Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

Sperm tail flexibility test: a simple test for selecting viable spermatozoa for intracytoplasmic sperm injection from semen samples without motile spermatozoa

PURPOSE: The objective was to describe the results of the injection of immotile spermatozoa with flexible tails when only immotile spermatozoa are present in the semen sample. METHODS: A retrospective study was conducted to analyze the procedure results for 10 couples who participated in our intracytoplasmic sperm injection program. The sperm tail was considered flexible when it moved up and down independently of the head movement, and it was considered inflexible when the movement occurred together (tail plus head). The fertilization and pregnancy rate were analyzed. RESULTS: The normal fertilization rate (presence of 2 pronuclei) was 30.3% (40/132), and the abnormal fertilization rate (presence of less than or more than 2 pronuclei) was 6.81% (9/132). A total of 52 embryos were obtained with 9 transfer procedures performed (pregnancy rate: 11.12%). CONCLUSIONS: The sperm tail flexibility test (STFT) is an easy and cost-effective way for selecting viable immotile spermatozoa and can be used as an alternative method for determining the viability of spermatozoa. This test seems to be a simple and risk-free method when compared to the swelling test.

Endothelium-dependent relaxation in response to poly-L-arginine in canine coronary arteries: implications about hyperpolarization as a mechanism of vasodilatation

OBJECTIVE: To study the mechanism by which poly-L-arginine mediates endothelium-dependent relaxation. METHODS: Vascular segments with and without endothelium were suspended in organ chambers filled with control solution maintained at 37ºC and bubbled with 95% O2 / 5% CO2. Used drugs: indomethacin, acetycholine, EGTA, glybenclamide, ouabain, poly-L-arginine, methylene blue, N G-nitro-L-arginine, and verapamil and N G-monomethyl-L-arginine. Prostaglandin F2á and potassium chloride were used to contract the vascular rings. RESULTS: Poly-L-arginine (10-11 to 10-7 M) induced concentration-dependent relaxation in coronary artery segments with endothelium. The relaxation to poly-L-arginine was attenuated by ouabain, but was unaffected by glybenclamide. L-NOARG and oxyhemoglobin caused attenuation, but did not abolish this relaxation. Also, the relaxations was unaffected by methylene blue, verapamil, or the presence of a calcium-free bathing medium. The endothelium-dependent to poly-L-arginine relaxation was abolished only in vessels contracted with potassium chloride (40 mM) in the presence of L-NOARG and indomethacin. CONCLUSION: These experiments indicate that poly-L-arginine induces relaxation independent of nitric oxide.

Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

The pathogenesis of diabetic complications: the role of DNA injury and poly(ADP-ribose) polymerase activation in peroxynitrite-mediated cytotoxicity

Recent work has demonstrated that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron-transport chain triggers several pathways of injury [(protein kinase C (PKC), hexosamine and polyol pathway fluxes, advanced glycation end product formation (AGE)] involved in the pathogenesis of diabetic complications by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Increased oxidative and nitrosative stress activates the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP). PARP activation, on one hand, depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport and ATP formation. On the other hand, PARP activation results in inhibition of GAPDH by poly-ADP-ribosylation. These processes result in acute endothelial dysfunction in diabetic blood vessels, which importantly contributes to the development of various diabetic complications. Accordingly, hyperglycemia-induced activation of PKC and AGE formation are prevented by inhibition of PARP activity. Furthermore, inhibition of PARP protects against diabetic cardiovascular dysfunction in rodent models of cardiomyopathy, nephropathy, neuropathy, and retinopathy. PARP activation is also present in microvasculature of human diabetic subjects. The present review focuses on the role of PARP in diabetic complications and emphasizes the therapeutic potential of PARP inhibition in the prevention or reversal of diabetic complications.

The effects of drugs, ions, and poly-l-lysine on the excretory system of Schistosoma mansoni

We have been able to label the excretory system of cercariae and all forms of schistosomula, immature and adult worms with the highly fluorescent dye resorufin. We have shown that the accumulation of the resorufin into the excretory tubules and collecting ducts of the male adult worm depends on the presence of extracellular calcium and phosphate ions. In the adult male worms, praziquantel (PZQ) prevents this accumulation in RPMI medium and disperses resorufin from tubules which have been prelabelled. Female worms and all other developmental stages are much less affected either by the presence of calcium and phosphate ions, or the disruption caused by PZQ. The male can inhibit the excretory system in paired female. Fluorescent PZQ localises in the posterior gut (intestine) region of the male adult worm, but not in the excretory system, except for the anionic carboxy fluorescein derivative of PZQ, which may be excreted by this route. All stages of the parasite can recover from damage by PZQ treatment in vitro. The excretory system is highly sensitive to damage to the surface membrane and may be involved in vesicle movement and damage repair processes. In vivo the adult parasite does not recover from PZQ treatment, but what is inhibiting recovery is unknown, but likely to be related to immune effector molecules.

Molecular characterization of soybean cultivars by microsatellite markers with universal tail sequence

The objective of this work was to standardize a semiautomated method for genotyping soybean, based on universal tail sequence primers (UTSP), and to compare it with the conventional genotyping method that uses electrophoresis in polyacrylamide gels. Thirty soybean cultivars were genotypically characterized by both methods, using 13 microsatellite loci. For the UTSP method, the number of alleles (NA) was 50 (2-7 per marker) and the polymorphic information content (PIC) ranged from 0.40 to 0.74. For the conventional method, the NA was 38 (2-5 per marker) and the PIC varied from 0.39 to 0.67. The genetic dissimilarity matrices obtained by the two methods were highly correlated with each other (0.8026), and the formed groups were coherent with the phenotypic data used for varietal registration. The 13 markers allowed the distinction of all analyzed cultivars. The low cost of the UTSP method, associated with its high accuracy, makes it ideal for the characterization of soybean cultivars and for the determination of genetic purity.

Synthesis and NMR characterization of aliphatic-aromatic copolyesters by reaction of poly(ethylene terephthalate) post-consumer and poly(ethylene adipate)

An aliphatic-aromatic copolyester of poly(ethylene terephthalate), PET, and poly(ethylene adipate), PEA, PET-co-PEA, was synthesized by the high temperature melt reaction of post-consumer PET and PEA. As observed by NMR spectroscopy, the reaction yielded random copolyesters in a few minutes through ester-interchange reactions, even without added catalyst. The copolyesters obtained in the presence of a catalyst presented higher intrinsic viscosity than that obtained without the addition of catalyst, due to simultaneous polycondensation and ester-interchange reactions. The structure of the aliphatic-aromatic copolyesters obtained in different PET/PEA ratio is random as observed by NMR analysis.

Effect of the structure of commercial poly(ethylene oxide-b-propylene oxide) demulsifier bases on the demulsification of water-in-crude oil emulsions: elucidation of the demulsification mechanism

Water-in-crude oil emulsions are formed during petroleum production and asphaltenes play an important role in their stabilization. Demulsifiers are added to destabilize such emulsions,however the demulsification mechanism is not completely known. In this paper, the performances of commercial poly(ethylene oxide-b-propylene oxide) demulsifiers were studied using synthetic water-in-oil emulsions and model-systems (asphaltenes in organic solvent). No change in the asphaltene aggregate size induced by the demulsifier was observed. The demulsification performance decreased as the asphaltene aggregate size increased, so it can be suggested that the demulsification mechanism is correlated to the voids between the aggregates adsorbed on the water droplets surface.

Poly(ethylene-co-methyl acrylate)/poly(caprolactone) triol blends for drug delivery systems: characterization and drug release

Poly(ethylene-co-methyl acrylate) (EMA) and poly (caprolactone) triol (PCL-T) blends, a biodegradable aliphatic polyester with low molecular weight and moderate water solubility containing diltiazem hydrochloride (DZ) were studied in terms of the thermal and morphological properties, and drug release mechanism. An increase in the PCL-T content in the EMA/PCL-T/DZ films decreased the degree of DZ crystallinity. Drug release from these films is temperature-dependent, and it is possible to modify the drug release rate by adjusting the EMA/PCL-T composition of the blends. The mechanism of drug release is governed by PCL-T melting and PCL-T leaching from EMA matrix.

Poly(o-methoxyaniline) modified electrode for detection of lithium ions

This paper reports the use of an electrode modified with poly(o-methoxyaniline) for detecting lithium ions. These ions are present in drugs used for treating bipolar disorder and that requires periodical monitoring of the concentration of lithium in blood serum. Poly(o-methoxyaniline) was obtained electrochemically by cyclic voltammetry on the surface of a gold electrode. The results showed that the electrode modified with a conducting polymer responded to lithium ions in the concentration range of 1 x 10-5 to 1 x 10-4 mol L-1 . The results also confirmed that the performance of the modified electrode was comparable to that of the standard method (atomic emission spectrophotometry).

Characterization and in vitro release of cyclosporine-A from poly(D,L-lactide-co-glycolide implants obtained by solvent/extraction evaporation

Cyclosporine-A-loaded PLGA implants were developed intended for ocular route. Implants were prepared using solvent extraction/evaporation technique followed by casting of the cake into rods in a heated surface. XRD patterns showed that cyclosporine-A was completely incorporated into PLGA. FTIR and DSC results indicated alterations on drug molecular conformation aiming to reach the most stable thermodynamic conformation at polymer/drug interface. Implants provided controlled/sustained in vitro release of the drug. During the first 7 weeks, the drug release was controlled by the diffusion of the cyclosporine-A; and between 7-23 week period, the drug diffusion and degradation of PLGA controlled the drug release.

Application of ftir in the determination of acrylate content in poly(sodium acrylate-co-acrylamide) superabsorbent hydrogels

Hydrogels have been prepared by free-radical solution copolymerization of acrylamide and sodium acrylate (NaAc), with molar ratio ranging from 25/75 to 80/20, respectively, using methylene bisacrylamide as the crosslinking agent. A FTIR spectroscopy procedure to determine the acrylate/acrylamide ratio in these hydrogels was proposed based on absorbance at 1410 cm-1 (nCOO-) and 2940 cm-1 (nCH and nCH2). A straight line with a good linear correlation coefficient (0.998) was obtained by plotting the acrylate content (Ac%) versus relative absorbance (Arel = A1410/A2940). Results were confirmed by the amount of sodium cation released in acid medium determined by atomic absorption spectrometry.

Synthesis, characterization and application of ion imprinted poly(vinylimidazole) for zinc ion extraction/preconcentration with faas determination

In this paper, we describe the synthesis of an ion imprinted polymer (IIP) by homogeneous polymerization and its use in solid-phase to extract and preconcentrate zinc ions. Under optimal conditions (pH 5.0, preconcentration flow rate of 12.0 mL min-1, and eluted with 1.0 mol L-1 HNO3) this procedure allows the determination of zinc with an enrichment factor of 10.2, and with limits of detection and quantification of 1.5 and 5.0 µg L-1, respectively. The accuracy of our results was confirmed by analysis of tap water and certified reference materials: NIST 1570a (Spinach leaves) and NIST 1515 (Apple leaves).

Starch/poly (butylene adipate-co-terephthalate)/montmorillonite films produced by blow extrusion

This study aims to prepare biodegradable films from cassava starch, poly (butylene adipate-co-terephthalate) (PBAT), and montmorillonite (MMT) using blow-extrusion process and analyze the effects of different types and concentrations of MMT on the microstructure, physicochemical, and mechanical properties of the resulting films. The films were produced by blending 30% of PBAT with glycerol (17.5%), starch (49.0-52.5%), and four different types of montmorillonite (Cloisite® Na+, 10A, 15A, and 30B) at two different concentrations (1.75% and 3.5%). All the films prepared in this study showed an increase in the basal spacing of MMT layers. In particular, the films with 10A and 30B showed the highest increase in intercalation basal spacing, suggesting the formation of intercalated composites. The addition of nanoclays decreased the elongation of films. The addition of Cloisite® 10A resulted in films with the lowest WVP values and the highest stability to water adsorption under different RH conditions.