Structure and Nanomechanics of Dry and Hydrated Intermediate Filament Films and Fibers Produced from Hagfish Slime Fibers.

Intermediate filaments (IFs) are known for their extensibility, flexibility, toughness, and their ability to hydrate. Using keratin-like IFs obtained from slime fibers from the invertebrate Atlantic hagfish ( Myxine glutinosa), films were produced by drop-casting and coagulation on the surface of a MgCl2 buffer. Drop-casting produced self-supporting, smooth, and dense films rich in ß-sheets (61%), whereas coagulation formed thin, porous films with a nanorough surface and a lower ß-sheet content (51%). The films hydrated and swelled immediately when immersed in water and did not dissolve. X-ray diffraction showed that the ß-crystallites remained stable upon hydration, that swelling presumably happens in the amorphous C-terminal tail-domains of the IFs, and that high salt conditions caused a denser network mesh size, suggesting polyelectrolyte behavior. Hydration resulted in a roughly 1000-fold decrease in apparent Young's modulus from 109 to 106 Pa as revealed by atomic force microscopy nanoindentation. Nanoindentation-based power-law rheology and stress-relaxation measurements indicated viscoelasticity and a soft-solid hydrogel character for hydrated films, where roughly 80% of energy is elastically stored and 20% is dissipated. By pulling coagulation films from the buffer interface, macroscopic fibers with highly aligned IF ß-crystals similar to natural hagfish fibers were produced. We propose that viscoelasticity and strong hydrogen bonding interactions with the buffer interface are crucial for the production of such long biomimetic fibers with aligned ß-sheets. This study demonstrates that hagfish fiber IFs can be reconstituted into functional biomimetic materials that are stiff when dry and retain the ability to hydrate to become soft and viscoelastic when in water.

FM19G11 reverses endothelial dysfunction in rat and human arteries through stimulation of the PI3K/Akt/eNOS pathway, independently of mTOR/HIF-1α activation.

BACKGROUND AND PURPOSE: FM19G11 up-regulates mammalian target of rapamycin (mTOR)/hypoxia inducible factor-1α (HIF-1α) and PI3K/Akt pathways, which are involved in endothelial function. We evaluated the effects of FM19G11 on defective endothelial vasodilatation in arteries from rats and humans and investigated the mechanisms involved. EXPERIMENTAL APPROACH: Effects of chronic in vivo administration of FM19G11 on aortic endothelial vasodilatation were evaluated together with ex vivo treatment in aortic and mesenteric arteries from control and insulin-resistant rats (IRR). Its effects on vasodilator responses of penile arteries (HPRAs) and corpus cavernosum (HCC) from men with vasculogenic erectile dysfunction (ED) (model of human endothelial dysfunction) were also evaluated. Vascular expression of phosphorylated-endothelial NOS (p-eNOS), phosphorylated-Akt (p-Akt) and HIF-1α was determined by immunodetection and cGMP by elisa. KEY RESULTS: Chronic administration of FM19G11 reversed the impaired endothelial vasodilatation in IRR. Ex vivo treatment with FM19G11 also significantly improved endothelium-dependent vasodilatation in aorta and mesenteric arteries from IRR. These effects were accompanied by the restoration of p-eNOS and cGMP levels in IRR aorta and were prevented by either NOS or PI3K inhibition. p-Akt and p-eNOS contents were increased by FM19G11 in aortic endothelium of IRR. FM19G11-induced restoration of endothelial vasodilatation was unaffected by mTOR/HIF-1α inhibitors. FM19G11 also restored endothelial vasodilatation in HPRA and HCC from ED patients. CONCLUSIONS AND IMPLICATIONS: Stimulation of the PI3K/Akt/eNOS pathway by FM19G11 alleviates impaired NO-mediated endothelial vasodilatation in rat and human arteries independently of mTOR/HIF-1α activation. This pharmacological strategy could be beneficial for managing pathological conditions associated with endothelial dysfunction, such as ED.

Recombinant S-adenosylhomocysteine hydrolase from Thermotoga maritima: cloning, overexpression, characterization, and thermal purification studies.

S-Adenosylhomocysteine hydrolase (SAHase) encoded by sahase gene is a determinant when catalyzing the reversible conversion of adenosine and homocysteine to S-adenosylhomocysteine in most living organisms. The sahase gene was isolated from the genome of the highly thermostable anaerobic bacteria Thermotoga maritima, and then it was cloned, characterized, overexpressed using Escherichia coli, and partially purified by thermal precipitation. The thermal purification of the recombinant SAHase resulted in changes in the circular dichroism spectra. As a result of this analysis, it was possible to determine the structural changes in the composition of the α-helix and ß-sheet content of the recombinant enzyme after purification. Moreover, a predicted secondary structure and 3D structural model was rendered by comparative molecular modeling to further understand the molecular function of this protein including its attractive biotechnological use.

Enzymatic synthesis of S-adenosylhomocysteine: immobilization of recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (ATCC 13032).

Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to Eupergit® C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD(+) release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 µM NAD(+) to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.

Inorganic-organic elastomer nanocomposites from integrated ellipsoidal silica-coated hematite nanoparticles as crosslinking agents.

We report on the synthesis of nanocomposites with integrated ellipsoidal silica-coated hematite (SCH) spindle type nanoparticles which can act as crosslinking agents within an elastomeric matrix. Influence of the surface chemistry of the hematite, leading either to dispersed particles or crosslinked particles to the elastomer matrix, was studied via swelling, scattering and microscopy experiments. It appeared that without surface modification the SCH particles aggregate and act as defects whereas the surface modified SCH particles increase the crosslinking density and thus reduce the swelling properties of the nanocomposite in good solvent conditions. For the first time, inorganic SCH particles can be easily dispersed into a polymer network avoiding aggregation and enhancing the properties of the resulting inorganic-organic elastomer nanocomposite (IOEN).

S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum: cloning, overexpression, purification, and biochemical characterization.

The S-adenosylhomocysteine hydrolase gene (sahase) was cloned from the Gram-positive soil bacterium Corynebacterium glutamicum (ATCC 13032) and sequenced. The sahase gene possesses an open reading frame, which consists of 1,434 nucleotides that encode 478 amino acids. The sahase gene from C. glutamicum was expressed in Escherichia coli Rosetta cells by inserting the 1,434-bp fragment downstream from the isopropyl-beta-D-thiogalactopyranoside-inducible promoter of the pET28a+ expression vector. The recombinant S-adenosylhomocysteine hydrolase from C. glutamicum (CgrSAHase) was purified efficiently by a two-step procedure, tangential ultrafiltration and affinity chromatography. The molecular weight of the CgrSAHase, estimated by gel filtration, was about 210 kDa, while sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded a relative molecular mass of 52 +/- 1 kDa. The Michaelis-Menten constants for the natural substrates of the enzyme, S-adenosylhomocysteine (SAH), adenosine, and homocysteine, were determined to be 12, 1.4, and 40 microM, respectively. The overexpression of CgrSAHase was achieved at high level (>40 mg protein/g wet cells). Because of its high capacity to synthesize SAH, this enzyme is of high biotechnological interest.

Cloning, overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Corynebacterium efficiens YS-314.

The gene encoding S-adenosylhomocysteine hydrolase activity (SAHase: EC 3.3.1.1) from Corynebacterium efficiens (YS-314) was cloned and expressed as a fusion protein in Escherichia coli Rosetta (DE3). The analyzed nucleotide sequence of the cloned gene proved to be identical to those reported on the NCBI database. The recombinant enzyme is a tetramer, showing a molecular weight of approximately 210 kDa, as estimated by gel filtration. The K(M) values of the enzyme for S-adenosylhomocysteine (SAH), adenosine (Ado), and homocysteine (Hcy), were determined to be 1.4, 10, and 45 microM. The overexpression of the recombinant enzyme produced a high level of protein (>40 mg of protein per gram of wet cells) and revealed certain thermostability when characterized at temperatures above 40 degrees C. It also showed a high capacity for the synthesis of SAH, thermal stability, and high kinetic similarity to human SAHase, indicating a high biotechnological and pharmacological potential.

Order and strain in main-chain smectic liquid-crystalline polymers and elastomers.

The layer correlations in main-chain smectic liquid-crystal polymer and elastomer systems have been studied using high-resolution X-ray scattering. In contrast to side-chain smectic polymers, in main-chain systems the polymer chains are oriented parallel to the layer normal. As a result they couple directly to the lamellar structure and any polymer defect is translated into layer distortions. For the homopolymers the resulting X-ray lineshapes are well described by Lorentzians. This is interpreted as an average of algebraically decaying order in domains with dimensions of hundreds of nm and a wide dispersion of sizes. The elastomers show much broader peaks than the correponding polymers. This is attributed to strong non-uniform strain within the finite-size domains due to defects of the layer structure.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

Prevention of endothelial dysfunction in streptozotocin-induced diabetic rats by gliclazide treatment.

The aim of the present work was to analyze whether the oral hypoglycemic drug gliclazide affects diabetic endothelial dysfunction in streptozotocin-induced diabetic rats. Gliclazide was compared with glibenclamide, ascorbic acid, and aminoguanidine. An insulin-dependent model of diabetes was selected to exclude insulin-releasing effects of the drugs. Both in isolated aortic segments and mesenteric microvessels, endothelium-dependent relaxation evoked by acetylcholine (ACh, 1 nM to 10 microM) was significantly reduced in vessels from diabetic animals. This impairment was reversed when the segments were previously incubated with 100 U/ml superoxide dismutase. When streptozotocin-induced diabetic rats were orally treated from the time of diabetes induction with gliclazide (10 mg/kg) or ascorbic acid (250 mg/kg), ACh-induced endothelium-dependent relaxation was well preserved both in aortic segments and mesenteric microvessels. In addition, the impaired vasodilatation to exogenous nitric oxide (NO) in aortic segments was also improved in gliclazide-treated diabetic rats. On the other hand, oral treatment with glibenclamide (1 and 10 mg/kg) or aminoguanidine (250 mg/kg) did not produce significant improvements in diabetic endothelial dysfunction. We conclude that gliclazide reverses the endothelial dysfunction associated with diabetes. This effect appears to be due not to the metabolic actions of the drug but rather to its antioxidant properties, as it can be mimicked by other antioxidants. We propose that the mechanism involved is the inactivation of reactive oxygen species, which are increased in diabetes probably as a result of increased early protein glycosylation products, such as glycosylated hemoglobin (HbA(1c)). These effects of gliclazide are not shared by other oral hypoglycemic agent such as glibenclamide, or by blockade of advanced glycosylation end product (AGE) generation with aminoguanidine.

Correction of glycosylated oxyhemoglobin-induced impairment of endothelium-dependent vasodilatation by gliclazide.

We have investigated whether gliclazide, a second-generation sulfonylurea hypoglycemic agent, interferes with the impairment of endothelium-dependent nitric-oxide-mediated relaxation produced by 14%-glycosylated human oxyhemoglobin (GHHb). For comparative purposes, other agents, like glibenclamide, aminoguanidine, ascorbic acid or superoxide dismutase (SOD), were also tested. GHHb (10 nM) caused a reduction in endothelium-dependent relaxation induced by acetylcholine (1 nM to 10 microM) in both isolated aortic segments and mesenteric microvessels from normoglycemic nondiabetic rats. Preincubation of the vessels with gliclazide (100 nM to 10 microM) prevented the impairment of endothelial relaxation, the threshold concentration of gliclazide being 300 nM. In addition, 10 microM gliclazide also prevented the reduction by 10 nM GHHb of the relaxation induced by exogenous nitric oxide (NO, 10 nM to 100 microM). Determination of superoxide anion release measured by the reduction in ferricytochrome c indicated that GHHb produced significant amounts of these free radicals that were concentration-dependently inhibited by gliclazide. The impairment of endothelium-mediated responses was also prevented by 100 U/ml SOD or 10 microM ascorbic acid, but not by 10 microM glibenclamide or 100 microM aminoguanidine. We conclude that gliclazide can reduce the impairment of nitric-oxide-mediated endothelium-dependent relaxation produced by GHHb. This reduction is likely related to the antioxidant properties of the drug, a mechanism suggested by these studies which demonstrate the inactivation of superoxide anions produced by the glycosylated protein by gliclazide.