Exogenous ornithine is an effective precursor and the delta-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves

The role of the delta-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Delta(1)-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS. GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Delta(1)-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves. (C) 2011 Elsevier GmbH. All rights reserved.

Genetic polymorphisms modulate the folate metabolism of Brazilian individuals with Down syndrome

Individuals with Down syndrome (DS) carry three copies of the Cystathionine beta-synthase (C beta S) gene. The increase in the dosage of this gene results in an altered profile of metabolites involved in the folate pathway, including reduced homocysteine (Hcy), methionine, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Furthermore, previous studies in individuals with DS have shown that genetic variants in genes involved in the folate pathway influence the concentrations of this metabolism's products. The purpose of this study is to investigate whether polymorphisms in genes involved in folate metabolism affect the plasma concentrations of Hcy and methylmalonic acid (MMA) along with the concentration of serum folate in individuals with DS. Twelve genetic polymorphisms were investigated in 90 individuals with DS (median age 1.29 years, range 0.07-30.35 years; 49 male and 41 female). Genotyping for the polymorphisms was performed either by polymerase chain reaction (PCR) based techniques or by direct sequencing. Plasma concentrations of Hcy and MMA were measured by liquid chromatography-tandem mass spectrometry as previously described, and serum folate was quantified using a competitive immunoassay. Our results indicate that the MTHFR C677T, MTR A2756G, TC2 C776G and BHMT G742A polymorphisms along with MMA concentration are predictors of Hcy concentration. They also show that age and Hcy concentration are predictors of MMA concentration. These findings could help to understand how genetic variation impacts folate metabolism and what metabolic consequences these variants have in individuals with trisomy 21.

N-4-Phenyl-substituted 2-acetylpyridine thiosemicarbazones: Cytotoxicity against human tumor cells, structure-activity relationship studies and investigation on the mechanism of action

N-4-Phenyl 2-acetylpyridine thiosemicarbazone (H2Ac4Ph; N-(phenyl)-2-(1-(pyridin-2-yl)ethylidene) hydrazinecarbothioamide) and its N-4-ortho-, -meta- and -para-fluorophenyl (H2Ac4oFPh, H2Ac4mFPh, H2Ac4pFPh), N-4-ortho-, -meta- and -para-chlorophenyl (H2Ac4oClPh, H2Ac4mClPh, H2Ac4pClPh), N-4-ortho-, -meta- and -para-iodophenyl (H2Ac4oIPh, H2Ac4mIPh, H2Ac4pIPh) and N-4-ortho-, -meta- and -para-nitrophenyl (H2Ac4oNO(2)Ph, H2Ac4mNO(2)Ph, H2Ac4pNO(2)Ph) derivatives were assayed for their cytotoxicity against human malignant breast (MCF-7) and glioma (T98G and U87) cells. The compounds were highly cytotoxic against the three cell lineages (IC50: MCF-7, 52-0.16 nM; T98G, 140-1.0 nM; U87, 160-1.4 nM). All tested thiosemicarbazones were more cytotoxic than etoposide and did not present any haemolytic activity at up to 10(-5) M. The compounds were able to induce programmed cell death. H2Ac4pClPh partially inhibited tubulin assembly at high concentrations and induced cellular microtubule disorganization. (C) 2012 Elsevier Ltd. All rights reserved.

Increasing the activity of copper(II) complexes against Leishmania through lipophilicity and pro-oxidant ability

Copper complexes with fluorinated beta-diketones were synthesized and characterized in terms of lipophilicity and peroxide-assisted oxidation of dihydrorhodamine as an indicator of redox activity. The biological activity of the complexes was tested against promastigotes of Leishmania amazonensis. Inhibition of trypanosomatid-specific trypanothione reductase was also tested. It was found that the highly lipophilic and redox-active bis(trifluoroacetylacetonate) derivative had increased toxicity towards promastigotes. These results indicate that it is possible to modulate the activity of metallodrugs based on redox-active metals through the appropriate choice of lipophilic chelators in order to design new antileishmanials. Further work will be necessary to improve selectivity of these compounds against the parasite.

Determination of the intrinsic kinetic parameters of sulfide-oxidizing autotrophic denitrification in differential reactors containing immobilized biomass

Nitrogen removal coupled with sulfide oxidation has potential for the treatment of effluents from anaerobic reactors because they contain sulfide, which can be used as an endogenous electron donor for denitrification. This work evaluated the intrinsic kinetics of sulfide-oxidizing autotrophic denitrification via nitrate and nitrite in systems containing attached cells. Differential reactors were fed with nitrified synthetic domestic sewage and different sulfide concentrations. The intrinsic kinetic parameters of nitrogen removal were determined when the mass transfer resistance was negligible. This bioprocess could be described by a half-order kinetic model for biofilms. The half-order kinetic coefficients ranged from 0.425 to 0.658 mg N-1/2 L-1/2 h(-1) for denitrification via nitrite and from 0.190 to 0.609 mg N-1/2 L-1/2 h(-1) for denitrification via nitrate. In this latter, the lower value was due to the use of electrons donated from intermediary sulfur compounds whose formation and subsequent consumption were detected. (C) 2011 Elsevier Ltd. All rights reserved.

Advice on the Management of Ambiguous Genitalia to a Young Endocrinologist from Experienced Clinicians

The birth of a child with ambiguous genitalia is a challenging and distressing event for the family and physician and one with life-long consequences. Most disorders of sexual differentiation (DSD) associated with ambiguous genitalia are the result either of inappropriate virilization of girls or incomplete virilization of boys. It is important to establish a diagnosis as soon as possible, for psychological, social, and medical reasons, particularly for recognizing accompanying life-threatening disorders such as the salt-losing form of congenital adrenal hyperplasia. In most instances, there is sufficient follow-up data so that making the diagnosis also establishes the appropriate gender assignment (infants with congenital adrenal hyperplasia, those with androgen resistance syndromes), but some causes of DSD such as steroid 5 alpha-reductase 2 deficiency and 17 beta-hydroxysteroid dehydrogenase deficiency are associated with frequent change in social sex later in life. In these instances, guidelines for sex assignment are less well established.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). Methods: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Results: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56-4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% alpha-helix, 39% beta-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys(52) residue and the amino acids Pro(45), Thr(49) and Arg(128) are conserved as in other 2-Cys-Prx. General significance: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. (C) 2012 Elsevier B.V. All rights reserved.

Radiation of the Tnt1 retrotransposon superfamily in three Solanaceae genera

Abstract Background Tnt1 was the first active plant retrotransposon identified in tobacco after nitrate reductase gene disruption. The Tnt1 superfamily comprises elements from Nicotiana (Tnt1 and Tto1) and Lycopersicon (Retrolyc1 and Tlc1) species. The study presented here was conducted to characterise Tnt1-related sequences in 20 wild species of Solanum and five cultivars of Solanum tuberosum. Results Tnt1-related sequences were amplified from total genomic DNA using a PCR-based approach. Purified fragments were cloned and sequenced, and clustering analysis revealed three groups that differ in their U3 region. Using a network approach with a total of 453 non-redundant sequences isolated from Solanum (197), Nicotiana (140) and Lycopersicon (116) species, it is demonstrated that the Tnt1 superfamily can be treated as a population to resolve previous phylogenetic multifurcations. The resulting RNAseH network revealed that sequences group according to the Solanaceae genus, supporting a strong association with the host genome, whereas tracing the U3 region sequence association characterises the modular evolutionary pattern within the Tnt1 superfamily. Within each genus, and irrespective of species, nearly 20% of Tnt1 sequences analysed are identical, indicative of being part of an active copy. The network approach enabled the identification of putative "master" sequences and provided evidence that within a genus these master sequences are associated with distinct U3 regions. Conclusion The results presented here support the hypothesis that the Tnt1 superfamily was present early in the evolution of Solanaceae. The evidence also suggests that the RNAseH region of Tnt1 became fixed at the host genus level whereas, within each genus, propagation was ensured by the diversification of the U3 region. Different selection pressures seemed to have acted on the U3 and RNAseH modules of ancestral Tnt1 elements, probably due to the distinct functions of these regions in the retrotransposon life cycle, resulting in both co evolution and adaptation of the element population with its host.

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

Abstract Background Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. Results In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation – cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-β-glucosidase) in mycelium cells; and ags (an α-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport – two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells – isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. Conclusion Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.

Seasonal variations of melatonin in ram seminal plasma are correlated to those of testosterone and antioxidant enzymes

Abstract Background Some breeds of sheep are highly seasonal in terms of reproductive capability, and these changes are regulated by photoperiod and melatonin secretion. These changes affect the reproductive performance of rams, impairing semen quality and modifying hormonal profiles. Also, the antioxidant defence systems seem to be modulated by melatonin secretion, and shows seasonal variations. The aim of this study was to investigate the presence of melatonin and testosterone in ram seminal plasma and their variations between the breeding and non-breeding seasons. In addition, we analyzed the possible correlations between these hormones and the antioxidant enzyme defence system activity. Methods Seminal plasma from nine Rasa Aragonesa rams were collected for one year, and their levels of melatonin, testosterone, superoxide dismutase (SOD), glutathione reductase (GRD), glutathione peroxidase (GPX) and catalase (CAT) were measured. Results All samples presented measurable quantities of hormones and antioxidant enzymes. Both hormones showed monthly variations, with a decrease after the winter solstice and a rise after the summer solstice that reached the maximum levels in October-November, and a marked seasonal variation (P < 0.01) with higher levels in the breeding season. The yearly pattern of GRD and catalase was close to that of melatonin, and GRD showed a significant seasonal variation (P < 0.01) with a higher activity during the breeding season. Linear regression analysis between the studied hormones and antioxidant enzymes showed a significant correlation between melatonin and testosterone, GRD, SOD and catalase. Conclusions These results show the presence of melatonin and testosterone in ram seminal plasma, and that both hormones have seasonal variations, and support the idea that seasonal variations of fertility in the ram involve interplay between melatonin and the antioxidant defence system.

The antioxidant response of the liver of male Swiss mice raised on a AIN 93 or commercial diet

Background: Reactive oxygen species (ROS) are formed under natural physiological conditions and are thought to play an important role in many human diseases. A wide range of antioxidants are involved in cellular defense mechanisms against ROS, which can be generated in excess during stressful conditions, these include enzymes and non-enzymatic antioxidants. The aim of this study was to evaluate the antioxidant responses of mice to two diets control, commercial and the purified AIN 93 diet, commonly used in experiments with rodents. Results: Malondialdehyde (MDA) and hydrogen peroxide (H2O2) concentrations and superoxide dismutase (SOD) and glutathione reductase (GR) activities determined in the liver were lower in the group of mice fed with the AIN 93 diet, while catalase (CAT) activity was higher in the same group, when compared to the group fed on the commercial diet. Liver glutathione peroxidase (GSH-Px) activity was similar in the groups fed on either AIN 93 or the commercial diets. Two SOD isoforms, Mn-SODII and a Cu/Zn-SODV, were specifically reduced in the liver of the AIN 93 diet fed animals. Conclusions: The clear differences in antioxidant responses observed in the livers of mice fed on the two diets suggest that the macro- and micro-nutrient components with antioxidant properties, including vitamin E, can promote changes in the activity of enzymes involved in the removal of the ROS generated by cell metabolism.

Plasmatic higher levels of homocysteine in Non-alcoholic fatty liver disease (NAFLD)

Background Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease, which includes a spectrum of hepatic pathology such as simple steatosis, steatohepatitis, fibrosis and cirrhosis. The increased serum levels of homocysteine (Hcy) may be associated with hepatic fat accumulation. Genetic mutations in the folate route may only mildly impair Hcy metabolism. The aim of this study was to investigate the relation between liver steatosis with plasma homocysteine level and MTHFR C677T and A1298C polymorphisms in Brazilian patients with NAFLD. Methods Thirty-five patients diagnosed with NAFLD by liver biopsy and forty-five healthy controls neither age nor sex matched were genotyped for C677T and A1298C MTHFR polymorphisms using PCR-RFLP and PCR-ASA, respectively, and Hcy was determined by HPLC. All patients were negative for markers of Wilson’s, hemochromatosis and autoimmune diseases. Their daily alcohol intake was less than 100 g/week. A set of metabolic and serum lipid markers were also measured at the time of liver biopsies. Results The plasma Hcy level was higher in NAFLD patients compared to the control group (p = 0.0341). No statistical difference for genotypes 677C/T (p = 0.110) and 1298A/C (p = 0.343) in patients with NAFLD and control subjects was observed. The genotypes distribution was in Hardy-Weinberg equilibrium (677C/T p = 0.694 and 1298 A/C p = 0.188). The group of patients and controls showed a statistically significant difference (p < 0.001) for BMI and HOMA_IR, similarly to HDL cholesterol levels (p < 0,006), AST, ALT, γGT, AP and triglycerides levels (p < 0.001). A negative correlation was observed between levels of vitamin B12 and Hcy concentration (p = 0.005). Conclusion Our results indicate that plasma Hcy was higher in NAFLD than controls. The MTHFR C677T and A1298C polymorphisms did not differ significantly between groups, despite the 677TT homozygous frequency was higher in patients (17.14%) than in controls (677TT = 4.44%) (p > 0.05). The suggested genetic susceptibility to the MTHFR C677T and A1298C should be confirmed in large population based studies.

Biochemical and histological characterization of tomato mutants

Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT), we observed that the malondialdehyde (MDA) content was enhanced in the diageotropica (dgt) and lutescent (l) mutants, whilst the highest levels of hydrogen peroxide (H2O2) were observed in high pigment 1 (hp1) and aurea (au) mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT) activity when compared to MT. Guaiacol peroxidase (GPOX) was enhanced in both sitiens (sit) and notabilis (not) mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX). Based on PAGE analysis, the activities of glutathione reductase (GR) isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD) isoform III was reduced in leaves of sit, epi, Never ripe (Nr) and green flesh (gf) mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.

Brazilian blood donation eligibility criteria for dermatologic patients

A focused and commented review on the impact of dermatologic diseases and interventions in the solidary act of donating blood is presented to dermatologists to better advise their patients. This is a review of current Brazilian technical regulations on hemotherapeutic procedures as determined by Ministerial Directive #1353/2011 by the Ministry of Health and current internal regulations of the Hemotherapy Center of Ribeirão Preto, a regional reference center in hemotherapeutic procedures. Criteria for permanent inaptitude: autoimmune diseases (>1 organ involved), personal history of cancer other than basal cell carcinoma, severe atopic dermatitis or psoriasis, pemphigus foliaceus, porphyrias, filariasis, leprosy, extra pulmonary tuberculosis or paracoccidioidomycosis, and previous use of etretinate. Drugs that impose temporary ineligibility: other systemic retinoids, systemic corticosteroids, 5-alpha-reductase inhibitors, vaccines, methotrexate, beta-blockers, minoxidil, anti-epileptic, and anti-psychotic drugs. Other conditions that impose temporary ineligibility: occupational accident with biologic material, piercing, tattoo, sexually transmitted diseases, herpes, and bacterial infections, among others. Discussion: Thalidomide is currently missing in the teratogenic drugs list. Although finasteride was previously considered a drug that imposed permanent inaptitude, according to its short halflife current restriction of 1 month is still too long. Dermatologists should be able to advise their patients about proper timing to donate blood, and discuss the impact of drug withdrawal on treatment outcomes and to respect the designated washout periods.

Oxidative processes during 'Golden' papaya fruit ripening

'Golden' papayas at maturity stage 1 (15% yellow skin) were chosen to study selected oxidative processes, the activity of antioxidant enzymes and lipid peroxidation in storage at 22°C, during the ripening of the fruit. An increase in ethylene production was observed on the second day of storage and it was followed by an increase in respiration. An increased activity of catalase, glutathione reductase and ascorbate peroxidase was observed concurrently or soon after this increase in ethylene production and respiration. The increased activity of these enzymes near the peaks of ethylene production and respiration is related to the production of oxidants accompanying the onset of ripening. On the fourth day of storage, there was an increased lipid peroxidation and decreased activities of catalase, glutathione reductase and superoxide dismutase. Lipid peroxidation induces the increase of antioxidant enzymes, which can be verified by further increases in the activities of catalase, glutathione reductase, superoxide dismutase and ascorbate peroxidase. Unlike the other antioxidant enzymes, the ascorbate peroxidase activity in the pulp increased continuously during ripening, suggesting its important role in combating reactive oxygen species during papaya ripening. With regard to physical-chemical characteristics, the soluble solids did not vary significantly, the acidity and ascorbic acid contents increased, and hue angle and firmness decreased during storage. The results revealed that there was variation in the activity of antioxidant enzymes, with peaks of lipid peroxidation during the ripening of 'Golden' papaya. These results provide a basis for future research, especially with regard to the relationships among the climacteric stage, the activation of antioxidant enzymes and the role of ascorbate peroxidase in papaya ripening.