Supercritical CO(2) extraction of carotenoids from pitanga fruits (Eugenia uniflora L.)

Supercritical carbon dioxide (SC-CO(2)) extraction was employed to extract carotenoids from the freeze-dried pulp of pitanga fruits (Eugenia uniflora L.), an exotic fruit, rich in carotenoids and still little explored commercially. The SC-CO(2) extraction was carried out at two temperatures, 40 and 60 degrees C, and seven pressures, 100, 150, 200, 250, 300, 350 and 400 bar. The carotenoids were determined by high-performance liquid chromatography connected to photodiode array and mass spectrometry detectors. Lycopene, rubixanthin and P-cryptoxanthin were the main carotenoids present in the freeze-dried pitanga pulp, whereas beta-cryptoxanthin concentration was negligible in the SC-CO(2) extracts, for all the investigated state conditions. The maximum recovery of carotenoids was obtained at 60 degrees C and 250 bar, extracting 55% of the total carotenoid content, 74% of the rubixanthin and 78% of the lycopene from the pulp. Under these state conditions, the total carotenoid concentration in the extract was 5474 mu g/g, represented by 66% lycopene and 32% rubixanthin. The experimental state conditions produced different SC-CO(2) extracts with respect to the extraction yield and concentration of different carotenoids, indicating that the supercritical carbon dioxide was selective in the extraction of the pitanga carotenoids as a function of temperature and pressure. (C) 2008 Elsevier B.V. All rights reserved.

Associations of dietary dark-green and deep-yellow vegetables and fruits with cervical intraepithelial neoplasia: modification by smoking

Smoking has been positively and fruit and vegetable intake has been negatively associated with cervical cancer, the second most common cancer among women worldwide. However, a lower consumption of fruits and reduced serum carotenoids have been observed among smokers. It is not known whether the smoking effect on the risk of cervical neoplasia is modified by a low intake of fruits and vegetables. The present study examined the combined effects of tobacco smoking and diet using a validated FFQ and serum carotenoid and tocopherol levels on cervical intraepithelial neoplasia grade 3 (CIN3) risk in a hospital-based case-control study conducted in Sao Paulo, Brazil, between 2003 and 2005. The sample comprised 231 incident, histologically confirmed cases of CIN3 and 453 controls. A low intake (<= 39 g) of dark-green and deep-yellow vegetables and fruits without tobacco smoking had a lesser effect on CIN3 (OR 1.14; 95% CI 0.49, 2.65) than among smokers with higher intake (>= 40 g; OR 1.83; 95% CI 0.73, 4.62) after adjusting for confounders. The OR for the joint exposure of tobacco smoking and low intake of vegetables and fruits was greater (3.86; 95% CI 1.74, 8.57; P for trend < 0.001) compared with non-smokers with higher intake after adjusting for confounding variables and human papillomavirus status. Similar results were observed for total fruit, serum total carotene (including beta-, alpha-and gamma-carotene) and tocopherols. These findings suggest that the effect of nutritional factors on CIN3 is modified by smoking.

Effects of N supply on the accumulation of photosynthetic pigments and photoprotectors in Gracilaria tenuistipitata (Rhodophyta) cultured under UV radiation

We have studied the effects of nitrate supply under photosynthetic active radiation (PAR) plus ultraviolet radiation (UVR) exposure on photosynthetic pigments (chlorophyll a and carotenoids), photoprotective UV screen mycosporine-like amino acids (MAAs), and photosynthetic parameters, including the maximum quantum yield (F(v)/F(m)) and electron transport rate (ETR) on the red agarophyte Gracilaria tenuistipitata. Apical tips of G. tenuistipitata were cultivated under ten different concentrations of NO(3)(-) for 7 days. It has been shown that G. tenuistipitata cultured under laboratory conditions has the ability to accumulate high amounts of MAAs following a nitrate concentration-dependent manner under PAR+UVR. Two MAAs were identified, shinorine and porphyra-334. The relative concentration of the first increased under high concentrations of nitrate, while the second one decreased. The presence of antheraxanthin is reported for the first time in this macro-algae, which also contains zeaxanthin, lutein, and beta-carotene. The accumulation of pigments, photoprotective compounds, and photosynthetic parameters of G. tenuistipitata is directly related to N availability. All variables decreased under low N supplies and reached constant maximum values with supplements higher than 0.5 mM NO(3)(-). Our results suggest a high potential to acclimation and photoprotection against stress factors (including high PAR and UVR) directly related to N availability for G. tenuistipitata.

Ethylene and polyamine interactions in morphogenesis of Passiflora cincinnata: effects of ethylene biosynthesis and action modulators, as well as ethylene scavengers

Ethylene is a plant hormone that is of fundamental importance to in vitro morphogenesis, but in many species, it has not been thoroughly studied. Its relationship with polyamines has been studied mainly because the two classes of hormones share a common biosynthetic precursor, S-adenosylmethionine (SAM). In order to clarify whether competition between polyamines and ethylene influences in vitro morphogenetic responses of Passiflora cincinnata Mast., a climacteric species, different compounds were used that act on ethylene biosynthesis and action, or as ethylene scavengers. Treatment with the ethylene inhibitor, aminoethoxyvinylglycine (AVG) caused a greater regeneration frequency in P. cincinnata, whereas treatment with the ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid (ACC) lessened regeneration frequencies. The data suggested that levels of polyamines and ethylene are not correlated with morphogenic responses in P. cincinnata. It was ascertained that neither the absolute ethylene and polyamine levels, nor competition between the compounds, correlated to the obtained morphogenic responses. However, sensitivity to, and signaling by, ethylene appears to play an important role in differentiation. This study reinforces previous reports regarding the requirement of critical concentrations and temporal regulation of ethylene levels for morphogenic responses. Temporal regulation also appeared to be a key factor in competition between the two biosynthetic pathways, without having any effects on morphogenesis. Further studies investigating the silencing or overexpression of genes related to ethylene perception, under the influence of polyamines in cell differentiation are extremely important for the complete understanding of this process.

Retinoic Acid and VEGF Delay Smooth Muscle Relative to Endothelial Differentiation to Coordinate Inner and Outer Coronary Vessel Wall Morphogenesis

Rationale: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. Objective: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. Methods and Results: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. Conclusion: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels. (Circ Res. 2010;107:204-216.)

Improved renal function after kidney transplantation is associated with heme oxygenase-1 polymorphism

Heme oxygenase-1 (HO-1) has a microsatellite polymorphism based on the number of guanosine-thymidine nucleotide repeats (GT) repeats that regulates expression levels and could have an impact on organ survival post-injury. We correlated HO-1 polymorphism with renal graft function. The HO-1 gene was sequenced (N = 181), and the allelic repeats were divided into subclasses: short repeats (S) (< 27 repeats) and long repeats (L) (>= 27 repeats). A total of 47.5% of the donors carried the S allele. The allograft function was statistically improved six months, two and three yr after transplantation in patients receiving kidneys from donors with an S allele. For the recipients carrying the S allele (50.3%), the allograft function was also better throughout the follow-up, but reached statistical significance only three yr after transplantation (p = 0.04). Considering only those patients who had chronic allograft nephropathy (CAN; 74 of 181), allograft function was also better in donors and in recipients carrying the S allele, two and three yr after transplantation (p = 0.03). Recipients of kidney transplantation from donors carrying the S allele presented better function even in the presence of CAN.

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)). Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA). Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l(-1)) were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l(-1) 2,4-D and 2 mg.l(-1) BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.

Microbial diversity associated with algae, ascidians and sponges from the north coast of Sao Paulo state, Brazil

Little is known about the microbial diversity associated with marine macroorganisms, despite the vital role microorganisms may play in marine ecosystems. The aim of the present study was to investigate the diversity of bacteria and fungi isolated from eight marine invertebrate and one algae samples. Data derived from ARDRA and sequencing analyses allowed the identification of marine-derived microorganisms isolated from those samples. Microbial strains identified up to the genus level revealed 144 distinct ribotypes out of 256 fungal strains and 158 distinct ribotypes out of 181 bacterial strains. Filamentous fungi were distributed among 24 different genera belonging to Ascomycota, Zygomycota and Basidiomycota, some of which had never been reported in the literature as marine invertebrate-inhabiting fungi (Pestalotiopsis, Xylaria, Botrysphaeria and Cunnninghamella). Bacterial isolates were affiliated to 41 different genera, being Bacillus, Ruegeria, Micrococcus, Pseudovibrio and Staphylococcus the most abundant ones. Results revealed an unexpected high microbial diversity associated to the macroorganisms which have been collected and suggested the selection of certain microbial taxonomic groups according to the host. The combined data gathered from this investigation contribute to broaden the knowledge of microbial diversity associated to marine macroorganisms, including as a promising source for the discovery of new natural products. (C) 2009 Elsevier GmbH. All rights reserved.

Diet and serum micronutrients in relation to cervical neoplasia and cancer among low-income Brazilian women

Cervical cancer is a leading cancer among women in developing countries. Infection with oncogenic human papillomavirus (HPV) types has been recognized as a necessary cause of this disease. Serum carotenoids and tocopherols have also been associated with risk for cervical neoplasia, but results from previous studies were not consistent. We evaluated the association of serum total carotene and tocopherols, and dietary intakes with the risk of newly diagnosed, histologically confirmed cervical intraepithelial neoplasia (CIN) grades 1, 2, 3 and invasive cancer in a hospital-based case-control study in Sao Paulo, Brazil. The investigation included 453 controls and 4 groups of cases (CIN1, n = 140; CIN2, n = 126; CIN3, n = 231; invasive cancer, n = 108) recruited from two major public clinics between 2003 and 2005. Increasing concentrations of serum lycopene were negatively associated with CIN1, CIN3 and cancer, with odds ratios (OR) (95% CI) for the highest compared to the lowest tertile of 0.53 (0.27-1.00, p for trend = 0.05), 0.48 (0.22-1.04, p for trend = 0.05) and 0.18 (0.06-0.52, p for trend = 0.002), respectively, after adjusting for confounding variables and HPV status. Increasing concentrations of serum alpha- and gamma-tocopherols, and higher dietary intakes of dark green and deep yellow vegetables/fruit were associated with nearly 50% decreased risk of CIN3. These results support the evidence that a healthy and balanced diet leading to provide high serum levels of antioxidants may reduce cervical neoplasia risk in low-income women.

Dietary predictors of serum total carotene in low-income women living in Sao Paulo, south-east Brazil

Objective Dietary intake and nutritional status of antioxidant vitamins have been reported to protect against some cancers The objective of the present study was to assess the correlations between serum levels of carotenoids (including beta-, alpha- and gamma-carotene), lycopene, retinol, alpha- and gamma-tocopherols, and dietary intakes estimated by an FFQ, among low-income women in the Brazilian Investigation into Nutrition and Cervical Cancer Prevention (BRINCA) study. Design Cross-sectional study of data for 918 women aged 21-65 years participating in the BRINCA study in Sao Paulo city. Multiple linear regression models were used with serum nutrient levels as the dependent variable and dietary intake levels as the independent variable, adjusted for confounding factors. Results In energy-adjusted analyses, the intakes of dark green and deep yellow vegetables and fruits (partial R(2) = 4.8%), total fruits and juices (partial R(2) = 1.8%), vegetables and fruits (partial R(2) = 1.8%), carrots (partial R(2) = 1.4%) and citrus fruits and juices only (partial R(2) = 0.8%) were positively correlated only with serum total carotene levels, after adjusting for serum total cholesterol concentration, age, hospital attended, smoking status. BMI and presence of cervical lesions Multiple-adjusted serum levels of carotenoids were positively correlated with intake quartiles of dark green and deep yellow vegetables and fruits and total fruits and juices independent of smoking status. Conclusions The intake of specific fruits and vegetables was an independent predictor of serum total carotene levels in low-income women living in Sao Paulo

The influence of maternal factors on the concentration of vitamin A in mature breast milk

Background & aims: This study evaluated the relationship between vitamin A concentration in maternal milk and the characteristics of the donors of a Brazilian human milk bank. Material and methods: A total of 136 donors were selected in 2003-2004 for micronutrient determinations in breast milk and blood, anthropometric measurements and investigation of obstetric, socioeconomic-demographic factors, and life style. Maternal serum/milk samples were obtained for vitamin A, iron, copper, and zinc determinations. Vitamin A concentrations in breast milk and blood were assessed by high-performance liquid chromatography. Copper, zinc and iron concentrations in breast milk, and copper and zinc concentrations in blood were detected by atomic emission spectrophotometry. Serum ceruloplasmin and serum iron were determined, respectively, by nephelometry and colorimetry. A linear regression model assessed the associations between milk concentrations of vitamin A and maternal factors. Results: Vitamin A in milk presented positive associations with iron in milk (p < 0.001), serum retinol (p = 0.03), maternal work (p = 0.02), maternal age (p = 0.02). and oral contraceptive use (p = 0.01), and a negative association with % body fat (p = 0.01) (R(2) = 0.47). Conclusion: These results suggest that some nutritional, obstetric, and socioeconomic-demographic factors may have an effect on mature breast milk concentrations of vitamin A in apparently healthy Brazilian mothers. (C) 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Deficiency of the Cytoskeletal Protein SPECC1L Leads to Oblique Facial Clefting

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L. are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Speed 1 1 is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.

Spider-fed bromeliads: seasonal and interspecific variation in plant performance

Background and Aims Several animals that live on bromeliads can contribute to plant nutrition through nitrogen provisioning (digestive mutualism). The bromeliad-living spider Psecas chapoda (Salticidae) inhabits and breeds on Bromelia balansae in regions of South America, but in specific regions can also appear on Ananas comosus (pineapple) plantations and Aechmea distichantha. Methods Using isotopic and physiological methods in greenhouse experiments, the role of labelled ((15)N) spider faeces and Drosophila melanogaster flies in the nutrition and growth of each host plant was evaluated, as well as seasonal variation in the importance of this digestive mutualism. Key Results Spiders contributed 0.6 +/- 0.2% (mean +/- s.e.; dry season) to 2.7 +/- 1% (wet season) to the total nitrogen in B. balansae, 2.4 +/- 0.4% (dry) to 4.1 +/- 0.3% (wet) in An. comosus and 3.8 +/- 0.4% (dry) to 5 +/- 1% (wet) in Ae. distichantha. In contrast, flies did not contribute to the nutrition of these bromeliads. Chlorophylls and carotenoid concentrations did not differ among treatments. Plants that received faeces had higher soluble protein concentrations and leaf growth (RGR) only during the wet season. Conclusions These results indicate that the mutualism between spiders and bromeliads is seasonally restricted, generating a conditional outcome. There was interspecific variation in nutrient uptake, probably related to each species` performance and photosynthetic pathways. Whereas B. balansae seems to use nitrogen for growth, Ae. distichantha apparently stores nitrogen for stressful nutritional conditions. Bromeliads absorbed more nitrogen coming from spider faeces than from flies, reinforcing the beneficial role played by predators in these digestive mutualisms.

Ethylene and polyamine production patterns during in vitro shoot organogenesis of two passion fruit species as affected by polyamines and their inhibitor

Levels of ethylene and polyamines (PAs) were measured during organogenesis of hypocotyl explants of two species of passion fruit (Passiflora cincinnata Masters and Passiflora edulis Sims f. flavicarpa Degener `FB-100`) to better understand the relationships of these regulators and their influence on cell differentiation and morphogenesis. Moreover, histological investigation of shoot ontogenesis was conducted to characterize the different events involved in cell redifferentiation and regulation of PA and ethylene levels. A delay was observed in morphogenic responses of P. edulis f. flavicarpa as compared to P. cincinnata, and these changes coincided with production of elevated levels of polyamine and ethylene levels. During differentiation, cells showed high rates of expansion and elongation, and high ethylene levels were associated with high PA levels, suggesting that the two biosynthesis pathways were highly regulated. Moreover, their interaction might be an important factor for determining cell differentiation. The addition of PAs to the culture medium did not promote organogenesis; however, the incorporation of the PA inhibitor methylglyoxal bisguanylhydrazone in the culture medium reduced shoot bud differentiation, suggesting the need to maintaining a minimum level of PAs for morphogenic events to take place.

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.