Biomedical subjects

A Sánchez-Ferrer

Publications and source records attributed to A Sánchez-Ferrer.

At least 19 recordsLinked to original sources

PubMed2006

Main-chain smectic liquid-crystalline polymers as randomly disordered systems.

We report a high-resolution X-ray lineshape study of main-chain smectic polymers. The results indicate that the layer ordering differs fundamentally from the algebraic decay typical for other smectic liquid-crystalline systems. The lineshapes are best described by broad squared Lorentzians indicating some form of short-range correlations. However, several higher harmonics are observed, which excludes simple liquid-like short-range order. This behaviour is tentatively attributed to a random field of defects associated with entangled hairpins in the main-chain polymer structure.

Complex Mixtures↗

PubMed2006

A colorimetric assay for S-adenosylhomocysteine hydrolase.

A colorimetric method for S-adenosyl-L-homocysteine hydrolase (SAHase) which uses S-adenosyl-L-homocysteine (SAH) as substrate is described. This method involves the hydrolytic conversion of SAH into adenosine (ADO) and L-homocysteine (HCY). The formation of HCY is quantified using Ellman's reagent and spectrophotometrical measured at 412 nm. Under these assay conditions, the product was followed continuously in a facile and quantitative manner until substrate conversion was complete. This method is an easy, cheap and shorter alternative to more complex methods and it is applicable to routine clinical analysis and in the assay and development of new S-nucleosidylhomocysteines to be used as therapeutic compounds.

Adenosine Deaminase↗

PubMed2005

Evidence of supercritical behavior in liquid single crystal elastomers.

Temperature profiles of the first and the second moment of the nematic order parameter distribution function, as determined from the deuteron nuclear magnetic resonance line shapes, as well as heat capacity response, provide support for the supercritical scenario of the nematic-paranematic phase transition in liquid single crystal elastomers. The relative strength of the locked-in internal mechanical field with respect to the critical field can be decreased by swelling the elastomer samples with low molecular mass nematogen. By increasing the concentration of the dopant, critical and below-critical behavior is promoted.

Journal Article↗

PubMed2003

Early and intermediate Amadori glycosylation adducts, oxidative stress, and endothelial dysfunction in the streptozotocin-induced diabetic rats vasculature.

AIMS/HYPOTHESIS: In a model of streptozotocin-induced Type 1 diabetes mellitus in rats of 9 weeks duration, we analysed time associations between the development of hyperglycaemia, early and intermediate glycosylation Amadori adducts, or AGE compared with enhancement of oxidative stress and endothelial dysfunction. METHODS: Endothelial function was tested at several stages of streptozotocin-induced diabetes and after treatment with insulin, resulting in different concentrations of blood glucose, glycosylated haemoglobin (an Amadori adduct), and AGE. Other animals were studied antagonising the formation of AGE with aminoguanidine. RESULTS: Relaxation in response to acetylcholine (1 nmol/l to 10 micro mol/l) was tested in isolated segments from aorta or mesenteric microvessels. Impairment of endothelium-dependent relaxations occurred after 2 weeks of untreated diabetes. Preincubation of vessels affected with 100 U/ml superoxide dismutase improved the relaxations to acetylcholine, along the time-course of the endothelial impairment. This indicates the participation of reactive oxygen species on diabetic endothelial dysfunction. The impairment of endothelium-dependent relaxations was recovered after 3 more weeks of insulin treatment. Aminoguanidine treatment did not modify this pattern of development. The time course of the rise and disappearance of endothelial dysfunction showed a higher correlation with glycosylated haemoglobin concentrations than with blood glucose or serum AGE. CONCLUSION/INTERPRETATION: Enhancement of early and intermediate Amadori adducts of protein glycosylation was the factor showing a better relation with the development of endothelium impairment. These results are consistent with a role for these products in the development of diabetic vasculopathy.

Acetylcholine↗

PubMed2002

Microencapsulation of Aerococcus viridans with catalase and its application for the synthesis of dihydroxyacetone phosphate.

Dihydroxyacetone phosphate is essential for the synthesis of polyhydroxylated compounds used as components or precursors of active pharmaceutical substances, such as antibiotics or glycosidase inhibitors. Dihydroxyacetone phosphate was produced by enzymatic oxidation of L-alpha-glycerophosphate in the presence of glycerophosphate oxidase or Aerococcus viridans coimmobilized with a hydrogen peroxide-decomposing enzyme. The microencapsulation of A. viridans with catalase in sodium alginate showed a conversion of 98.5%; the conversion percentage remained constant in all five runs. Liquid chromatography of the product revealed that the product peak corresponded to that of the dihydroxyacetone phosphate internal standard. This indicated a high degree of product purity.

Alginates↗

PubMed2001

Cyclodextrin biospecific-like displacement in dye-affinity chromatography.

Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-beta-CD revealed only two absorbing species, indicating a host-guest ratio of 1:1. The dissociation constant for this HP-beta-CD-CB F3GA complex, Kd, was found to be 0.43 mM. The data for HP-beta-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution strategy. It provided a 15-fold purification factor with 89% recovery and sharp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1.8 column volumes). In addition, the new elution strategy showed a better purification factor and sharper elution profile than traditional non-specific elution with KCl (4.5-fold, and 1.4 column volumes). Hence, competitive elution by inclusion complexes may be a promising strategy for eluting proteins with high recoveries and purification factors in dye-affinity chromatography.

Chromatography, Affinity↗

PubMed2001

Highly efficient Aerococcus viridans L-alpha-glycerophosphate oxidase production in the presence of H2O2-decomposing agent: purification and kinetic characterization.

Glycerophosphate oxidase was purified from Aerococcus viridans cells by phase partitioning in Triton X-114, ammonium sulfate fractionation, FPLC ion-exchange chromatography and FPLC hydrophobic-interaction chromatography. The purification achieved from a crude extract of A. viridans was 38-fold with a 32% recovery of activity. Under the growth conditions used, A. viridans strain CECT 978 proved to be an excellent glycerophosphate-oxidase producer, with enzyme production 2,800-fold greater than that described in the literature for the same microorganism. The culture medium used in the present work is that commonly used for cultivation of this microorganism, except that an H2O2-decomposing enzyme was added. The addition of catalase to the growth medium had a clear effect on the growth rate. Furthermore, methylglyoxal, a metabolite that is formed enzymatically from triose phosphates, was found to be an inactivator of glycerophosphate oxidase activity.

Catalase↗

PubMed2001

Proteolytic activation of latent Paraguaya peach PPO. Characterization of monophenolase activity.

The kinetics of the activation process of latent peach PPO by trypsin was studied. By coupling this activation process to the oxidation of 4-tert-butylcatechol (TBC) to its corresponding quinone, it was possible to evaluate the specific rate constant of active PPO formation, k(3), which showed a value of 0.04 s(-1). This proteolytic activation of latent peach PPO permitted us to characterize the monophenolase activity of peach PPO for the first time using p-cresol as substrate, and it showed the characteristic lag period of the kinetic mechanism of monophenols hydroxylation, which depended on the enzyme and substrate concentration, the pH and the presence of catalytic amounts of o-diphenol (4-methylcatechol). The enzyme activation constant, k(act), was 2 microM.

Catechol Oxidase↗

PubMed2001

Non-linear slow-binding inhibition of Aerococcus viridans lactate oxidase by Cibacron Blue 3GA.

Lactate oxidase (LOD) was purified from cells of Aerococcus viridans by phase partitioning in Triton X-114 (TX-114), ammonium sulphate fractionation and FPLC ion exchange chromatography. The purification achieved from a crude extract of A. viridans was 32-fold with a 60% recovery of activity. The isolated enzyme was a true FMN-containing LOD in tetrameric form with a subunit molecular weight of 48,000. The KM for L-lactate was 175 microM, a 6-fold less value than described in the literature. Among the inhibitors tested, Cibacron Blue 3GA showed the lowest Ki. At low concentrations, Cibacron Blue 3GA behaved as a dye-, pH- and time-dependent inhibitor. A Dixon plot of the steady-state rate showed the time-dependent inhibition to be non-linear, contrary to that described for other slow-binding inhibitors. A model to explain this phenomenon was proposed. The model implies the binding of Cibacron Blue 3GA to the isomerised form of the initial enzyme-inhibition complex (E'I).

Apoenzymes↗

PubMed2000

Reversible sodium dodecyl sulfate activation of latent peach polyphenol oxidase by cyclodextrins.

The reversibility of the SDS-mediated activation of latent peach PPO has been studied using cyclodextrins as strip detergent agent. Cyclodextrins produced a combined inhibitory effect on enzymatic activity of latent peach PPO due to the complexation of detergent and the hydrophobic substrate 4-tert-butylcatechol (TBC) molecules. To study the reversibility of the activation process, this combined effect has to be separated. On the one hand, the enzyme was activated by acid-shocking and the activity was measured in the presence of cyclodextrins, using TBC as substrate. The inhibition curves obtained permitted study of the complexation of TBC into cyclodextrins. On the other hand, the enzyme was activated by SDS and the activity in the presence of cyclodextrins was measured using the highly hydrophilic o-diphenol dopamine as substrate. In this case, the inhibition curves obtained indicated the reversibility of the activation process when SDS was trapped by cyclodextrins. In addition, the complexation constant between SDS and 2-hydroxypropyl-beta-cyclodextrins was calculated by measuring conductivity (K(s) = 3500 M(-1)).

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2000

Highly glycated oxyhaemoglobin impairs nitric oxide relaxations in human mesenteric microvessels.

AIMS/HYPOTHESIS: It has been recently shown that glycated human haemoglobin induces endothelial dysfunction in rat vessels by generating superoxide anions that interfere with nitric oxide mediated responses. Our study analysed the effect of glycated human haemoglobin on the endothelium-dependent relaxations of human vessels. METHODS: Omental microvessels were obtained from patients (without diabetes, hypertension or vascular disease) during surgery and mounted in a small vessel myograph to study their vasoactive responses (vessels from 3-7 patients for each set of experiments). RESULTS: Cumulative vasodilatory responses to bradykinin (10 nmol/l to 3 mumol/l) were induced in vessels precontracted with 35-50 mmol/l potassium chloride. Addition of 100 mumol/l NG-nitro-L-arginine methyl ester reduced the relaxation evoked by bradykinin, but preincubation with both NG-nitro-L-arginine methyl ester and 10 mumol/l indomethacin was needed to abolish it. Bradykinin-induced responses were inhibited by 1 mumol/l non-glycated oxyhaemoglobin whereas no effect was obtained with 10 nmol/l oxyhaemoglobin. At these low concentrations (10 nmol/l), glycated human oxyhaemoglobin caused an impairment of bradykinin-induced relaxation when the percentage of glycation was 10% or higher. This effect was prevented by preincubating the vessels with ascorbic acid (10 mumol/l), superoxide dismutase (100 U/ml) and gliclazide (1 and 10 mumol/l), but not with indomethacin (10 mumol/l), catalase (400-600 U/ml), dimethylthiourea (1 mmol/l) or glibenclamide (10 mumol/l). In vessels preincubated with NG-nitro-L-arginine methyl ester (100 mumol/l), glycohaemoglobin did not add any additional effect. CONCLUSION/INTERPRETATION: Highly glycated human oxyhaemoglobin, at physiological plasmatic concentrations, impairs nitric oxide-mediated responses by a mechanism involving superoxide anions but not cyclooxygenase derivatives.

Animals↗

PubMed2000

Oxidation of salsolinol by banana pulp polyphenol oxidase and its kinetic synergism with dopamine.

Salsolinol, a tethrahydroisoquinoline present in banana and biosynthesized from dopamine, was oxidized by banana pulp polyphenol oxidase to its corresponding salsolinol-o-quinone. This oxidation was pH-dependent and showed a maximum at acidic pH values. At physiological pH of 5.0, the values obtained for the kinetic parameter (V(m) and K(m)) were 62.5 microM/min and 1.7 mM, respectively. When dopamine was added to the reaction medium to imitate physiological conditions, salsolinol was co-oxidized by dopamine-quinone. When this phenomenon was studied oxygraphically, an unexpected activation of dopamine oxidation was found in the presence of salsolinol. This activation was related with the enzyme's kinetic mechanism and was named "kinetic synergism", because a bad substrate activated a good one. A possible physiological role is discussed.

Catechol Oxidase↗

PubMed2000

Treatment with acarbose may improve endothelial dysfunction in streptozotocin-induced diabetic rats.

We sought to determine whether a single reduction of hyperglycemia and those derivatives from nonenzymatic protein glycosylation may be effective in reducing the development of diabetic endothelial dysfunction. Therefore, we investigated how acarbose, an inhibitor of intestinal alpha-glucosidase that reduce hyperglycemia by lowering glucose absorption, may prevent the impairment of acetylcholine (ACh)-induced endothelium-dependent relaxations observed in isolated vascular segments from untreated streptozotocin-induced diabetic rats. When administered after diabetes induction, 10 mg/kg acarbose decreased modestly the enhancement of blood glucose and glycosylated hemoglobin (HbA1c) levels, but not those of advanced glycosylation end products (AGEs). This effect was linked to a partial improvement of ACh-induced responses both in conductance vessels, such as aortic segments, and resistance vasculature, like mesenteric microvessels. When acarbose was introduced after 6 weeks of untreated diabetes, blood glucose, HbA1c, and AGE levels were not affected and endothelial dysfunction remained unchanged in mesenteric microvessels, whereas a small improvement was observed in aortic segments. The addition of 100 U/ml superoxide dismutase enhanced the impaired relaxations to values similar to vessels from nondiabetic rats, indicating a main role for superoxide anions in diabetes-induced endothelial dysfunction. We conclude that hyperglycemia itself or elevated HbA1c, but not plasma AGEs, are related to enhanced oxidative stress and to the impairment of endothelium function associated to diabetes. This process can be partially prevented by reducing glucose absorption with acarbose.

Acarbose↗

PubMed1999

Hydroperoxidase activity of lipoxygenase in the presence of cyclodextrins.

The oxidation of xenobiotics by the hydroperoxidase activity of lipoxygenase in the presence of cyclodextrins was studied. These produced an inhibitory effect on xenobiotics oxidation, based on their degree of hydrophobicity and the charge (isoproterenol < 4-methyl-catechol (4MC) < 4-tert-butylcatechol (TBC) < 4-tert-octylcatechol (TOC)). This inhibitory effect was due to the complexation of xenobiotics in the hydrophobic cavity of cyclodextrins. The complexation constant Kc was calculated by nonlinear regression of the inhibition curves obtained in the presence of cyclodextrins, and the values obtained were 400, 16,250, and 35,127 M-1 for 4MC, TBC, and TOC, respectively. The validity of these values was checked at different points of the Michaelis-Menten saturation curve, and a sigmoidal inhibition curve was obtained at the saturating concentration of the o-diphenol, TBC, with no change in the Kc value. This demonstrates the validity of the equations used to calculate Kc for the complete range of the Michaelis-Menten equation.

Catechols↗

PubMed1999

Impairment of endothelial relaxations by glycosylated human oxyhemoglobin depends on the oxidative state of the heme group.

While nanomolar met- or cyanomethemoglobin, either non-glycosylated or glycosylated, did not alter endothelial function, glycosylated oxyhemoglobin induced contractile responses and caused an impairment of endothelium-dependent relaxations in rat aortic segments. The vascular effects induced by glycosylated oxyhemoglobin were prevented by superoxide dismutase. Furthermore, glycosylated oxyhemoglobin produced higher amounts of superoxide anions than other hemoglobin derivatives. These results suggest that glycosylated hemoglobin requires the existence of a functional heme group containing iron in ferrous state to interfere with the endothelial function at nanomolar concentrations. This effect is mediated by generation of superoxide anions.

Acetylcholine↗

PubMed1999

Cyclodextrins as activator and inhibitor of latent banana pulp polyphenol oxidase.

The effect of cyclodextrins (CDs) on o-diphenol oxidation catalyzed by banana polyphenol oxidase (PPO) was studied. The oxidation of dopamine, the natural substrate of banana, in the presence of cyclodextrins was unaffected, because this hydrophilic phenol does not form inclusion complexes with CDs. However, when a hydrophobic phenol such as tert-butylcatechol (TBC) was used, a marked inhibition was observed with beta-, hydroxypropyl-beta-, and maltosyl-beta- CDs. This inhibition was due to the complexation of TBC in the CD core, demonstrating that banana pulp PPO worked only toward free substrate and not toward the complex TBC-CDs. In addition, the effect of some inhibitors in the presence of CDs and dopamine as substrate was studied. Increasing concentrations of CDs, in the presence of two inhibitors (4-iodophenol and cinnamic acid) were able to activate the inhibited enzyme to reach the noninhibited level by complexing the inhibitors in the hydrophobic core of the CDs. This dual effect of CDs as activator and inhibitor was tested in crude banana pulp extracts, with surprising activation effects never before described being observed.

Catechol Oxidase↗

PubMed1999

Cyclodextrins as diethylstilbestrol carrier system: characterization of diethylstilbestrol-cyclodextrins complexes.

PURPOSE: The in vitro formation of DES-cyclodextrins inclusion complexes was characterized using lipoxygenase as enzymatic system. METHODS: DES-cyclodextrins complexes were obtained in aqueous solution. RESULTS: The addition of cyclodextrins to the reaction medium had an inhibitory effect on DES oxidation by lipoxygenase due to the drug's complexation into the cyclodextrin cavity. This inhibitory effect depends on the complexation constant between DES and the cyclodextrins type used. In this case, beta-, 2-hydroxypropyl-beta- and gamma-cyclodextrins have similar complexation constants and therefore produce the same inhibitory effect. Moreover, depending on the type of cyclodextrins used, the solubility of DES can be enhanced up to 956 times, while the lipoxygenase activity remains constant. CONCLUSIONS: These results suggest that the system described may be used as a controlled-release delivery system for DES, since it may diminish the local and systemic adverse side effects caused by high concentrations of the drug.

Cyclodextrins↗

PubMed1998

Endothelial dysfunction and metabolic control in streptozotocin-induced diabetic rats.

1. The aim of this work was to study the influence of the metabolic control, estimated by the levels of glycosylated haemoglobin in total blood samples (HbA1c), in developing vascular endothelial dysfunction in streptozotocin-induced diabetic rats. Four groups of animals with different levels of insulin treatment were established, by determining HbA1c values in 5.5 to 7.4%, 7.5 to 9.4%, 9.5 to 12% and > 12%, respectively. 2. The parameters analysed were: (1) the endothelium-dependent relaxations to acetylcholine (ACh) in isolated aorta and mesenteric microvessels; (2) the vasodilator responses to exogenous nitric oxide (NO) in aorta: and (3) the existence of oxidative stress by studying the influence of the free radical scavenger superoxide dismutase (SOD) on the vasodilator responses to both ACh and NO. 3. In both isolated aortic segments and mesenteric microvessels, the endothelium-mediated concentration-dependent relaxant responses elicited by ACh were significantly decreased when the vessels were obtained from diabetic animals but only with HbA1c values higher than 7.5%. There was a high correlation between HbA1c levels and the impairment of ACh-induced relaxations, measured by pD2 values. 4. The concentration-dependent vasorelaxant responses to NO in endothelium-denuded aortic segments were significantly reduced only in vessels from diabetic animals with HbA1c values higher than 7.5%. Again, a very high correlation was found between the HbA1c values and pD2 for NO-evoked responses. 5. In the presence of SOD, the responses to ACh or NO were only increased in the segments from diabetic rats with HbA1c levels higher than 7.5%, but not in those from non-diabetic or diabetic rats with a good metabolic control (HbA1c levels <7.5%). 6. These results suggest the existence of: (1) a close relation between the degree of endothelial dysfunction and the metabolic control of diabetes, estimated by the levels of HbA1c; and (2) an increased production of superoxide anions in the vascular wall of the diabetic rats, which is also related to the metabolic control of the disease.

Acetylcholine↗