Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) with a broad range of 3HV content at high yields by Burkholderia sacchari IPT 189

The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from sucrose and propionic acid by Burkholderia sacchari IPT 189 was studied using a two-stage bioreactor process. In the first stage, this bacterium was cultivated in a balanced culture medium until sucrose exhaustion. In the second stage, a solution containing sucrose and propionic acid as carbon source was fed to the bioreactor at various sucrose/propionic acid (s/p) ratios at a constant specific flow rate. Copolymers with 3HV content ranging from 40 down to 6.5 (mol%) were obtained with 3HV yield from propionic acid (Y-3HV/prop) increasing from 1.10 to 1.34 g g(-1). Copolymer productivity of 1 g l(-1) h(-1) was obtained with polymer biomass content rising up to 60% by increasing a specific flow rate at a constant s/p ratio. Increasing values of 3HV content were obtained by varying the s/p ratios. A simulation of production costs considering Y-3HV/prop obtained in the present work indicated that a reduction of up to 73% can be reached, approximating US$ 1.00 per kg which is closer to the value to produce P3HB from sucrose (US$ 0.75 per kg).

Poly(lactic acid-glycolic acid) nanoparticles markedly improve immunological protection provided by peptide P10 against murine paracoccidioidomycosis

Background and purpose: The present study reports on the preparation and testing of a sustained delivery system for the immunomodulatory peptide P10 aimed at reducing the in vivo degradation of the peptide and the amount required to elicit a protective immune response against paracoccidioidomycosis. Experimental approach: BALB/c mice were infected with the yeast Paracoccidioides brasiliensis to mimic the chronic form of paracoccidioidomycosis. The animals were treated daily with sulfamethoxazole/trimethoprim alone or combined with peptide P10, either emulsified in Freund`s adjuvant or entrapped in poly(lactic acid-glycolic acid) (PLGA) nanoparticles at different concentrations (1 mu g, 5 mu g, 10 mu g, 20 mu g or 40 mu g center dot 50 mu L-1). Therapeutic efficacy was assessed as fungal burden in tissues and the immune response by quantitative determination of cytokines. Key results: Animals given combined chemotherapy and P10 nanotherapy presented a marked reduction of fungal load in the lungs, compared with the non-treated animals. After 30 days of treatment, P10 entrapped within PLGA (1 mu g center dot 50 mu L-1) was more effective than `free` P10 emulsified in Freund`s adjuvant (20 mu g center dot 50 mu L-1), as an adjuvant to chemotherapy. After treatment for 90 days, the higher doses of P10 entrapped within PLGA (5 or 10 mu g center dot 50 mu L-1) were most effective. Treatment with P10 emulsified in Freund`s adjuvant (20 mu g center dot 50 mu L-1) or P10 entrapped within PLGA (1 mu g center dot 50 mu L-1) were accompanied by high levels of interferon-gamma in lung. Conclusions and implications: Combination of sulfamethoxazole/trimethoprim with the P10 peptide entrapped within PLGA demonstrated increased therapeutic efficacy against paracoccidioidomycosis. P10 incorporation into PLGA nanoparticles dramatically reduced the peptide amount necessary to elicit a protective effect.

Disruption of the 2-methylcitric acid cycle and evaluation of poly-3-hydroxybutyrate-co-3-hydroxyvalerate biosynthesis suggest alternate catabolic pathways of propionate in Burkholderia sacchari

The objective of the present work was to evaluate the relevance of the 2-methylcitric acid cycle (2MCC) to the catabolism of propionate in Burkholderia sacchari. Two B. sacchari mutants unable to grow on propionate were obtained: one disrupted in acnM, and the other in acnM and prpC deleted. An operative 2MCC significantly reduces the bacterial ability to incorporate 3-hydroxyvalerate (3HV) into a biodegradable copolyester accumulated from carbohydrates plus propionate. The efficiency of the mutants in converting propionate to 3HV units (Y(3HV/prp)) increased from 0.09 g.g(-1) to 0.81-0.96 g.g(-1), indicating that acnM and prpC are both essential for growth on propionate. None of the mutations resulted in achievement of the maximum theoretical Y(3HV/prp) (1.35 g.g(-1)). When increasing concentrations of propionate were supplied, decreasing values of Y(3HV/prp) were observed. The results obtained corroborate the hypothesis of the presence of other propionate catabolic pathways in B. sacchari. The 2MCC would be the more operative pathway, but a second pathway, which remains to be elucidated, would assume more importance under propionate concentrations of 1 g.L(-1) or higher. The efficiency in converting propionate to 3HV units can be improved by decreasing the propionate concentrations, owing to the role of the 2MCC.

Sialic Acid Residues Are Essential for the Anaphylactic Activity of Murine IgG1 Antibodies

Glycosylation of the Ab molecule is essential for maintaining the functional structure of Fc region and consequently for Ab-mediated effector functions, such as binding to cells or complement system activation. Alterations in the composition of the sugar moiety can dramatically influence Ab activity; however, it is not completely clear how differences in the N-linked oligosaccharide structure impact the biological function of Abs. We have described that murine IgG1 Abs can be separated according to their ability to elicit in vivo anaphylaxis in a fraction of anaphylactic and other of non-anaphylactic molecules. Furthermore, we showed that the N-linked oligosaccharide chain is essential for the structural conformation of the anaphylactic IgG1, the binding to Fc gamma RIII on mast cells, and, consequently, for the ability to mediate anaphylactic reactions. In this study, we evaluated the contribution of individual sugar residues to this biological function. Differences in the glycan composition were observed when we analyzed oligosaccharide chains from anaphylactic or non-anaphylactic IgG1, mainly the presence of more sialic acid and fucose residues in anaphylactic molecules. Interestingly, the enzymatic removal of terminal sialic acid residues in anaphylactic IgG1 resulted in loss of the ability to trigger mast cell degranulation and in vivo anaphylactic reaction, similarly to the deglycosylated IgG1 Ab. In contrast, fucose removal did not affect the anaphylactic function. Therefore, we demonstrated that the ability of murine IgG1 Abs to mediate anaphylaxis is directly dependent on the amount of sialic acid residues associated to the oligosaccharide chain attached to the Fc region of these molecules. The Journal of Immunology, 2008, 181: 8308-8314.

Retinoic acid inhibits dendritic cell differentiation driven by interleukin-4

All-trans-retinoic acid (atRA) appears to affect Th1-Th2 differentiation and its effects on immune responses might also be mediated by dendritic cell (DC). Nonetheless, studies have been showing contradictory results since was observed either induction or inhibition of DC differentiation. Our aim was to investigate atRA action on human monocyte derived DC differentiation. For this purpose we tested pharmacological and physiological doses of atRA with or without cytokines. Cell phenotypes were analyzed by flow cytometry and function was investigated by phagocytosis and respiratory burst. DC, positive control group, was differentiated with GM-CSF and IL-4 and maturated with TNF-alpha. We demonstrated that atRA effects depend on the dose used as pharmacological doses inhibited expression of all phenotypic markers tested while a physiological dose caused cell differentiation. However, atRA combined or not with cytokines did not promote DC differentiation. In fact, atRA was detrimental on IL-4 property as a DC inductor. (C) 2009 Elsevier Inc. All rights reserved.

Gamma-linolenic Acid Alters Ku80, E2F1, and Bax Expression and Induces Micronucleus Formation in C6 Glioma Cells In Vitro

Gamma-linolenic acid (GLA) is an inhibitor of tumor cell proliferation in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of 150 mu M GLA on the expression of E2F1, cyclin D1, bax, bcl2, Ku70, and Ku80 in C6 rat glioma cells. The Ku proteins were chosen as previous studies have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation. The fact that GLA exposure is known to enhance the efficacy of radiation treatment raised the question whether the Ku proteins could be involved in this effect as seen for other molecules such as roscovitine and flavopiridol. GLA altered the mRNA expression of E2F1, cyclin D1, and bax, but no changes were found for bcl2, Ku70, and Ku80. Alterations in protein expression were observed for bax, Ku80, and E2F1. The 45% decrease in E2F1 expression was proportional to decreased cell proliferation (44%). Morphological analysis found a 25% decrease in mitotic activity in the GLA-treated cells, which was accompanied by a 49% decrease in S-phase by FACS analysis. A 39% increase in the number of micronuclei detected by Hoechst fluorescence points to GLA`s effects on cell division even at concentrations that do not produce significant increases in apoptosis. Most important was the finding that Ku80 expression, a critical protein involved in DNA repair as a heterodimer with Ku70, was decreased by 71%. It is probable that reduced Ku80 is responsible for the increase in micronucleus formation in GLA-treated cells in a similar manner to that found in Ku80 null cells exposed to radiation. The decreased expression of Ku80 and E2F1 could make cells more susceptible to radiotherapy and chemotherapy. (C) 2009 IUBMB

Strategies to Optimize Biosensors Based on Impedance Spectroscopy to Detect Phytic Acid Using Layer-by-Layer Films

Impedance spectroscopy has been proven a powerful tool for reaching high sensitivity in sensor arrays made with nanostructured films in the so-called electronic tongue systems, whose distinguishing ability may be enhanced with sensing units capable of molecular recognition. In this study we show that for optimized sensors and bio-sensors the dielectric relaxation processes involved in impedance measurements should also be considered, in addition to an adequate choice of sensing materials. We used sensing units made from layer-by-layer (LbL) films with alternating layers of the polyeletrolytes, poly(allylamine) hydrochloride (PAH) and poly(vinyl sulfonate) (PVS), or LbL films of PAH alternated with layers of the enzyme phytase, all adsorbed on gold interdigitate electrodes. Surprisingly, the detection of phytic acid was as effective in the PVS/PAH sensing system as with the PAH/phytase system, in spite of the specific interactions of the latter. This was attributed to the dependence of the relaxation processes on nonspecific interactions such as electrostatic cross-linking and possibly on the distinct film architecture as the phytase layers were found to grow as columns on the LbL film, in contrast to the molecularly thin PAH/PVS films. Using projection techniques, we were able to detect phytic acid at the micromolar level with either of the sensing units in a data analysis procedure that allows for further optimization.

Adsorption of Pb(2+), Cu(2+) and Cd(2+) in FDU-1 silica and FDU-1 silica modified with humic acid

Ordered mesoporous silica with cubic structure, type FDU-1, was synthesized under strong acid media using B-50-6600 poly(ethylene oxide)-poly(butilene oxide)-poly(ethylene oxide) triblock copolymer (EO(39)BO(47)EO(39)) and tetraethyl orthosilicate (TEOS). Humic acid (HA) was modified to the synthesis process at a concentration of 1.5 mmol per gram of SiO(2). Thermogravimetry, small angle X-ray diffraction, nitrogen adsorption and high resolution transmission electron microscopy were used to characterize the samples. The pristine FDU-1 and FDU-1 with incorporated 1.5 mmol of HA were tested for adsorption of Pb(2+), Cu(2+) and Cd(2+) in aqueous solution. Incorporation of humic acid into the FDU-1 silica afforded an adsorbent with strong affinity for Cd(2+), Cu(2+) and Pb(2+) from single ion solutions. Adsorption of Cu(2+) was significantly enhanced after incorporation of humic acid, a fact that can be explained by the formation of complexes with carboxylic and phenolic groups at low concentrations of the metal cation. The results demonstrated the potential applicability of FDU-1 with incorporated HA in the removal of low concentrations of heavy metal cations from aqueous solution, such as wastewaters, after usual precipitation of metal hydroxides in alkaline medium and proper pH conditioning in the range between 6 and 7. (C) 2007 Elsevier Inc. All rights reserved.

Continuum, Discrete, and Explicit Solvation Models for Describing the Low-Lying Absorption Spectrum of the Pterin Acid in Aqueous Environment

The absorption spectrum of the acid form of pterin in water was investigated theoretically. Different procedures using continuum, discrete, and explicit models were used to include the solvation effect on the absorption spectrum, characterized by two bands. The discrete and explicit models used Monte Carlo simulation to generate the liquid structure and time-dependent density functional theory (B3LYP/6-31G+(d)) to obtain the excitation energies. The discrete model failed to give the correct qualitative effect on the second absorption band. The continuum model, in turn, has given a correct qualitative picture and a semiquantitative description. The explicit use of 29 solvent molecules, forming a hydration shell of 6 angstrom, embedded in the electrostatic field of the remaining solvent molecules, gives absorption transitions at 3.67 and 4.59 eV in excellent agreement with the S(0)-S(1) and S(0)-S(2) absorption bands at of 3.66 and 4.59 eV, respectively, that characterize the experimental spectrum of pterin in water environment. (C) 2010 Wiley Periodicals, Inc. Int J Quantum Chem 110: 2371-2377, 2010

Electronic transport properties of ascorbic acid and nicotinamide adsorbed on single-walled carbon nanotubes

Ab initio simulations of carbon nanotubes interacting with ascorbic acid and nicotinamide are reported. The electronic transport properties of these systems are studied using a combination of density functional theory and non-equilibrium Green`s functions methods. The adsorptions of both molecules are observed to depend strongly on their functionalization. The interaction through the appropriate functionalized species modifies the structural and electronic properties of the original system, resulting in a chemisorption regime. Changes in the electronic transport properties are also observed, with reductions on the total electronic transmission probabilities. Nevertheless, when the molecules interact through the pristine form, a physisorption interaction is observed with insignificant structural and electronic transport changes. (c) 2011 Elsevier B.V. All rights reserved.

Long-term follow-up of topical 5-aminolaevulinic acid photodynamic therapy diode laser single session for non-melanoma skin cancer

Photodynamic therapy (PDT) is based on the association of a light source and tight sensitive agents in order to cause the selective death of tumor cells. To evaluate topical 5-aminolaevulinic acid (5-ALA) and diode laser photodynamic single session therapy single session for non-melanoma skin cancer (NMSC), a long-term follow-up was performed. Nineteen Bowen`s disease (BD) and 15 basal cell. carcinoma (BCC) lesions were submitted to 6-h topical and occlusive 20% 5-ALA plus DMSO and EDTA, and later were exposed to 630 nm diode laser, 100 or 300 J cm(-2) dose. At 3 months tumor-free rate was 91.2% (31/34) whereas at 60 months, 57.7% (15/26), slightly higher in BCC (63.6%; 7/11). The relation between the reduction of the clinical response and the increase of tumor dimension observed at 18 months was lost at 60 months. The sBCC recurrence was earlier compared to the nBCC one. ALA-PDT offered important advantages: it is minimally invasive, an option for patients under risk of surgical complications; clinical feasibility; treatment of multiple lesions in only one session or lesions in poor heating sites and superior esthetical results. However, the recurrence rate increase after ALA-PDT diode laser single session can be observed at tong-term follow-up, and the repetitive sessions, an additional. advantage of the method, is strongly recommended. The clinical response and recurrence time seem to be related to the laser light dose and NMSC types/sub-types, thickness and dimension, which must be considered for the choice of the ALA-PDT. (C) 2009 Elsevier B.V. All rights reserved.

From Rational Design of Drug Crystals to Understanding of Nucleic Acid Structures: Lamivudine Duplex

A DNA-like duplex of nucleosides is probable to exist even without the 5`-phosphate groups needed to assemble the chain backbone. However, double-stranded helical structures of nucleosides are unknown. Here, we report a duplex of nucleoside analogs that is spontaneously assembled due to stacking of the neutral and protonated molecules of lamivudine, a nucleoside reverse transcriptase inhibitor (NTRI) widely used in anti-HIV drug combinatory medication. The left-handed lamivudine duplex has features similar to those of i-motif DNA, as the face-to-face base stacking and the helix rise per base pair. Furthermore, the protonation pattern on alternate bases expected for it DNA-like duplex stabilized by pairing of neutral and protonated cytosine fragments was observed for the first time in the lamivudine double-stranded helix. This structure demonstrates that hydrogen bonds can substitute for covalent phosphodiester linkage in the stabilization of the duplex backbone. This interesting example of spontaneous molecular self-organization indicates that the 5`-phosphate group could not be a requirement for duplex assembly.

Synthesis and structural characterization of a new polymeric zinc(II) complex with thiophene-2-carboxylic acid

A new polymeric zinc(II) complex with thiophene-2-carboxylic acid (-tpc) of composition [Zn2(C20H12O8S4)]n was obtained and structurally characterized by X-ray diffraction, thermal analysis, nuclear magnetic resonance (NMR), and infrared spectroscopies. Upfield shift in the 1H-NMR spectrum is explained by the crystalline structure, which shows the thiophene rings overlapping each other in parallel pairs. The compound crystallizes in the monoclinic system, space group P21/c, with a = 9.7074(4) angstrom, b = 13.5227(3) angstrom, c = 18.9735(7) angstrom, = 95.797(10)degrees, and Z = 4. Three -tpc groups bridge between two Zn(II) ions through oxygens and the fourth one bridges between one of these ions and the third one, symmetry related by a twofold screw axis. This arrangement gives rise to infinite chains along the crystallographic a direction. The metal atoms display an approximate tetrahedral configuration. The complex is insoluble in water, ethanol, and acetone, but soluble in dimethyl sulfoxide.

Synthesis, characterization, crystal structure, and biological studies of vanadium complexes with glycolic acid

Synthesis, characterization, crystal structure, and biological studies of two complexes with glycolic acid are described. The solid complexes were formulated as K2[VO(C2H2O3)(C2H3O3)2] H2O (1) and K2[{VO2(C2H2O3)}2] (2) and characterized by X-ray studies, Fourier transform infrared spectroscopy (FTIR), Electron paramagnetic resonance (EPR), and magnetic susceptibility. Conversion of 1 to 2 was studied in aqueous solution by UV-Vis spectroscopy and in the solid state by diffuse reflectance spectroscopy. Complex 2 contains dinuclear [{VO2(C2H2O3)}2]2- anions in which glycolate(2-) is a five-membered chelating ring formed by carboxylate and -hydroxy groups. The geometry around the vanadium in 2 was interpreted as intermediate between a trigonal bipyramid and a square pyramid. Vanadium(IV) is pentacoordinate in 1 as a distorted square pyramid. Complex 1 contains a vanadyl group (V=O) surrounded by two oxygens from deprotonated carboxylate and hydroxy groups forming a five-membered ring. Two oxygens from different glycolates(1-) are bonded to the (V=O) also. Biological analysis for potential cytotoxic effects of 1 was performed using Human Cervix Adenocarcinoma (HeLa) cells, a human cervix adenocarcinoma-derived cell line. After incubation for 48 h, 1 causes 90 and 95% of HeLa cells death at 20 and 200 mol L-1, respectively.

Anacardic acid derivatives as inhibitors of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Chagas` disease, a parasitic infection caused by the flagellate protozoan Trypanosoma cruzi, is a major public health problem affecting millions of individuals in Latin America. On the basis of the essential role in the life cycle of T. cruzi, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been considered an attractive target for the development of novel antitrypanosomatid agents. In the present work, we describe the inhibitory effects of a small library of natural and synthetic anacardic acid derivatives against the target enzyme. The most potent inhibitors, 6-n-pentadecyl-(1) and 6-n-dodecylsalicilic acids (10e), have IC(50) values of 28 and 55 mu M, respectively. The inhibition was not reversed or prevented by the addition of Triton X-100, indicating that aggregate-based inhibition did not occur. In addition, detailed mechanistic characterization of the effects of these compounds on the T. cruzi GAPDH-catalyzed reaction showed clear noncompetitive inhibition with respect to both substrate and cofactor. (C) 2008 Elsevier Ltd. All rights reserved.