Diagnosis of Smith-Lemli-Opitz syndrome by ultraviolet spectrophotometry

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder due to an inborn error of cholesterol metabolism, characterized by congenital malformations, dysmorphism of multiple organs, mental retardation and delayed neuropsychomotor development resulting from cholesterol biosynthesis deficiency. A defect in 3ß-hydroxysteroid-delta7-reductase (delta7-sterol-reductase), responsible for the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol, causes an increase in 7-DHC and frequently reduces plasma cholesterol levels. The clinical diagnosis of SLOS cannot always be conclusive because of the remarkable variability of clinical expression of the disorder. Thus, confirmation by the measurement of plasma 7-DHC levels is needed. In the present study, we used a simple, fast, and selective method based on ultraviolet spectrophotometry to measure 7-DHC in order to diagnose SLOS. 7-DHC was extracted serially from 200 µl plasma with ethanol and n-hexane and the absorbance at 234 and 282 nm was determined. The method was applied to negative control plasma samples from 23 normal individuals and from 6 cases of suspected SLOS. The method was adequate and reliable and 2 SLOS cases were diagnosed.

The secondary alcohol and aglycone metabolites of doxorubicin alter metabolism of human erythrocytes

Anthracyclines, a class of antitumor drugs widely used for the treatment of solid and hematological malignancies, cause a cumulative dose-dependent cardiac toxicity whose biochemical basis is unclear. Recent studies of the role of the metabolites of anthracyclines, i.e., the alcohol metabolite doxorubicinol and aglycone metabolites, have suggested new hypotheses about the mechanisms of anthracycline cardiotoxicity. In the present study, human red blood cells were used as a cell model. Exposure (1 h at 37ºC) of intact human red blood cells to doxorubicinol (40 µM) and to aglycone derivatives of doxorubicin (40 µM) induced, compared with untreated red cells: i) a ~2-fold stimulation of the pentose phosphate pathway (PPP) and ii) a marked inhibition of the red cell antioxidant enzymes, glutathione peroxidase (~20%) and superoxide dismutase (~60%). In contrast to doxorubicin-derived metabolites, doxorubicin itself induced a slighter PPP stimulation (~35%) and this metabolic event was not associated with any alteration in glutathione reductase, glutathione peroxidase, catalase or superoxide dismutase activity. Furthermore, the interaction of hemoglobin with doxorubicin and its metabolites induced a significant increase (~22%) in oxygen affinity compared with hemoglobin incubated without drugs. On the basis of the results obtained in the present study, a new hypothesis, involving doxorubicinol and aglycone metabolites, has been proposed to clarify the mechanisms responsible for the doxorubicin-induced red blood cell toxicity.

Cholesterol induces fetal rat enterocyte death in culture

The effect of cholesterol on fetal rat enterocytes and IEC-6 cells (line originated from normal rat small intestine) was examined. Both cells were cultured in the presence of 20 to 80 µM cholesterol for up to 72 h. Apoptosis was determined by flow cytometric analysis and fluorescence microscopy. The expression of HMG-CoA reductase and peroxisome proliferator-activated receptor gamma (PPARgamma) was measured by RT-PCR. The addition of 20 µM cholesterol reduced enterocyte proliferation as early as 6 h of culture. Reduction of enterocyte proliferation by 28 and 41% was observed after 24 h of culture in the presence and absence of 10% fetal calf serum, respectively, with the effect lasting up to 72 h. Treatment of IEC-6 cells with cholesterol for 24 h raised the proportion of cells with fragmented DNA by 9.7% at 40 µM and by 20.8% at 80 µM. When the culture period was extended to 48 h, the effect of cholesterol was still more pronounced, with the percent of cells with fragmented DNA reaching 53.5% for 40 µM and 84.3% for 80 µM. Chromatin condensation of IEC-6 cells was observed after treatment with cholesterol even at 20 µM. Cholesterol did not affect HMG-CoA reductase expression. A dose-dependent increase in PPARgamma expression in fetal rat enterocytes was observed. The expression of PPAR-gamma was raised by 7- and 40-fold, in the presence and absence of fetal calf serum, respectively, with cholesterol at 80 mM. The apoptotic effect of cholesterol on enterocytes was possibly due to an increase in PPARgamma expression.

Advanced biofuel production: Engineering metabolic pathways for butanol and propane biosynthesis.

Greenhouse gases emitted from energy production and transportation are dramatically changing the climate of Planet Earth. As a consequence, global warming is affecting the living conditions of numerous plant and animal species, including ours. Thus the development of sustainable and renewable liquid fuels is an essential global challenge in order to combat the climate change. In the past decades many technologies have been developed as alternatives to currently used petroleum fuels, such as bioethanol and biodiesel. However, even with gradually increasing production, the market penetration of these first generation biofuels is still relatively small compared to fossil fuels. Researchers have long ago realized that there is a need for advanced biofuels with improved physical and chemical properties compared to bioethanol and with biomass raw materials not competing with food production. Several target molecules have been identified as potential fuel candidates, such as alkanes, fatty acids, long carbon‐chain alcohols and isoprenoids. The current study focuses on the biosynthesis of butanol and propane as possible biofuels. The scope of this research was to investigate novel heterologous metabolic pathways and to identify bottlenecks for alcohol and alkane generation using Escherichia coli as a model host microorganism. The first theme of the work studied the pathways generating butyraldehyde, the common denominator for butanol and propane biosynthesis. Two ways of generating butyraldehyde were described, one via the bacterial fatty acid elongation machinery and the other via partial overexpression of the acetone‐butanol‐ethanol fermentation pathway found in Clostridium acetobutylicum. The second theme of the experimental work studied the reduction of butyraldehyde to butanol catalysed by various bacterial aldehyde‐reductase enzymes, whereas the final part of the work investigated the in vivo kinetics of the cyanobacterial aldehyde deformylating oxygenase (ADO) for the generation of hydrocarbons. The results showed that the novel butanol pathway, based on fatty acid biosynthesis consisting of an acyl‐ACP thioesterase and a carboxylic acid reductase, is tolerant to oxygen, thus being an efficient alternative to the previous Clostridial pathways. It was also shown that butanol can be produced from acetyl‐CoA using acetoacetyl CoA synthase (NphT7) or acetyl‐CoA acetyltransferase (AtoB) enzymes. The study also demonstrated, for the first time, that bacterial biosynthesis of propane is possible. The efficiency of the system is clearly limited by the poor kinetic properties of the ADO enzyme, and for proper function in vivo, the catalytic machinery requires a coupled electron relay system.

Effect of (-)-∆9-tetrahydrocannabinoid on the hepatic redox state of mice

(-)-∆9-Tetrahydrocannabinol (∆9-THC), a psychoactive component of marijuana, has been reported to induce oxidative damage in vivo and in vitro. In this study, we administered ∆9-THC to healthy C57BL/6J mice aged 15 weeks in order to determine its effect on hepatic redox state. Mice were divided into 3 groups: ∆9-THC (N = 10), treated with 10 mg/kg body weight ∆9-THC daily; VCtrl (N = 10), treated with vehicle [1:1:18, cremophor EL® (polyoxyl 35 castor oil)/ethanol/saline]; Ctrl (N = 10), treated with saline. Animals were injected ip twice a day with 5 mg/kg body weight for 10 days. Lipid peroxidation, protein carbonylation and DNA oxidation were used as biomarkers of oxidative stress. The endogenous antioxidant defenses analyzed were glutathione (GSH) levels as well as enzyme activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and glutathione peroxidase (GPx) in liver homogenates. The levels of mRNA of the cannabinoid receptors CB1 and CB2 were also monitored. Treatment with ∆9-THC did not produce significant changes in oxidative stress markers or in mRNA levels of CB1 and CB2 receptors in the liver of mice, but attenuated the increase in the selenium-dependent GPx activity (Δ9-THC: 8%; VCtrl: 23% increase) and the GSH/oxidized GSH ratio (Δ9-THC: 61%; VCtrl: 96% increase), caused by treatment with the vehicle. Δ9-THC administration did not show any harmful effects on lipid peroxidation, protein carboxylation or DNA oxidation in the healthy liver of mice but attenuated unexpected effects produced by the vehicle containing ethanol/cremophor EL®.

In vitro activity of hypnophilin from Lentinus strigosus: a potential prototype for Chagas disease and leishmaniasis chemotherapy

Hypnophilin and panepoxydone, terpenoids isolated from Lentinus strigosus, have significant inhibitory activity onTrypanosoma cruzi trypanothione reductase (TR). Although they have similar TR inhibitory activity at 10 μg/mL (40.3 μM and 47.6 μM for hypnophilin and panepoxydone, respectively; ~100%), hypnophilin has a slightly greater inhibitory activity (~71%) on T. cruzi amastigote (AMA) growth in vitro as well as on in vitro phytohemagglutinin (PHA)-induced peripheral blood mononuclear (PBMC) proliferation (~70%) compared to panepoxydone (69% AMA inhibition and 91% PBMC inhibition). Hypnophilin and panepoxydone at 1.25 μg/mL had 67% inhibitory activity onLeishmania (Leishmania) amazonensis amastigote-like (AMA-like) growth in vitro. The panepoxydone activity was accompanied by a significant inhibitory effect on PHA-induced PBMC proliferation, suggesting a cytotoxic action. Moreover, incubation of human PBMC with panepoxydone reduced the percentage of CD16+ and CD14+ cells and down-regulated CD19+, CD4+ and CD8+ cells, while hypnophilin did not alter any of the phenotypes analyzed. These data indicate that hypnophilin may be considered to be a prototype for the design of drugs for the chemotherapy of diseases caused by Trypanosomatidae.

Long-term melatonin treatment reduces ovarian mass and enhances tissue antioxidant defenses during ovulation in the rat

Melatonin regulates the reproductive cycle, energy metabolism and may also act as a potential antioxidant indoleamine. The present study was undertaken to investigate whether long-term melatonin treatment can induce reproductive alterations and if it can protect ovarian tissue against lipid peroxidation during ovulation. Twenty-four adult female Wistar rats, 60 days old (± 250-260 g), were randomly divided into two equal groups. The control group received 0.3 mL 0.9% NaCl + 0.04 mL 95% ethanol as vehicle, and the melatonin-treated group received vehicle + melatonin (100 µg·100 g body weight-1·day-1) both intraperitoneally daily for 60 days. All animals were killed by decapitation during the morning estrus at 4:00 am. Body weight gain and body mass index were reduced by melatonin after 10 days of treatment (P < 0.05). Also, a marked loss of appetite was observed with a fall in food intake, energy intake (melatonin 51.41 ± 1.28 vs control 57.35 ± 1.34 kcal/day) and glucose levels (melatonin 80.3 ± 4.49 vs control 103.5 ± 5.47 mg/dL) towards the end of treatment. Melatonin itself and changes in energy balance promoted reductions in ovarian mass (20.2%) and estrous cycle remained extensive (26.7%), arresting at diestrus. Regarding the oxidative profile, lipid hydroperoxide levels decreased after melatonin treatment (6.9%) and total antioxidant substances were enhanced within the ovaries (23.9%). Additionally, melatonin increased superoxide dismutase (21.3%), catalase (23.6%) and glutathione-reductase (14.8%) activities and the reducing power (10.2% GSH/GSSG ratio). We suggest that melatonin alters ovarian mass and estrous cyclicity and protects the ovaries by increasing superoxide dismutase, catalase and glutathione-reductase activities.

Lactobacillus plantarum strains isolated from naturally fermented sausages and their technological properties for application as starter cultures

In the present study, technological properties of L. plantarum strains isolated from naturally fermented sausages manufactured in the South region of Brazil were investigated in order to obtain a starter culture. The technological properties evaluated were the following: ability to growth at different pH values, at different temperatures, in different salt concentrations and in the presence of commercial curing salt, fast production of acid, determination of D - and L - lactic acid; nitrate reductase activity; antagonistic activity and stability of the isolated cultures after fermentation, concentration, and freeze-drying process. The isolated strains showed effectiveness to improve technological properties as starter cultures.

Microbiological quality of hamburgers sold in the streets of Cuiabá - MT, Brazil and vendor hygiene-awareness

This study evaluated the microbiological quality of hamburgers and the microbe community on the hands of vendors in Cuiabá, Mato Grosso, Brazil, in relation to vendors´ awareness as to what constitute acceptable food-handling practices as part of a broad-spectrum research programme on street foods in Brazil . Sale of the hamburger known as the 'baguncinha' is common and widespread in urban Cuiabá, Mato Grosso, Brazil. Food inspectors encounter various difficulties in carrying out inspections. One hundred and five hamburgers samples were evaluated using conventional methods including tests for facultative aerobic and/or anaerobic mesophytic bacteria, coliform counts at 45 °C, the coagulase test for Staphylococcus, Gram-staining for the presence of Bacillus cereus, Clostridium sulphite reductase and Salmonella spp. The hamburgers were categorized as unsuitable for human consumption in 31.4% of samples, with those testing positive for coliforms and Staphylococcus at unacceptably high levels by Brazilian standards. High levels of microbiological contamination were detected on the hands of the food handlers and mesophytic bacterial counts reached 1.8 × 10(4) CFU/hand. Interviews were carried out by means of questionnaires to evaluate levels of awareness as to acceptable food handling practices and it was found that 80,1% of vendors had never participated in any kind of training.

Effect of extracts from araticum (Annona crassiflora) on CCl4-induced liver damage in rats

The influence of ethanolic extracts of Annona crassiflora on the activities of hepatic antioxidant enzymes was examined. Extracts of A. crassiflora seeds and peel were administered orally (50 mg of galic acid equivalents.kg-1) to Wistar rats for 14 consecutive days followed by a single oral dose of carbon tetrachloride (CCl4, 2 g.kg-1). Lipid peroxidation and the activities of hepatic catalase (CAT), cytochromes P450 (CP450) and b5, glutathione peroxidase (GPx), glutathione reductase (GRed), superoxide dismutase (SOD), and the content of glutathione equivalents (GSH) were evaluated. The treatment with CCl4 increased lipid peroxidation, the level of GSH equivalents and the content of cytochrome b5 by 44, 140 and 32%, respectively, with concomitant reductions of 23, 34 and 39% in the activities of CAT, SOD, and CP450, respectively. The treatment with A. crassiflora seeds and peel extracts alone inhibited lipid peroxidation by 27 and 22%, respectively without affecting the CP450 content. The pretreatment with the A. crassiflora extracts prevented the lipid peroxidation, the increase in GSH equivalents and the decrease in CAT activity caused by CCl4, but it had no effect on the CCl4-mediated changes in CP450 and b5 and SOD. These results show that A. crassiflora seeds and peel contain antioxidant activity in vivo that could be of potential therapeutic use.

Deoxynivalenol (DON) degradation and peroxidase enzyme activity in submerged fermentation

This work aims to evaluate deoxynivalenol degradation by Aspergillus oryzae and Rhizopus oryzae in a submerged fermentation system and to correlate it to the activity of oxydo-reductase enzymes. The submerged medium consisted of sterile distilled water contaminated with 50 μg of DON and 4 × 10(6) spore.mL-1 inoculum of Aspergillus oryzae and Rhizopus oryzae species, respectively in each experiment. Sampling was performed every 24 hours for monitoring the peroxidase specific activity, and every 48 hours for determining mycotoxin levels. Results showed that the fungi species were able to decrease DON levels as the peroxidase activity increased. The 48 hours fermentation interval presented the highest peroxidase specific activity (ΔABS/minute.μg.protein-1), 800 and 198, while the highest DON degradation velocity was 10.8 and 12.4 ppb/hour, respectively in both cases for Rhizopus oryzae and Aspergillus oryzae.

Monooxygenase Diversity and Oxygen Activation within Polyketide Antibiotic Biosynthesis

Molecular oxygen (O2) is a key component in cellular respiration and aerobic life. Through the redox potential of O2, the amount of free energy available to organisms that utilize it is greatly increased. Yet, due to the nature of the O2 electron configuration, it is non-reactive to most organic molecules in the ground state. For O2 to react with most organic compounds it must be activated. By activating O2, oxygenases can catalyze reactions involving oxygen incorporation into organic compounds. The oxygen activation mechanisms employed by many oxygenases to have been studied, and they often include transition metals and selected organic compounds. Despite the diversity of mechanisms for O2 activation explored in this thesis, all of the monooxygenases studied in the experimental part activate O2 through a transient carbanion intermediate. One of these enzymes is the small cofactorless monooxygenase SnoaB. Cofactorless monooxygenases are unusual oxygenases that require neither transition metals nor cofactors to activate oxygen. Based on our biochemical characterization and the crystal structure of this enzyme, the mechanism most likely employed by SnoaB relies on a carbanion intermediate to activate oxygen, which is consistent with the proposed substrate-assisted mechanism for this family of enzymes. From the studies conducted on the two-component system AlnT and AlnH, both the functions of the NADH-dependent flavin reductase, AlnH, and the reduced flavin dependent monooxygenase, AlnT, were confirmed. The unusual regiochemistry proposed for AlnT was also confirmed on the basis of the structure of a reaction product. The mechanism of AlnT, as with other flavin-dependent monooxygenases, is likely to involve a caged radical pair consisting of a superoxide anion and a neutral flavin radical formed from an initial carbanion intermediate. In the studies concerning the engineering of the S-adenosyl-L-methionine (SAM) dependent 4-O-methylase DnrK and the homologous atypical 10-hydroxylase RdmB, our data suggest that an initial decarboxylation of the substrate is catalyzed by both of these enzymes, which results in the generation of a carbanion intermediate. This intermediate is not essential for the 4-O-methylation reaction, but it is important for the 10-hydroxylation reaction, since it enables substrate-assisted activation of molecular oxygen involving a single electron transfer to O2 from a carbanion intermediate. The only role for SAM in the hydroxylation reaction is likely to be stabilization of the carbanion through the positive charge of the cofactor. Based on the DnrK variant crystal structure and the characterizations of several DnrK variants, the insertion of a single amino acid in DnrK (S297) is sufficient for gaining a hydroxylation function, which is likely caused by carbanion stabilization through active site solvent restriction. Despite large differences in the three-dimensional structures of the oxygenases and the potential for multiple oxygen activation mechanisms, all the enzymes in my studies rely on carbanion intermediates to activate oxygen from either flavins or their substrates. This thesis provides interesting examples of divergent evolution and the prevalence of carbanion intermediates within polyketide biosynthesis. This mechanism appears to be recurrent in aromatic polyketide biosynthesis and may reflect the acidic nature of these compounds, propensity towards hydrogen bonding and their ability to delocalize π-electrons.

Identification and characterization of a Catharanthus roseus mutant altered in monoterpenoid indole alkaloid biosynthesis

The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is the only source for several pharmaceutically valuable monoterpenoid indole alkaloids (MIAs), including the powerful antihypertensive ajmalicine and the antineoplastic agents vincristine and vinblastine. While biosynthesis of MIA precursors has been elucidated, conversion of the common MIA precursor strictosidine to MIAs of different families, for example ajmalicine, catharanthine or vindoline, remains uncharacterized. Deglycosylation of strictosidine by the key enzyme Strictosidine beta-glucosidase (SGD) leads to a pool of uncharacterized reaction products that are diverted into the different MIA families, but the downstream reactions are uncharacterized. Screening of 3600 EMS (ethyl methane sulfonate) mutagenized C. roseus plants to identify mutants with altered MIA profiles yielded one plant with high ajmalicine, and low catharanthine and vindoline content. RNA sequencing and comparative bioinformatics of mutant and wildtype plants showed up-regulation of SGD and the transcriptional repressor Zinc finger Catharanthus transcription factor (ZCT1) in the mutant line. The increased SGD activity in mutants seems to yield a larger pool of uncharacterized SGD reaction products that are channeled away from catharanthine and vindoline towards biosynthesis of ajmalicine when compared to the wildtype. Further bioinformatic analyses, and crossings between mutant and wildtype suggest a transcription factor upstream of SGD and ZCT1 to be mutated, leading to up-regulation of Sgd and Zct1. The crossing experiments further show that biosynthesis of the different MIA families is differentially regulated and highly complex. Three new transcription factors were identified by bioinformatics that seem to be involved in the regulation of Zct1 and Sgd expression, leading to the high ajmalicine phenotype. Increased cathenamine reductase activity in the mutant converts the pool of SGD reaction products into ajmalicine and its stereoisomer tetrahydroalstonine. The stereochemistry of ajmalicine and tetrahydroalstonine biosynthesis in vivo and in vitro was further characterized. In addition, a new clade of perakine reductase-like enzymes was identified that reduces the SGD reaction product vallesiachotamine in a stereo-specific manner, characterizing one of the many reactions immediately downstream of SGD that determine the different MIA families. This study establishes that RNA sequencing and comparative bioinformatics, in combination with molecular and biochemical characterization, are valuable tools to determine the genetic basis for mutations that trigger phenotypes, and this approach can also be used for identification of new enzymes and transcription factors.

Chemical, biochemical, and molecular characterization of a low vindoline Catharanthus roseus mutant.

The Madagascar periwinkle (Catharanthus roseus) is the sole source of the anticancer drug vinblastine, which is formed via the coupling of monoterpenoid indole alkaloids (MIAs) catharanthine and vindoline. A mutant line of C. roseus (M2-1865) with an altered MIA profile was identified in a screen of 4000 M2 lines generated by ethylmethanesulfonate (EMS) chemical mutagenesis. While this line did not accumulate vinblastine due to reduced levels of vindoline within the leaves, significant levels of 2,3-epoxide derivatives of tabersonine accumulated on the leaf surface. Detailed nucleotide, amino acid, and enzyme activity analyses of tabersonine 3-reductase in the M2-1865 line showed that a single amino acid substitution (H189Y) diminished the biochemical activity of T3R by 95%. Genetic crosses showed the phenotype to be recessive, exhibiting standard Mendelian single-gene inheritance. The usefulness of EMS mutagenesis in elucidating MIA biosynthesis is highlighted by the results of this study.

Régulation de la proprotéine convertase subtilisine / kexine type 9 (PCSK9) dans les cellules intestinales Caco-2/15

La proprotéine convertase subtilisine/kexine type 9 (PCSK9) favorise la dégradation post-transcriptionnelle du récepteur des lipoprotéines de faible densité (LDLr) dans les hépatocytes et augmente le LDL-cholestérol dans le plasma. Cependant, il n’est pas clair si la PCSK9 joue un rôle dans l’intestin. Dans cette étude, nous caractérisons les variations de la PCSK9 et du LDLr dans les cellules Caco-2/15 différentiées en fonction d’une variété d’effecteurs potentiels. Le cholestérol (100 µM) lié à l’albumine ou présenté en micelles a réduit de façon significative l’expression génique (30%, p<0,05) et l’expression protéique (50%, p<0,05) de la PCSK9. Étonnamment, une diminution similaire dans le LDLr protéique a été enregistrée (45%, p<0,05). Les cellules traitées avec le 25-hydroxycholestérol (50 µM) présentent également des réductions significatives dans l’ARNm (37%, p<0,01) et la protéine (75%, p<0,001) de la PCSK9. Une baisse des expressions génique (30%, p<0,05) et protéique (57%, p<0,01) a également été constatée dans le LDLr. Des diminutions ont aussi été observées pour la HMG CoA réductase et la protéine liant l’élément de réponse aux stérols SREBP-2. Il a été démontré que le SREBP-2 peut activer transcriptionnellement la PCSK9 par le biais de la liaison de SREBP-2 à son élément de réponse aux stérols situé dans la région proximale du promoteur de la PCSK9. Inversement, la déplétion du contenu cellulaire en cholestérol par l’hydroxypropyl-β-cyclodextrine a augmenté l’expression génique de la PCSK9 (20%, p<0,05) et son contenu protéique (540%, p<0,001), en parallèle avec les niveaux protéiques de SREBP-2. L’ajout des acides biliaires taurocholate et déoxycholate dans le milieu apical des cellules intestinales Caco-2/15 a provoqué une baisse d’expression génique (30%, p<0,01) et une hausse d’expression protéique (43%, p<0,01) de la PCSK9 respectivement, probablement via la modulation du FXR (farnesoid X receptor). Ces données combinées semblent donc indiquer que la PCSK9 fonctionne comme un senseur de stérols dans le petit intestin.