Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: Evaluation of reduced glutathione levels and matrix metalloproteinase secretion

Background and purpose: Calendula officinalis flowers have long been employed time in folk therapy, and more than 35 properties have been attributed to decoctions and tinctures from the flowers. The main uses are as remedies for burns (including sunburns), bruises and cutaneous and internal inflammatory diseases of several origins. The recommended doses are a function both of the type and severity of the condition to be treated and the individual condition of each patient. Therefore, the present study investigated the potential use of Calendula officinalis extract to prevent UV irradiation-induced oxidative stress in skin. Methods: Firstly, the physico-chemical composition of marigold extract(ME) (hydroalcoholic extract)was assessed and the in vitro antioxidant efficacy was determined using different methodologies. Secondly, the cytotoxicity was evaluated in L929 and HepG2 cells with the MTT assay. Finally, the in vivo protective effect of ME against UVB-induced oxidative stress in the skin of hairless mice was evaluated by determining reduced glutathione (GSH) levels and monitoring the secretion/activity of metalloproteinases. Results and conclusions: The polyphenol, flavonoid, rutin and narcissin contents found in ME were 28.6 mg/g, 18.8 mg/g, 1.6 mg/g and 12.2 mg/g, respectively and evaluation of the in vitro antioxidant activity demonstrated a dose-dependent effect of ME against different radicals. Cytoxicity experiments demonstrated that ME was not cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of 15 mg/mL However, concentrations greater than or equal to 30 mg/mL, toxic effects were observed. Finally, oral treatment of hairless mice with 150 and 300 mg/kg of ME maintained GSH levels close to non-irradiated control mice. In addition, this extract affects the activity/secretion of matrix metalloproteinases 2 and 9 (MMP-2 and -9) stimulated by exposure to UVB irradiation. However, additional studies are required to have a complete understanding of the protective effects of ME for skin. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Efficacy of Marigold Extract-Loaded Formulations Against UV-induced Oxidative Stress

The present study investigated the potential use of topical formulations containing marigold extract (ME) (Calendula officinalis extract) against ultraviolet (UV) B irradiation-induced skin damage. The physical and functional stabilities, as well as the skin penetration capacity, of the different topical formulations developed were evaluated. In addition, the in vivo capacity to prevent/treat the UVB irradiation-induced skin damage, in hairless mice, of the formulation with better skin penetration capacity was investigated. All of the formulations were physically and functionally stable. The gel formulation [Formulation 3 (F3)] was the most effective for the topical delivery of ME, which was detected as 0.21 mu g/cm(2) of narcissin and as 0.07 mu g/cm(2) of the rutin in the viable epidermis. This formulation was able to maintain glutathione reduced levels close to those of nonirradiated animals, but did not affect the gelatinase-9 and myeloperoxidase activities increased by exposure to UVB irradiation. In addition, F3 reduced the histological skin changes induced by UVB irradiation that appear as modifications of collagen fibrils. Therefore, the photoprotective effect in hairless mice achieved with the topical application of ME in gel formulation is most likely associated with a possible improvement in the collagen synthesis in the subepidermal connective tissue. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:2182-2193, 2011

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle. (C) 2008 Elsevier Ltd. All rights reserved.

Micro Far-Infrared Reflectivity of CaNb(2)O(6) Single Crystal Fibers Grown by the Laser-Heated Pedestal Growth Technique

CaNb(2)O(6) single crystal fibers were grown by the laser-heated pedestal growth technique, directly from the starting reagents. Optically transparent fibers were obtained in the form of rods with elliptical cross-section, free from cracks, impurities, and secondary phases, with an average diameter of 0.4 mm and about 20 mm of length. The fibers grew within the orthorhombic Pbcn columbite structure, with the growth axis nearly parallel to the crystallographic a-direction. The parameters b and c were parallel to the shorter and larger ellipsis axes. A special setup using a microscope was developed to obtain the far-infrared reflectivity spectra of these micrometer-sized fibers, allowing the identification and assignment of 34 of the 38 polar phonons foreseen for the material. From these phonons, the intrinsic dielectric constant ( = 18.2) and quality factor ( of 185 THz) could be estimated, showing the potential of the material for applications in microwave circuitry. These results, along with previous polarized Raman data (Cryst. Growth Des. 2010, 10, 1569), allow us to present a comprehensive set of optical phonon modes and to discuss the potential use of designed CaNb(2)O(6) microcrystals in compact optical devices.

Thermodynamic characterization of the palm tree Roystonea regia peroxidase stability

The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23 kcal per mol of monomer at 25 degrees C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N ->(k) D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25 degrees C is ca. 8 kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization. (c) 2008 Elsevier Masson SAS. All rights reserved.

Culture and exopolysaccharide production from sugarcane molasses by Gordonia polyisoprenivorans CCT 7137, isolated from contaminated groundwater in Brazil

Gordonia polyisoprenivorans CCT 7137 was isolated from groundwater contaminated with leachate in an old controlled landfill (Sauo Paulo, Brazil), and cultured in GYM medium at different concentrations of sugarcane molasses (2%, 6%, and 10%). The strain growth was analyzed by monitoring the viable cell counts (c.f.u. mL(-1)) and optical density and EPS production was evaluated at the end of the exponential phase and 24 h after it. The analysis of the viable cell counts showed that the medium that most favored bacterial growth was not the one that favored EPS production. The control medium (GYM) was the one that most favored the strain growth, at the maximum specific growth rate of 0.232 h(-1). Differences in bacterial growth when cultured at three different concentrations of molasses were not observed. Production of EPS, in all culture media used, began during the exponential phase and continued during the growth stationary phase. The highest total EPS production, after 24 h of stationary phase, was observed in 6% molasses medium (172.86 g L(-1)) and 10% (139.47 g L(-1)) and the specific total EPS production was higher in 10% molasses medium (39.03 x10(-11)g c.f.u.(-1)). After the exponential phase, in 2%, 6%, and 10% molasses media, a higher percentage of free exopolysaccharides (EPS) was observed, representing 88.4%, 62.4%, and 64.2% of the total, respectively. A different result was observed in pattern medium, which presented EPS made up of higher percentage of capsular EPS (66.4% of the total). This work is the first study on EPS production by G. polyisoprenivorans strain in GYM medium and in medium utilizing sugarcane molasses as the sole nutrient source and suggests its potential use for EPS production by G. polyisoprenivorans CCT 7137 aiming at application in biotechnological processes.

Inhibition of Mycobacterium tuberculosis tyrosine phosphatase PtpA by synthetic chalcones: Kinetics, molecular modeling, toxicity and effect on growth

Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, and it is estimated that one-third of the world`s population is infected with Mycobacterium tuberculosis. Among a series of tested compounds, we have recently identified five synthetic chalcones which inhibit the activity of M. tuberculosis protein tyrosine phosphatase A (PtpA), an enzyme associated with M. tuberculosis infectivity. Kinetic studies demonstrated that these compounds are reversible competitive inhibitors. In this work we also carried out the analysis of the molecular recognition of these inhibitors on their macromolecular target, PtpA, through molecular modeling. We observed that the predominant determinants responsible for the inhibitory activity of the chalcones are the positions of the two methoxyl groups at the A-ring, that establish hydrogen bonds with the amino acid residues Arg17, His49, and Thr12 in the active site of PtpA, and the substitution of the phenyl ring for a 2-naphthyl group as B-ring, that undergoes p stacking hydrophobic interaction with the Trp48 residue from PtpA. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics. The biological activity, synthetic versatility, and low cost are clear advantages of this new class of potential tuberculostatic agents. (C) 2010 Elsevier Ltd. All rights reserved.

Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts

Schistosoma mansoni is a well-adapted blood-dwelling parasitic helminth, persisting for decades in its human host despite being continually exposed to potential immune attack. Here, we describe in detail micro-exon genes (MEG) in S. mansoni, some present in multiple copies, which represent a novel molecular system for creating protein variation through the alternate splicing of short (<= 36 bp) symmetric exons organized in tandem. Analysis of three closely related copies of one MEG family allowed us to trace several evolutionary events and propose a mechanism for micro-exon generation and diversification. Microarray experiments show that the majority of MEGs are up-regulated in life cycle stages associated with establishment in the mammalian host after skin penetration. Sequencing of RT-PCR products allowed the description of several alternate splice forms of micro-exon genes, highlighting the potential use of these transcripts to generate a complex pool of protein variants. We obtained direct evidence for the existence of such pools by proteomic analysis of secretions from migrating schistosomula and mature eggs. Whole-mount in situ hybridization and immunolocalization showed that MEG transcripts and proteins were restricted to glands or epithelia exposed to the external environment. The ability of schistosomes to produce a complex pool of variant proteins aligns them with the other major groups of blood parasites, but using a completely different mechanism. We believe that our data open a new chapter in the study of immune evasion by schistosomes, and their ability to generate variant proteins could represent a significant obstacle to vaccine development.

1,4-Dioxane enhances properties and biocompatibility of polyanionic collagen for tissue engineering applications

Polyanionic collagen obtained from bovine pericardial tissue submitted to alkaline hydrolysis is an acellular matrix with strong potential in tissue engineering. However, increasing the carboxyl content reduces fibril formation and thermal stability compared to the native tissues. In the present work, we propose a chemical protocol based on the association of alkaline hydrolysis with 1,4-dioxane treatment to either attenuate or revert the drastic structural modifications promoted by alkaline treatments. For the characterization of the polyanionic membranes treated with 1,4-dioxane, we found that (1) scanning electron microscopy (SEM) shows a stronger reorientation and aggregation of collagen microfibrils; (2) histological evaluation reveals recovering of the alignment of collagen fibers and reassociation with elastic fibers; (3) differential scanning calorimetry (DSC) shows an increase in thermal stability; and (4) in biocompatibility assays there is a normal attachment, morphology and proliferation associated with high survival of the mouse fibroblast cell line NIH3T3 in reconstituted membranes, which behave as native membranes. Our conclusions reinforce the ability of 1,4-dioxane to enhance the properties of negatively charged polyanionic collagen associated with its potential use as biomaterials for grafting, cationic drug- or cell-delivery systems and for the coating of cardiovascular devices.

Chemiluminescence as a PDT light source for microbial control

The photodynamic therapy (PDT) is a combination of using a photosensitizer agent, light and oxygen that can cause oxidative cellular damage. This technique is applied in several cases, including for microbial control. The most extensively studied light sources for this purpose are lasers and LED-based systems. Few studies treat alternative light sources based PDT. Sources which present flexibility, portability and economic advantages are of great interest. In this study, we evaluated the in vitro feasibility for the use of chemiluminescence as a PDT light source to induce Staphylococcus aureus reduction. The Photogem (R) concentration varied from 0 to 75 mu g/ml and the illumination time varied from 60 min to 240 min. The long exposure time was necessary due to the low irradiance achieved with chemiluminescence reaction at mu W/cm(2) level. The results demonstrated an effective microbial reduction of around 98% for the highest photosensitizer concentration and light dose. These data suggest the potential use of chemiluminescence as a light source for PDT microbial control, with advantages in terms of flexibility, when compared with conventional sources. (C) 2011 Elsevier B.V. All rights reserved.

Sugarcane spirit extracts of oak and Brazilian woods: antioxidant capacity and activity

Sugarcane spirit extracts of six different Brazilian woods for potential use in manufacturing aging casks were compared with similar extracts of five oak samples from different geographic origin and heat treatment regarding: (1) content of phenolics and copper; (2) radical reducing capacity and reactivity toward 2,2-diphenyl-1-picrylhydrazyl radical (DPPH center dot); and (3) effect on the rate of oxygen depletion rate in a peroxidating lipid model system. Total phenolic contents of the Brazilian wood extracts ranged from 0.65 (canela-sassafras) to 6.4 (jatoba) mmol(GAE) L(-1) and from 1.39 to 2.87 mmol(GAE) L(-1) for oak extracts. Flavonoids ranged from 1.54 x 10(-4) (ipe) to 6.5 x 10(-2) (oak) mmol(rutin) L(-1), and tannins from below the detection limit to 0.22 (jatoba) mmol(tannic acid) L(-1). Correlation was observed for the antioxidant capacity versus phenolics/flavonoids/tannins content, where oak extracts exhibit the highest radical scavenging capacity compared to Brazilian woods. Rate constant for radical scavenging by the extracts ranged from 4.9 x 10(3) M(-1) s(-1)(canela-sassafras) to 9.7 x 10(4) M(-1) s(-1) (oak). The oxygen consumption index showed the Brazilian woods amendoim and jatoba to be more efficient inhibitors than the oak extracts for lipid autoxidation initiated by metmyoglobin, despite that the oak extracts seem to be more efficient to scavenge DPPH center dot. No simple correlation with phenolics or copper content could be established, and a prooxidative tendency was observed for the extracts of canela-sassafras, castanheira, and louro-canela.

Use of correlated potential harmonic basis functions for the description of the (4)He trimer and small clusters

A correlated many-body basis function is used to describe the (4)He trimer and small helium clusters ((4)HeN) with N = 4-9. A realistic helium dimer potential is adopted. The ground state results of the (4)He dimer and trimer are in close agreement with earlier findings. But no evidence is found for the existence of Efimov state in the trimer for the actual (4)He-(4)He interaction. However, decreasing the potential strength we calculate several excited states of the trimer which exhibit Efimov character. We also solve for excited state energies of these clusters which are in good agreement with Monte Carlo hyperspherical description. (C) 2011 American Institute of Physics. [doi:10.1063/1.3583365]

A spatial approach for the epidemiology of antibiotic use and resistance in community-based studies: the emergence of urban clusters of Escherichia coli quinolone resistance in Sao Paulo, Brasil

Background: Population antimicrobial use may influence resistance emergence. Resistance is an ecological phenomenon due to potential transmissibility. We investigated spatial and temporal patterns of ciprofloxacin (CIP) population consumption related to E. coli resistance emergence and dissemination in a major Brazilian city. A total of 4,372 urinary tract infection E. coli cases, with 723 CIP resistant, were identified in 2002 from two outpatient centres. Cases were address geocoded in a digital map. Raw CIP consumption data was transformed into usage density in DDDs by CIP selling points influence zones determination. A stochastic model coupled with a Geographical Information System was applied for relating resistance and usage density and for detecting city areas of high/low resistance risk. Results: E. coli CIP resistant cluster emergence was detected and significantly related to usage density at a level of 5 to 9 CIP DDDs. There were clustered hot-spots and a significant global spatial variation in the residual resistance risk after allowing for usage density. Conclusions: There were clustered hot-spots and a significant global spatial variation in the residual resistance risk after allowing for usage density. The usage density of 5-9 CIP DDDs per 1,000 inhabitants within the same influence zone was the resistance triggering level. This level led to E. coli resistance clustering, proving that individual resistance emergence and dissemination was affected by antimicrobial population consumption.

Preliminary biocompatibility investigation of magnetic albumin nanosphere designed as a potential versatile drug delivery system

Background: The magnetic albumin nanosphere (MAN), encapsulating maghemite nanoparticles, was designed as a magnetic drug delivery system (MDDS) able to perform a variety of biomedical applications. It is noteworthy that MAN was efficient in treating Ehrlich's tumors by the magnetohyperthermia procedure. Methods and materials: In this study, several nanotoxicity tests were systematically carried out in mice from 30 minutes until 30 days after MAN injection to investigate their biocompatibility status. Cytometry analysis, viability tests, micronucleus assay, and histological analysis were performed. Results: Cytometry analysis and viability tests revealed MAN promotes only slight and temporary alterations in the frequency of both leukocyte populations and viable peritoneal cells, respectively. Micronucleus assay showed absolutely no genotoxicity or cytotoxicity effects and histological analysis showed no alterations or even nanoparticle clusters in several investigated organs but, interestingly, revealed the presence of MAN clusters in the central nervous system (CNS). Conclusion: The results showed that MAN has desirable in vivo biocompatibility, presenting potential for use as a MDDS, especially in CNS disease therapy.

New Source of Muscle-Derived Stem Cells with Potential for Alveolar Bone Reconstruction in Cleft Lip and/or Palate Patients

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in non-immunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.