Spot urine porphyrins/creatinine ratio profile of healthy Brazilian individuals adjusted for personal habits

Changes in urinary porphyrin excretion may be the result of hereditary causes and/or from environmental or occupational exposure. The objective of this study was to measure the amount of some porphyrins in spot urine samples obtained from volunteers randomly selected from a healthy adult population of São Paulo with a sensitive HPLC method and to estimate normal ranges for a non-exposed population. Spot urine samples were collected from 126 subjects (both genders, 18 to 65 years old) not occupationally exposed to porphyrinogenic agents. Porphyrin fractions were separated on RP-18 HPLC column eluted with a methanol/ammonium acetate buffer gradient, pH 4.0, and measured fluorometrically (excitation 405 nm/emission 620 nm). The amount of porphyrins was corrected for urinary creatinine excretion. Only 8-carboxyl (uro) and 4-carboxyl (copro) porphyrins were quantified as µg/g creatinine. Data regarding age, gender, occupational activities, smoking and drinking habits were analyzed by Mann-Whitney and Kruskal-Wallis tests. Uroporphyrin results did not differ significantly between the subgroups studied. Copro and uro + copro porphyrins were significantly different for smokers (P = 0.008) and occupational activities (P = 0.004). With respect to alcohol consumption, only men drinking >20 g/week showed significant differences in the levels of copro (P = 0.022) and uro + copro porphyrins (P = 0.012). The 2.5-97.5th percentile limit values, excluding those for subjects with an alcohol drinking habit >20 g/week, were 0-20.8, 11.7-93.1, and 15.9-102.9 µg/g creatinine for uro, copro and uro + copro porphyrins, respectively. These percentile limit values can be proposed as a first attempt to provide urinary porphyrin reference values for our population, serving for an early diagnosis of porphyrinopathies or as biomarkers of exposure to porphyrinogenic agents.

cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA2). The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.

Streptococcus mutans GlnK protein: an unusual PII family member

Streptococcus mutans is a Gram-positive bacterium present in the oral cavity, and is considered to be one of the leading causes of dental caries. S. mutans has a glnK gene, which codes for a PII-like protein that is possibly involved in the integration of carbon, nitrogen and energy metabolism in several organisms. To characterize the GlnK protein of S. mutans, the glnK gene was amplified by PCR, and cloned into the expression vectors pET29a(+) and pET28b(+). The native GlnK-Sm was purified by anion exchange (Q-Sepharose) and affinity (Hi-Trap Heparin) chromatography. The GlnK-His-Sm protein was purified using a Hi-Trap Chelating-Ni2+ column. The molecular mass of the GlnK-His-Sm proteins was 85 kDa as determined by gel filtration, indicating that this protein is a hexamer in solution. The GlnK-His-Sm protein is not uridylylated by the Escherichia coli GlnD protein. The activities of the GlnK-Sm and GlnK-His-Sm proteins were assayed in E. coli constitutively expressing the Klebsiella pneumoniae nifLA operon. In K. pneumoniae, NifL inhibits NifA activity in the presence of high ammonium levels and the GlnK protein is required to reduce the inhibition of NifL in the presence of low ammonium levels. The GlnK-Sm protein was unable to reduce NifL inhibition of NifA protein. Surprisingly, the GlnK-His-Sm protein was able to partially reduce NifL inhibition of the NifA protein under nitrogen-limiting conditions, in a manner similar to the GlnK protein of E. coli. These results suggested that S. mutans GlnK is functionally different from E. coli PII proteins.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Sedative and anesthetic activities of the essential oils of Hyptis mutabilis (Rich.) Briq. and their isolated components in silver catfish (Rhamdia quelen)

This study evaluated the sedative and anesthetic effects of the essential oils (EO) of Hyptis mutabilis (Rich.) Briq. and their isolated components on silver catfish (Rhamdia quelen). Quantitative chemical differences between the EOs obtained from leaves and inflorescences were verified, and a new chemotype rich in globulol was described. Although there were no significant differences in the time of induction for sedation and anesthesia between the EOs, only the leaf EO at 344 mg/L anesthetized all fish without side effects. Fractionation of the leaf EO was carried out by column chromatography. The isolated compounds [(+)-1-terpinen-4-ol and (-)-globulol] showed different activity from that detected for the leaf EO in proportional concentrations and similar sedation to a eugenol control at 10 mg/L. However, fish exposed to 1-terpinen-4-ol (3 and 10 mg/L) did not remain sedated for 30 min. Anesthesia was obtained with 83-190 mg/L globulol, but animals showed loss of mucus during induction and mortality at these concentrations. Synergism of the depressor effects was detected with the association of globulol and benzodiazepine (BDZ), compared with either drug alone. Fish exposed to BDZ or globulol+BDZ association showed faster recovery from anesthesia in water containing flumazenil, but the same did not occur with globulol. In conclusion, the use of globulol in aquaculture procedures should be considered only at sedative concentrations of 10 and 20 mg/L, and its mechanism of action seems not to involve the GABAA-BDZ system.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus

Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.

HIGH PRESSURE VAPOR-LIQUID EQUILIBRIA OF PALM FATTY ACIDS DISTILLATES-CARBON DIOXIDE SYSTEM

Vapor-Liquid equilibria of palm fatty acids distillates/carbon dioxide system has been investigated experimentally at temperatures of 333, 353, and 373 K and pressures of 20, 23, 26, and 29 MPa using the static method. Experimental data for the quasi-binary system palm fatty acids distillates/carbon dioxide has been correlated with Redlich-Kwong-Aspen equation of state. Modeling shows good agreement with experimental data. Selectivity obtained indicates that supercritical carbon dioxide is a reasonable solvent for separating saturated (palmitic acid) and unsaturated (oleic+linoleic acids) fatty acids from palm fatty acids distillates in a continuous multistage countercurrent column.

Evaluation of total carotenoids, alpha- and beta-carotene in carrots (Daucus carota L.) during home processing

This study aims to analyze the influence of dehydration and different preparation methods during home processing related toalpha-carotene, beta-carotene and total carotenoids stability in carrots. Vitamin A values were evaluated after different treatments. Thus, carrots were submitted to steam cooking, water cooking with and without pressure, moist/dry cooking and conventional dehydration. Determination of alpha- and beta-carotenes was made by High-Performance Liquid Chromatography (HPLC) (conditions were developed by us) using spectrophotometric detection visible-UV at 470 nm; a RP-18 column and methanol: acetonitrile: ethyl acetate (80: 10: 10) as mobile phase. Total carotenoids quantification was made by 449 nm spectrophotometer. The retention of the analyzed carotenoids ranged from 60.13 to 85.64%. Water cooking without pressure promoted higher retention levels of alpha- and beta-carotene and vitamin A values, while water cooking with pressure promoted higher retention levels of total carotenoids. Dehydration promoted the highest carotenoid losses. The results showed that, among the routinely utilized methods under domestic condition, cooking without pressure, if performed under controlled time and temperature, is the best method as it reduces losses in the amount of alpha- and beta-carotene, the main carotenoids present in the carrots. Despite the significant carotenoid losses, carrots prepared through domestic methods, remain a rich source of provitamin A.

Organic acid profile of commercial sour cassava starch

Organic acids are present in sour cassava starch ("polvilho azedo") and contribute with organoleptic and physical characteristics like aroma, flavor and the exclusive baking property, that differentiate this product from the native cassava starch. Samples of commercial sour cassava starch collected in South and Southeast Brazil were prepared for high performance liquid chromatography (HPLC) analysis. The HPLC equipment had a Biorad Aminex HPX-87H column for organic acid analysis and a refractometric detector. Analysis was carried out with 0.005M sulfuric acid as mobile phase, 0.6ml/min flow rate and column temperature of 60° C. The acids quantified were lactic (0.036 to 0.813 g/100g), acetic (0 to 0.068 g/100g), propionic (0 to 0.013 g/100g) and butyric (0 to 0.057 g/100g), that are produced during the natural fermentation of cassava starch. Results showed large variation among samples, even within the same region. Some samples exhibited high acid levels, mainly lactic acid, but in these neither propionic nor butyric acids were detected. Absence of butyric acid was not expected because this is an important component of the sour cassava starch aroma, and the lack of this acid may suggest that such samples were produced without the natural fermentation step.

Aflatoxin M1 in samples of "minas" cheese commercialized in the city of Belo Horizonte - Minas Gerais/Brazil

Milk products such as cheeses may be contaminated by aflatoxin M1 when dairy cattle have consumed feeds contaminated with aflatoxin B1. Samples of "Minas" cheeses (fresh, canastra and standard) were collected by the Inspection Service in the Mercado Central in Belo Horizonte city, Minas Gerais - Brazil. A purified extract was obtained by extraction with dichloromethane followed by a washing with n-hexane and immunoaffinity column purification. The quantification of aflatoxin M1 was done by high performance liquid chromatography (HPLC) using a fluorescence detector. Recoveries were about 75%. In 56 of the 75 samples (74.7%), the presence of aflatoxin M1 was detected in concentrations ranging between 0.02 and 6.92ng/g of cheese. In the positive cases ( > or = 0.02ng/g) the mean contamination level of aflatoxin M1 was 0.08ng/g in fresh cheese, 0.36ng/g in canastra cheese and 0.62ng/g in standard cheese. No aflatoxin M1 maximum tolerance level in cheese has been established in Brazil.

SURVEY OF DEOXYNIVALENOL, DIACETOXYSCIRPENOL, AND T2 TOXIN IN POPCORN HYBRIDS PLANTED IN THE STATE OF SÃO PAULO AND IN POPCORN COMMERCIALIZED IN THE CITY OF CAMPINAS, SP

The incidence of the trichothecenes deoxynivalenol (DON), diacetoxyscirpenol (DAS), and T2 toxin (T2) in popcorn was investigated in 90 samples, belonging to 2 commercial and 28 experimental hybrids planted at experimental stations of the Agronomic Institute of Campinas at the locations of Campinas, Mococa, and Capão Bonito, and in 15 samples of popcorn, 9 branded and 6 unbranded, acquired from commercial outlets in the city of Campinas, SP. The samples were analyzed by gas chromatography with flame ionization detector combined with an alumina:carbon cleanup column. The detection limits were 30ng/g for DON, 50ng/g for DAS, and 40ng/g for T2. Five samples were contaminated with DON, four of them commercial and one from an experimental cultivar. The level of contamination in the commercial samples ranged from 30 to 40ng/g. The sample from the experimental cultivar contained 770ng/g DON. DAS and T2 were not detected in any of the popcorn samples analyzed.

Determination of trans-10-hydroxy-2-decenoic acid (10-HDA) in royal jelly from São Paulo State, Brazil

The 10-HDA content in Brazilian samples (São Paulo State) of royal jelly (RJ) was analyzed using an HPLC method based on the work by BLOODWORTH et al. [2]. The chromatographic conditions were: isocratic system, reversed phase C18-H column, auto sampler, diode array UV-VIS detector adjusted to 225nm, mobile phase composed by methanol/water (45:55) at pH= 2.5 adjusted with phosphoric acid; a-naphtol was used as internal standard, and the running time was 30min. By statistical analysis of the results, the 10-HDA contents of the samples analyzed seem to have two ranges: 1.8% and 3% (w/w), that would be useful to qualify the RJ. This is the first data regarding 10-HDA content of Brazilian RJ.

A method for fast determination of epigallocatechin gallate (EGCG), epicatechin (EC), catechin (C) and caffeine (CAF) in green tea using HPLC

Tea has been considered a medicine and a healthy beverage since ancient times, but recently it has received a great deal of attention because of its antioxidant properties. Green tea polyphenols have demonstrated to be an effective chemopreventive agent. Recently, investigators have found that EGCG, one of the green tea catechins, could have anti-HIV effects when bound to CD4 receptor. Many factors can constitute important influences on the composition of tea, such as species, season, age of the leaf, climate, and horticultural practices (soil, water, minerals, fertilizers). This paper presents an HPLC analytical methodology development, using column RP-18 and mobile phase composed by water, acetonitrile, methanol, ethyl acetate, glacial acetic acid (89:6:1:3:1 v/v/v/v/v) for simultaneous determination and quantification of caffeine (CAF), catechin (C), epicatechin (EC) and epigallocatechin gallate (EGCG) in samples of Camellia sinensis (green tea) grown in Brazil and harvested in spring, in summer and in autumn, in comparison to Brazilian black tea, to samples of Japanese and Chinese green tea and to two standardized dry extracts of green tea. The method has been statistically evaluated and has proved to be adequate to qualitative and quantitative determination of the samples.