SacRALF1, a peptide signal from the grass sugarcane (Saccharum spp.), is potentially involved in the regulation of tissue expansion

Rapid alkalinization factor (RALF) is part of a growing family of small peptides with hormone characteristics in plants. Initially isolated from leaves of tobacco plants, RALF peptides can be found throughout the plant kingdom and they are expressed ubiquitously in plants. We took advantage of the small gene family size of RALF genes in sugarcane and the ordered cellular growth of the grass sugarcane leaves to gain information about the function of RALF peptides in plants. Here we report the isolation of two RALF peptides from leaves of sugarcane plants using the alkalinization assay. SacRALF1 was the most abundant and, when added to culture media, inhibited growth of microcalli derived from cell suspension cultures at concentrations as low as 0.1 mu M. Microcalli exposed to exogenous SacRALF1 for 5 days showed a reduced number of elongated cells. Only four copies of SacRALF genes were found in sugarcane plants. All four SacRALF genes are highly expressed in young and expanding leaves and show a low or undetectable level of expression in expanded leaves. In half-emerged leaf blades, SacRALF transcripts were found at high levels at the basal portion of the leaf and at low levels at the apical portion. Gene expression analyzes localize SacRALF genes in elongation zones of roots and leaves. Mature leaves, which are devoid of expanding cells, do not show considerable expression of SacRALF genes. Our findings are consistent with SacRALF genes playing a role in plant development potentially regulating tissue expansion.

Hydrolysis Influence on Phytochemical Composition, Antioxidant Activity, Plasma Concentration, and Tissue Distribution of Hydroethanolic Ilex paraguariensis Extract Components

The infusion of aerial parts of Ilex paraguariensis is widely consumed. Its antioxidant activity suggests an important role of this plant in the treatment/prevention of oxidative stress related diseases. Plant extract active compounds are frequently found in esterified form that may be poorly absorbed. Hydrolysis of the extract is a possible approach to increase its bioavailability. The aim of this study was to perform a phytochemical analysis and evaluate in rats the plasma concentration and tissue distribution of antioxidant compounds in the hydroethanolic extract of Ilex paraguariensis, before and after enzymatic hydrolysis. Both extracts presented high antioxidant activity and phenolic content. Rats given single or repeated doses of the hydrolyzed extract showed increased plasma antioxidant activity and higher plasma levels of caffeic acid. However, no changes of endogenous antioxidants were observed. In conclusion, hydrolysis of the extract of Ilex paraguariensis is a strategy to improve its bioavailability and in vivo antioxidant activity.

Cytotoxicity and Endothelial Cell Adhesion of Lyophilized and Irradiated Bovine Pericardium Modified With Silk Fibroin and Chitosan

Grafts of biological tissues have been used since the 1960s as an alternative to the mechanical heart prostheses. Nowadays, the most consolidated treatment to bovine pericardial (BP) bioprostheses is the crosslinking with glutaraldehyde (GA), although GA may induce calcification in vivo. In previous work, our group demonstrated that electron beam irradiation applied to lyophilized BP in the absence of oxygen promoted crosslinks among collagen fibers of BP tissue. In this work, the incorporation of silk fibroin (SF) and chitosan (CHIT) in the BP not treated with GA was studied. The samples were irradiated and then analyzed for their cytotoxicity and the ability of adhesion and growth of endothelial cells. Initially, all samples showed cytotoxicity. However, after a few washing cycles, the cytotoxicity due to acetic acid and ethanol residues was removed from the biomaterial making it suitable for the biofunctional test. The samples modified with SF/CHIT and electron beam irradiated favored the adhesion and growth of endothelial cells throughout the tissue.

Care during freeze-drying of bovine pericardium tissue to be used as a biomaterial: A comparative study

Bovine pericardium (BP) tissue is widely used in the manufacture of bioprosthetics. The effects of freeze-drying on the BP tissue have been studied by some researchers in order to decrease their cytotoxicity due to preservation in formaldehyde solution, and to increase the lifetime of the product in storage. This study was undertaken in order to study the effect of freeze-drying in the structure of BP. To perform this study BP samples were freeze-dried in two different types of freeze-dryers available in our laboratory: a laboratory freeze-dryer, in which it was not possible to control parameters and a pilot freeze-dryer, wherein all parameters during freezing and drying were controlled. After freeze-drying processes, samples were analyzed by SEM, Raman spectroscopy, tensile strength, water uptake tests and TEM. In summary, it has been demonstrated that damages occur in collagen fibers by the loss of bulk water of collagen structure implicating in a drastic decreasing of BP mechanical properties due to its structural alterations. Moreover, it was proven that the collagen fibrils suffered breakage at some points, which can be attributed to the uncontrolled parameters during drying. (C) 2011 Elsevier Inc. All rights reserved.

A New Method to Prepare Porous Silk Fibroin Membranes Suitable for Tissue Scaffolding Applications

A new method to prepare porous silk fibroin (SF) membranes without dialysis proposed. Silk fibers were degummed to remove sericin and the resultant fibroin was dissolved in a CaCl(2)-CH(3)CH(2)OH-H(2)O ternary solvent. Rather than undergoing dialysis, a fibroin salty solution was diluted in water and then submitted to a mechanical agitation that led to a phase separation through foam formation on the solution surface. This foam was continually collected and then compacted between plates to remove the excess of water. The membranes presented large pores with diameters of greater than 100 pm (as shown by scanning electron microscopy - SEM), porosity of 68% and water content of 91% w/w. X-ray diffraction (XRD) and infrared spectroscopy (FTIR-ATR) indicated that the membranes present SF in a beta-sheet structure even before the ethanol treatment. A typical elastic deformation profile and degradation under temperature were observed using calorimetric analysis (DSC), thermal gravimetric analysis (TGA) and mechanical tests. As indicated by the in vitro cytotoxicity tests, these membranes present potential for use as scaffolds. (C) 2009 Wiley Periodicals, Inc. J Appl Polym Sci 114: 617-623, 2009

Prospects in Lyophilization of Bovine Pericardium

Almost 30 years after the introduction of heart valve prostheses patients worldwide are benefiting from the implant of these devices. Among the various types of heart valves, the ones made of treated bovine pericardium have become a frequently used replacement of the heart`s native valve. Lyophilization, also known as freeze-drying, is an extremely useful technique for tissue storage for surgical applications. This article gives a brief overview on the current bovine pericardium lyophilization development, including the chemical modification to improve physical-chemical characteristics and the advanced technologies used to guarantee a high-quality product. It was shown that lyophilization process can be successfully applied as a method of bovine pericardium preservation and also as a technological tool to prepare new materials obtained by chemical modification of native tissues.

Application of hydrophobic resin adhesives to acid-etched dentin with an alternative wet bonding technique

Hydrophilic dentin adhesives are prone to water sorption that adversely affects the durability of resin-dentin bonds. This study examined the feasibility of bonding to dentin with hydrophobic resins via the adaptation of electron microscopy tissue processing techniques. Hydrophobic primers were prepared by diluting 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane/triethyleneglycol dimethacrylate resins with known ethanol concentrations. They were applied to acid-etched moist dentin using an ethanol wet bonding technique that involved: (1) stepwise replacement of water with a series of increasing ethanol concentrations to prevent the demineralized collagen matrix from collapsing; (2) stepwise replacement of the ethanol with different concentrations of hydrophobic primers and subsequently with neat hydrophobic resin. Using the ethanol wet bonding technique, the experimental primer versions with 40, 50, and 75% resin exhibited tensile strengths which were not significantly different from commercially available hydrophilic three-step adhesives that were bonded with water wet bonding technique. The concept of ethanol wet bonding may be explained in terms of solubility parameter theory. This technique is sensitive to water contamination, as depicted by the lower tensile strength results from partial dehydration protocols. The technique has to be further improved by incorporating elements of dentin permeability reduction to avoid water from dentinal tubules contaminating water-free resin blends during bonding. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res 84A: 19-29, 2008.

Ruthenium-nitrite complex as pro-drug releases NO in a tissue and enzyme-dependent way

Nitric oxide (NO) plays an important role in the control of the vascular tone and the most often employed NO donors have limitations due to their harmful side-effects. In this context, new NO donors have been prepared, in order to minimize such undesirable effects. cis-[Ru(bpy)(2)(py)NO(2)](PF(6)) (RuBPY) is a new nitrite complex synthesized in our laboratory that releases NO in the presence of the vascular tissue only. In this work the vasorelaxation induced by this NO donor has been studied and compared to that obtained with the well known NO donor SNP. The relaxation induced by RuBPY is concentration-dependent in denuded rat aortas pre-contracted with phenylephrine (EC(50)). This new compound induced relaxation with efficacy similar to that of SNP, although its potency is lower. The time elapsed until maximum relaxation is achieved (E(max) = 240 s) is similar to measured for SNP (210 s). Vascular reactivity experiments demonstrated that aortic relaxation by RuBPY is inhibited by the soluble guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiozolo[4,3-a]quinoxaline-1-one (ODQ 1 mu M). In a similar way, 1 mu M ODQ also reduces NO release from the complex as measured with DAF-2 DA by confocal microscopy. These findings suggest that this new complex RuBPY that has nitrite in its structure releases NO inside the vascular smooth muscle cell. This ruthenium complex releases significant amounts of NO only in the presence of the aortic tissue. Reduction of nitrite to NO is most probably dependent on the soluble guanylyl-cyclase enzyme, since NO release is inhibited by ODQ. (C) 2011 Elsevier Inc. All rights reserved.

Preparation and characterization of D, L-PLA loaded 17-beta-Estradiol valerate by emulsion/evaporation methods

PLA microparticles containing 17-beta-estradiol valerate were prepared by an emulsion/evaporation method in order to sustain drug release. This system was characterized concerning particle size, particle morphology and the influence of formulation and processing parameters on drug encapsulation and in vitro drug release. The biodegradation of the microparticles was observed by tissue histological analysis. Scanning electron microscopy and particle size analysis showed that the microparticles were spherical, presenting non-aggregated homogeneous surface and had diameters in the range of 718-880 nm (inert microparticles) and 3-4 mu m (drug loaded microparticles). The encapsulation efficiency was similar to 80%. Hormone released from microparticles was sustained. An in vivo degradation experiment confirmed that microparticles are biodegradable. The preparation method was shown to be suitable, since the morphological characteristics and efficiency yield were satisfactory. Thus, the method of developed microparticles seems to be a promising system for sustained release of 17-beta-estradiol.

Reduction of parasitism tissue by treatment of mice chronically infected with Trypanosoma cruzi with lignano lactones

The reduction of parasitism tissue upon treatment with two lignano lactones, namely (-)- cubebin (CUB) and (-)-hinokinin (HNK), was evaluated in the chronic phase of Chagas` disease by quantifying the enzyme beta-galactosidase expressed by the CL B5 clone strain of Trypanosoma cruzi. Tissue karyometry was also performed. Treatment with the assessed lignans led to a larger reduction in parasitism tissue in all evaluated organs, compared with benznidazole (BZN). Oral treatment with CUB or HNK was more effective. Karyometry results demonstrated that the infected control animals had increased nuclear area compared with uninfected controls, indicating cellular hypertrophy. Results also revealed that use of CUB or HNK was able to significantly prevent this increase, and a slight decrease in the nuclear area was observed, compared with mice treated with BZN. Taken together, these data demonstrate that CUB and HNK could be considered as potential compounds for the development of new drugs for treatment of Chagas` disease.

Topical Delivery of Lycopene Using Microemulsions: Enhanced Skin Penetration and Tissue Antioxidant Activity

Topical delivery of lycopene is a convenient way to supplement cutaneous levels of antioxidants. In this study, lycopene was incorporated (0.05%, w/w) in two microemulsions containing BRIJ-propylene glycol (2:1, w/w, surfactant blend) but different oil phases: mono/diglycerides of capric and caprylic acids (MG) or triglycerides of the same fatty acids (TG). Microemulsions containing MG and TG were isotropic, fluid, and clear, with internal phase diameters of 27 and 52 nm, respectively. Both MG- or TG-containing microemulsions markedly increased lycopene penetration in the stratum corneum, (6- and 3.6-fold, respectively) and in viable layers of porcine ear skin 2 (from undetected to 172.6 +/- 41.1 and 103.1 +/- 7.2 ng/cm(2), respectively) compared to a control solution. To assure that lycopene delivered to the skin was active, the antioxidant activity of skin treated with MG-containing microemulsion was determined by CUPRAC assay, and found to be 10-fold higher than untreated skin. The cytotoxicity of MG-containing microemulsion in cultured fibroblasts was similar to propylene glycol (considered safe) and significantly less than of sodium lauryl sulfate (a moderate-to-severe irritant) at 1-50 mu g/mL. These results demonstrate that the MG-containing microemulsion is an efficient and safe system to increase lycopene delivery to the skin and the antioxidant activity in the tissue. (C) 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1346-1357, 2010

New method for quantification of Trypanosoma cruzi in animal`s tissue in the chronic phase of experimental Chagas` disease

We developed a new method for the quantification of parasites in tissue. Trypanosoma cruzi strain CL parasites were genetically engineered to express the Escherichia coli beta-galactosidase gene, lacZ and this enzyme is able to catalyze a colorimetric reaction with chlorophenol red beta-d galactopyranoside (CPRG) as the substrate. The animals were infected with clone CL Brener strain B5 of T. cruzi and treated with benznidazole in order to verify the reduction in the number of parasites in tissue study by quantifying the enzyme beta-galactosidase. The assay demonstrates a reduction in the number of parasites in the groups treated. Thus, this test can be used to test other substances with the aim of verifying the effectiveness in the chronic phase of experimental Chagas` disease.

HPLC-FLD methods to quantify chloroaluminum phthalocyanine in nanoparticles, plasma and tissue: application in pharmacokinetic and biodistribution studies

Analytical and bioanalytical methods of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) were developed and validated for the determination of chloroaluminum phthalocyanine in different formulations of polymeric nanocapsules, plasma and livers of mice. Plasma and homogenized liver samples were extracted with ethyl acetate, and zinc phthalocyanine was used as internal standard. The results indicated that the methods were linear and selective for all matrices studied. Analysis of accuracy and precision showed adequate values, with variations lower than 10% in biological samples and lower than 2% in analytical samples. The recoveries were as high as 96% and 99% in the plasma and livers, respectively. The quantification limit of the analytical method was 1.12 ng/ml, and the limits of quantification of the bioanalytical method were 15 ng/ml and 75 ng/g for plasma and liver samples, respectively. The bioanalytical method developed was sensitive in the ranges of 15-100 ng/ml in plasma and 75-500 ng/g in liver samples and was applied to studies of biodistribution and pharmacokinetics of AlClPc. (C) 2011 Elsevier B.V. All rights reserved.

Preliminary study of human breast tissue using synchrotron radiation combining WAXS and SAXS techniques

Using synchrotron radiation, we combined simultaneously wide angle X-ray scattering (WAXS) and small angle X-ray scattering (SAXS) techniques to obtain the scattering profiles of normal and neoplastic breast tissu-es samples at the momentum transfer range 6.28 nm(-1) <= Q(=4 pi.sin(theta/2)lambda) <= 50.26 nm(-1) and 0.15 nm(-1) <= Q <= 1.90 nm(-1), respectively. The results obtained show considerable differences between the scattering profiles of these tissues. We verified that the combination of some parameters (ratio between glandular and adipose peak intensity and third-order axial peak intensity) extracted from scattering profiles can be used for identifying breast cancer. (c) 2009 Elsevier Ltd. All rights reserved.

Effect of the immunosuppressants on hepatocyte proliferation and apoptosis in a young animal model of liver regeneration: An immunohistochemical study using tissue microarrays

Hepatocyte proliferation and apoptosis (programmed cell death) occur during the liver parenchyma regeneration and the liver size modeling is mainly controlled by hepatocyte apoptosis. The purpose of the present study was to verify the influence of immunosuppressant drugs on these phenomena by utilizing tissue microarray techniques. Thirty-six weaning rats (age 21-23 days, weight 30-50 g) were divided into six groups: control, sham, hepatectomy, hepatectomy plus solumedrol, hepatectomy plus CsA, and hepatectomy plus Tac. The animals were killed one day after hepatectomy, and the remnant livers were weighed and harvested for tissue microarray sections. Liver cell proliferation was evaluated by staining for PCNA and apoptosis was detected by the TUNEL method. It was verified that CsA promoted a decrease in the liver weight, Tac and CsA decreased the proliferation index of hepatocytes, and glucocorticoid had no significant effects. The apoptosis index was not altered by hepatectomy or immunosuppressants. Our data indicate that, in the growing rat, CsA and Tac have negative effects on hepatocyte proliferation and have no effect on the hepatocyte apoptosis.