Chiral separations of mandelic acid by HPLC using molecularly imprinted polymers

Styrene is used in a variety of chemical industries. Environmental and occupational exposures to styrene occur predominantly through inhalation. The major metabolite of styrene is present in two enantiomeric forms, chiral R- and S- hydroxy-1-phenyl-acetic acid (R-and S-mandelic acid, MA). Thus, the concentration of MA, particularly of its enantiomers, has been used in urine tests to determine whether workers have been exposed to styrene. This study describes a method of analyzing mandelic acid using molecular imprinting techniques and HPLC detection to perform the separation of diastereoisomers of mandelic acid. The molecularly imprinted polymer (MIP) was prepared by non-covalent molecular imprinting using (+) MA, (-) MA or (+) phenylalanine, (-) phenylalanine as templates. Methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) were copolymerized in the presence of the template molecules. The bulk polymerization was carried out at 4ºC under UV radiation. The resulting MIP was grounded into 25~44¼m particles, which were slurry packed into analytical columns. After the template molecules were removed, the MIP-packed columns were found to be effective for the chromatographic resolution of (±)-mandelic acid. This method is simpler and more convenient than other chromatographic methods.

Morphological and thermal analyses of flexible polyurethane foams containing commercial calcium carbonate

One filler often utilized in flexible polyurethane foams is calcium carbonate (CaCO3) because it is non-abrasiveness, non-toxicity and facilitated pigmentation. However, it is observed that the excess of commercial CaCO3 utilized in industry possibly causing permanent deformations and damaging the quality of the final product. The effect of different concentrations of commercial CaCO3, in flexible foams, was studied. Different concentrations of CaCO3 were used for the synthesis of flexible polyurethane foams, which were submitted to morphological and thermal analyses to verify the alterations provoked by the progressive introduction of this filler.

Evaluation of group electronegativities and hardness (softness) of group 14 elements and containing functional groups through density functional theory and correlation with NMR spectra data

Quantum Chemical calculations for group 14 elements of Periodic Table (C, Si, Ge, Sn, Pb) and their functional groups have been carried out using Density Functional Theory (DFT) based reactivity descriptors such as group electronegativities, hardness and softness. DFT calculations were performed for a large series of tetracoordinated Sn compounds of the CH3SnRR'X type, where X is a halogen and R and R' are alkyl, halogenated alkyl, alkoxy, or alkyl thio groups. The results were interpreted in terms of calculated electronegativity and hardness of the SnRR'X groups, applying a methodology previously developed by Geerlings and coworkers (J. Phys. Chem. 1993, 97, 1826). These calculations allowed to see the regularities concerning the influence of the nature of organic groups RR' and inorganic group X on electronegativities and hardness of the SnRR'X groups; in this case, it was found a very good correlation between the electronegativity of the fragment and experimental 119Sn chemical shifts, a property that sensitively reflects the change in the valence electronic structure of molecules. This work was complemented with the study of some compounds of the EX and ER types, where E= C, Si, Ge, Sn and R= CH3, H, which was performed to study the influence that the central atom has on the electronegativity and hardness of molecules, or whether these properties are mainly affected for the type of ligand bound to the central atom. All these calculations were performed using the B3PW91 functional together with the 6-311++G** basis set level for H, C, Si, Ge, F, Cl and Br atoms and the 3-21G for Sn and I atoms.

Photo-Fenton process for treating biological laboratory wastewater containing formaldehyde

Laboratories consume great amounts of hazardous chemicals substances and consequently generate wastewater containing them, for example formaldehyde. This substance is widely utilized to preserve biological samples generating many liters of this residue every year. The present work proposes the use of the photo-Fenton process to treat formaldehyde wastewater using sunlight irradiation. Some aspects were investigated such as the iron source, sample and hydrogen peroxide concentration and also the use of stirred systems. The use of ferrioxalate (0.5 mmol L-1) improved the efficiency of the process in relation to the use of iron nitrate, while at least 1.0 mol L-1 H2O2 is necessary to treat the sample of the 500 mg C L-1. Under these conditions, every formaldehyde detectable was degradeted and 89% of the dissolved organic carbon was removed in two hours of exposure to sunlight. These results are satisfaction considerate for São Paulo State Environmental Agency.

Optimization of NMP extraction in 1, 3-butadiene production line

N-methylpyrrolidone is a powerful solvent for variety of chemical processes due to its vast chemical properties. It has been used in manufacturing processes of polymers, detergents, pharmaceuticals rubber and many more chemical substances. However, it creates large amount of residue in some of these processes which has to be dealt with. Many well known methods such as BASF in rubber producing units have tried to regenerate the solvent at the end of each run, however, there is still discarding of large amount of residue containing NMP, which over time, could cause environmental concerns. In this study, we have tried to optimize regeneration of the NMP extraction from butadiene production. It is shown that at higher temperatures NMP is separated from the residue with close to 90% efficiency, and the solvent residue proved to be the most effective with a 6: 1 ratio.

Crystal-associated cholangiopathy in sheep grazing Brachiaria decumbens containing the saponin protodioscin

An outbreak of hepatogenous photosensitization is reported in a flock of 28 sheep grazing Brachiaria decumbens in Mato Grosso do Sul State, Central-Western Brazil. Seven lambs and an adult sheep were affected and 6 of them died. Two surviving affected lambs and one lamb without clinical signs had increased serum values of gamma glutamyltransferase, bilirubin, and cholesterol. In two adult unaffected sheep those parameters were within normal values. An adult sheep submitted to necropsy presented moderate body condition, unilateral corneal opacity, drying of the muzzle, moderate jaundice, increased lobular pattern of the liver, and a distended gallbladder. Histological lesions were epithelial degeneration, necrosis, and hyperplasia of small bile ducts. Mild amounts of foamy macrophages were observed, mainly in the centroacinar zone. Diffuse swelling and vacuolation were observed in hepatocytes. Crystal negative images were found within bile ducts, foamy macrophages, and the lumen of some renal tubules. The heart showed multifocal areas of degeneration and necrosis of the muscle fibers. Pasture samples (Brachiaria decumbens) contained 2.36% of protodioscin. No Pithomyces chartarum spores were found in the pasture. Samples from a similar neighboring B. decumbens pasture grazed by cattle without photosensitization contained 1.63% of protodioscin isomers. Outbreaks of photosensitization caused by Brachiaria spp. are common in cattle in the Brazilian Cerrado (savanna) with about 51 million hectares of Brachiaria spp pastures. Sheep farming has been recently developed in this region, and the number of sheep is increasing significantly. Because sheep are more susceptible than cattle to lithogenic saponins, poisoning by Brachiaria should be an important limiting factor for the sheep industry.

Occurrence of xyloglucan containing protuberances in the storage cell walls of cotyledons of Hymenaea courbaril L.

Despite the suggestions of its pectic composition, no clear evidence for this has been presented. Here we show the occurrence of such a structure in walls of cells from cotyledons of Hymenaea courbariI L. These cells are known to accumulate large amounts of storage xyloglucan in the wall and, in this case, the protuberances seem to contain this storage polysaccharide rather than pectin. A hypothetical sequence of events leading from wall strands to protuberances was assembled based on scanning electron microscopy observations. On this basis, a tentative model for how polysaccharides are distributed into the wall, near the regions where protuberances are found, is proposed to explain the presence of storage xyloglucan in their composition.

Effect of abscisic acid on the mobilisation of galactomannan and embryo development of Sesbania virgata (Cav.) Pers. (Leguminosae - Faboideae)

Galactomannans (GM) are storage cell wall polysaccharides present in endospermic seeds of legumes. They are thought to be storage polymers, since it has been observed for a few species (among them Sesbania virgata) that they are completely broken down after germination and their products are transferred to the growing embryo. We examined the effect of 10-4 M abscisic acid (ABA) on the degradation of galactomannan in isolated endosperms and intact seeds of S. virgata. We found that after seed germination the initial embryo growth was retarded. Ultrastructural analysis showed that the embryo is completely surrounded by an endosperm which displays very thick galactomannan-containing cell walls. Although an inhibitory effect has been observed on the increase of fresh mass of the embryo, the effect of ABA on the dry mass was weaker and transitory (from 48 to 96 h). Endosperm dry mass and galactomannan degradation were significantly inhibited and the activity of alpha-galactosidase was strongly affected. The addition of ABA before and/or after the start of mobilisation in intact seeds or isolated endosperms, showed that whereas addition before mobilisation did not affect dry mass decrease in intact seeds, it was strongly affected in isolated endosperms. On the other hand, whereas it affected embryo fresh mass increase in intact seeds, but not in isolated embryos, no significant effect was observed on dry mass. These results suggest that ABA affects galactomannan degradation and by doing so, prevents water absorption by the embryo, rather than affect its dry mass. As ABA has been detected in the endosperm of seeds of S. virgata, it is proposed that it probably acts as a modulator of galactomannan mobilisation and consequently synchronises it with early growth of the embryo.

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

Purification of human albumin by the combination of the method of Cohn with liquid chromatography

Large volumes of plasma can be fractionated by the method of Cohn at low cost. However, liquid chromatography is superior in terms of the quality of the product obtained. In order to combine the advantages of each method, we developed an integrated method for the production of human albumin and immunoglobulin G (IgG). The cryoprecipitate was first removed from plasma for the production of factor VIII and the supernatant of the cryoprecipitate was fractionated by the method of Cohn. The first precipitate, containing fractions (F)-I + II + III, was used for the production of IgG by the chromatographic method (see Tanaka K et al. (1998) Brazilian Journal of Medical and Biological Research, 31: 1375-1381). The supernatant of F-I + II + III was submitted to a second precipitation and F-IV was obtained and discarded. Albumin was obtained from the supernatant of the precipitate F-IV by liquid chromatography, ion-exchange on DEAE-Sepharose FF, filtration through Sephacryl S-200 HR and introduction of heat treatment for fatty acid precipitation. Viral inactivation was performed by pasteurization at 60ºC for 10 h. The albumin product obtained by the proposed procedure was more than 99% pure for the 15 lots of albumin produced, with a mean yield of 25.0 ± 0.5 g/l plasma, containing 99.0 to 99.3% monomer, 0.7 to 1.0% dimers, and no polymers. Prekallikrein activator levels were <=5 IU/ml. This product satisfies the requirements of the 1997 Pharmacopée Européenne.

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.

Toxicity of a cyanobacterial extract containing microcystins to mouse lungs

Toxic cyanobacteria in drinking water supplies can cause serious public health problems. In the present study we analyzed the time course of changes in lung histology in young and adult male Swiss mice injected intraperitoneally (ip) with a cyanobacterial extract containing the hepatotoxic microcystins. Microcystins are cyclical heptapeptides quantified by ELISA method. Ninety mice were divided into two groups. Group C received an injection of saline (300 µl, ip) and group Ci received a sublethal dose of microcystins (48.2 µg/kg, ip). Mice of the Ci group were further divided into young (4 weeks old) and adult (12 weeks old) animals. At 2 and 8 h and at 1, 2, 3, and 4 days after the injection of the toxic cyanobacterial extract, the mice were anesthetized and the trachea was occluded at end-expiration. The lungs were removed en bloc, fixed, sectioned, and stained with hematoxylin-eosin. The percentage of the area of alveolar collapse and the number of polymorphonuclear (PMN) and mononuclear cell infiltrations were determined by point counting. Alveolar collapse increased from C to all Ci groups (123 to 262%) independently of time, reaching a maximum value earlier in young than in adult animals. The amount of PMN cells increased with time of the lesion (52 to 161%). The inflammatory response also reached the highest level earlier in young than in adult mice. After 2 days, PMN levels remained unchanged in adult mice, while in young mice the maximum number was observed at day 1 and was similar at days 2, 3, and 4. We conclude that the toxins and/or other cyanobacterial compounds probably exert these effects by reaching the lung through the blood stream after ip injection.

In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229). We infer that an identical new gene (RING) finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D) model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3) as a binding site for ubiquitin-conjugating enzyme (E2). Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4) may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744). Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734). The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ).

Preparation and cytotoxicity of cisplatin-containing liposomes

We encapsulated cisplatin into stealth pH-sensitive liposomes and studied their stability, cytotoxicity and accumulation in a human small-cell lung carcinoma cell line (GLC4) and its resistant subline (GLC4/CDDP). Since reduced cellular drug accumulation has been shown to be the main mechanism responsible for resistance in the GLC4/CDDP subline, we evaluated the ability of this new delivery system to improve cellular uptake. The liposomes were composed of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol 2000 (DSPE-PEG2000) and were characterized by determining the encapsulation percentage as a function of lipid concentration. Among the different formulations, DOPE/CHEMS/DSPE-PEG liposomes (lipid concentration equal to 40 mM) encapsulated cisplatin more efficiently than other concentrations of liposomes (about 20.0%, mean diameter of 174 nm). These liposomes presented good stability in mouse plasma which was obtained using a 0.24-M EDTA solution (70% cisplatin was retained inside the liposomes after 30 min of incubation). Concerning cytotoxic effects, they are more effective (1.34-fold) than free cisplatin for growth inhibition of the human lung cancer cell line A549. The study of cytotoxicity to GLC4 and GLC4/CDDP cell lines showed similar IC50 values (approximately 1.4 µM), i.e., cisplatin-resistant cells were sensitive to this cisplatin formulation. Platinum accumulation in both sensitive and resistant cell lines followed the same pattern, i.e., approximately the same intracellular platinum concentration (4.0 x 10-17 mol/cell) yielded the same cytotoxic effect. These results indicate that long-circulating pH-sensitive liposomes, also termed as stealth pH-sensitive liposomes, may present a promising delivery system for cisplatin-based cancer treatment. This liposome system proved to be able to circumvent the cisplatin resistance, whereas it was not observed when using non-long-circulating liposomes composed of phosphatidylcholine, phosphatidylserine, and cholesterol.

The adherence of Pseudomonas fluorescens to marble, granite, synthetic polymers, and stainless steel

The adherence of Pseudomonas fluorescens cells to nine food-processing contact surfaces was evaluated using the plate-count method. The surfaces include marble, granite, stainless steel, polyvinyl chloride, polyurethane, and silicone-coated cloth, which have been used only in a few studies concerning bacterial adherence. The number of cells adhered to the surfaces increased with contact time reaching 5.0-6.1 log CDM.cm-2 after 10 hours, which can be considered a well established adherence process. The number of adhered cells doubled in 29.5 minutes and 23.5 minutes on stainless steel and thin polyvinyl chloride-coated cloth, respectively. For the other surfaces, this value was 9.8 minutes on average. Marble, granite, thick polyvinyl-coated cloth, double-faced rugous polyurethane, and silicone-coated cloth were not different (p < 0.05) in their ability to adhere cells (CFU/cm²) after 2 and 10 hours. The surfaces that had higher percentage of similarity in the adhesion level and higher log CFU/cm² of adhered cells were double-faced rugous polyurethane, silicone-coated cloth, and granite. The surfaces showed very different microtopography characteristics when viewed using scanning electron microscopy. This experiment showed the importance of using appropriate materials for food contact during processing, which will affect the cleaning and sanitation procedures.