactors: Design of a reactor with naringinase covalently immobilized into glycophase-coated porous glass.

A design equation is presented for packed-bed reactors containing immobilized enzymes in spherical porous particles with internal diffusion effects and obeying reversible one-intermediate Michaelis-Menten kinetics. The equation is also able to explain irreversible and competitive product inhibition kinetics. It allows the axial substrate profiles to be calculated and the dependence of the effectiveness factor along the reactor length to be continuously evaluated. The design equation was applied to explain the behavior of naringinase immobilized in Glycophase-coated porous glass operating in a packed-bed reactor and hydrolyzing both p-nitrophenyl-alpha-L-rhamnoside and naringin. The theoretically predicted results were found to fit well with experimentally measured values.

Activity of soluble and immobilized hesperidinase on insoluble hesperidin.

A michaelian kinetic behaviour was found when the α-ramnosidase activity, both of native and immobilized hesperidinase, was determined on hesperidin suspensions. In spite of the low hesperidin solubility in the reaction medium, the maximum rates overtook the expected values, thus pointing to the enzyme ability to degrade insoluble hesperidin.

A cross-flow reactor with immobilized pectolytic enzymes for juice clarification.

A new system for continuous juices clarification is presented. The bioreactor combines microporous plates commercially available and industrial pectinases immobilized on nylon membranes in a cross-flow configuration. The kinetic behaviour of the reactor for different recirculation flow rates has been determined. Fresh apricot juice has been continuously clarified in the bioreactor with excellent results.

A method for assaying the rhamnosidase activity of naringinase.

The use of the p-nitrophenyl-alpha-L-rhamnopyranoside for the specific measurement of the alpha-rhamnosidase activity of naringinase, by colorimetrically following the appearance of p-nitrophenolate anion, is proposed. Use of this synthetic substrate did not change the pH, temperature, or ionic strength optima of the enzyme. It did, however, result in (a) a decrease of the Michaelis constant of the enzyme, allowing the Vmax to be measured, this being impossible to accomplish with naringin, (b) an increase in the sensitivity of the assay to the presence of inhibitors in the reaction media, (c) an increase in the sensitivity which enabled measurement of low levels of naringinase due to the high absorptivity of p-nitrophenolate, and (d) a quick and cheap method of evaluating the alpha-rhamnosidase activity of naringinase.

Tyrosine hydroxylase activity of immobilized tyrosinase on enzacryl-AA and CPG-AA supports: Stabilization and properties.

Frog epidermis tyrosinase has been immobilized on Enzacryl-AA (a polyacrylamide-based support) and CPG(zirclad)-Arylamine (a controlled pore glass support) in order to stabilize the tyrosine hydroxylase activity of the enzyme; in this way, the immobilized enzyme could be used to synthesize L-dopa from L-tyrosine. The activity immobilization yield Y(IME) (act) (higher than 86%), coupling efficiency (up to 90%), storage stability (no loss in 120 days), and reaction stability (t(1/2) was higher than 20 h in column reactors) were measured for tyrosinase after its immobilization. The results showed a noticeable improvement (in immobilization yield, coupling efficiency, and storage and operational stabilities) over previous reports in which tyrosinase was immobilized for L-dopa production. The activity and stability of immobilized enzyme preparations working in three different reactor types have been compared when used in equivalent conditions with respect to a new proposed parameter of the reactor (R(p)), which allows different reactor configurations to be related to the productivity of the reactor during its useful life time. The characteristic reaction inactivation which soluble tyrosinase shows after a short reaction time has been avoided by immobilization, and the stabilization was enhanced by the presence of ascorbate. However, another inactivation process appeared after a prolonged use of the immobilized enzyme. The effects of reactor type and operating conditions on immobilized enzyme activity and stability are discussed.

Conformational studies of soluble and immobilized frog epidermis tyrosinase by fluorescence.

Fluorescence spectra and soluble quenching of intrinsic protein fluorescence were used as indexes of conformational changes suffered by frog epidermis tyrosinase. The activation process and the immobilization of the enzyme involving either free amino groups or its carbohydrate moiety were studied. The conformational changes resulting from denaturation of each one of the protein derivatives, as well as the effect of active center copper extraction, were followed by fluorescence studies. The results showed that: both activation and immobilization were accompanied by conformational changes of the protein leading to more unfolded states; neither enzyme nor immobilized enzyme were fully unfolded upon denaturation although enzymic activity was lost; the enzyme immobilized through its carbohydrate moiety was more unfolded upon denaturation than the enzyme immobilized through amino groups, thus pointing to a higher conformational stabilization in the last situation; and that tryptophyl residues moved to a localization near the active site upon activation.

Incorporation of bovine thyroid peroxidase in liposomes.

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropriate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (Tc) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37 degrees C, the temperature at which thyroid peroxidase works 'in vivo'. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.

Acid proteinase activity in fish--I. Comparative study of extraction of cathepsins B and D from Mujil auratus.

Acid catheptic activity was measured in crude extracts of muscle, liver, heart, spleen and gonads from the fishes Mujil auratus, Sparus aurata and Lightonatus mormyrus. The spleen was the organ which showed the highest activity. A comparative study of the seven most commonly used extraction methods was made. Some were modified to account for the characteristics of the fish organs and the activity extracted from them. The Siebert method resulted as the best extraction method only if 1 mM EDTA was present in the medium. The activity from Mujil auratus muscle was strongly inhibited by iodoacetate, N-ethylmaleimide, p-hydroxy mercuribenzoate, and diazo-acetyl-DL-norleucine methyl ester. The results indicated the presence of a carboxyl-proteinase and a thiol-proteinase. According to inhibition studies, the levels of proteinase and amidase activities shown by different organs of Mujil auratus were re-examined. The spleen extract showed the maximum activity for both cathepsins, but muscle extract accounted for more than 95% of total catheptic activity.

Acid proteinase activity in fish II. Purification and characterization of cathepsins B and D from Mujil auratus muscle.

Two cathepsins were detected in Mujil auratus muscle extracts. They were classified as a thiol- and aspartyl-proteinase (cathepsins B and D, respectively) on the basis of their catalytic behaviour in presence of specific inhibitors. Following extraction in 1% KCl, the proteinases were purified by autolysis, acetone fractionation, affinity chromatography, and gel permeation chromatography. The haemoglobin-agarose column chromatography allowed us to separate the two activities. Sephadex G-75 column chromatography resulted in apparent molecular weights of 25,000 (cathepsin B) and 35,000 (cathepsin D). The molecular size, together with pH-activity profiles and kinetic parameters are similar to those reported for mammalian cathepsins B and D. This was not the case with the temperature-activity profiles, the optimum temperature as well as the heat stability being higher for fish cathepsins than for those obtained from other sources. Cathepsin B was characterized by its ability to inactivate aldolase. Fluorescence quenching experiments showed that tryptophyl residues of cathepsin B were less occluded and located in a more electronegative microenvironment that those pertaining to cathepsin D.

[Dehydrogenase denaturation by guanidine hydrochloride measured by fluorescence (author's transl)]. / La desnaturalización con clorhidrato de guanidina de algunas deshidrogenasas seguida por medidas de fluorescencia

Denaturation and subsequent renaturation of the enzymes Lactate, Glucose-6-phosphate, Glutamate and Alcohol dehydrogenases, by means of fluorescence spectra and the variation of enzyme activity in each conformational state, have been studied. The denaturating agent has been Guanidine chloride in a range of concentration from 0.5 to 6 M. Special behaviour has been observed in each enzyme in the presence of the denaturating agent. The action of this agent is compared with that of urea. The renaturation percentages obtained are relatively low. Interaction between the denaturating agent and the aminoacids producing the fluorescence of the enzymes is observed.