Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp citri

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and spray-inoculated with an Xcc suspension (10(6) cfu mL(-1)). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (non-transgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptI1 or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease. (C) 2009 Elsevier B.V. All rights reserved.

Fatty acid profile and qualitative characteristics of meat from zebu steers fed with different oilseeds

The objective of this study was to evaluate the effects of dietary addition of ground oilseed sources on the quality, fatty acid profile, and CLA content of meat from zebu steers. Thirty-one zebu steers with an initial average age of 23 mo and an initial BW of 365 kg were used in this study. The experimental period was 84 d, which was preceded by an adaption period of 28 d. The diet was provided ad libitum with a forage: concentrate ratio of 40:60. Corn silage was used as the forage source. Four different concentrates were formulated for each treatment: without additional lipids (control) or with ground soybeans (SB), ground cottonseed (CS), or ground linseed (LS). The SB, CS, and LS diets were formulated to have 6.5% ether extract on a total dietary DM basis. The experiment was set up as a completely randomized design. After slaughter, samples were taken from the longissimus thoracis muscle for the measurement of fatty acid concentration and the evaluation of meat quality. The luminosity index was greater in the control and LS diets (P < 0.01). The greatest percentages of myristic acid (C14:0), palmitic acid (C16:0), trans octadecenoic acid (C18:1 trans-10, trans-11, or trans-12), and SFA in the subcutaneous fat were observed in the CS treatment (P < 0.01). Moreover, the least percentages of oleic acid (C18:1 cis-9) and total unsaturated fatty acids in the subcutaneous fat were observed in the CS diet (P < 0.01). The meat linoleic acid and a-linolenic acid percentages were greatest in the SB and LS treatments, respectively (P < 0.001). The unsaturated fatty acid: SFA ratio was smallest for the CS diet (P < 0.01). A gradual increase in oxidation was observed as a function of storage time; however, the diets did not affect the rancidity of the meat (P > 0.05). The fatty acid profile of subcutaneous fat was impaired by the addition of CS. Supplying ground oilseeds did not increase the content of CLA in the meat.

Effect of the Gamma Radiation Dose Rate on Psychrotrophic Bacteria, Thiobarbituric Acid Reactive Substances, and Sensory Characteristics of Mechanically Deboned Chicken Meat

Frozen samples of mechanically deboned chicken meat (MDCM) with skin were irradiated with gamma radiation doses of 0.0 kGy (control) and 3 kGy at 2 different radiation dose rates: 0.32 kGy/h (3 kGy) and 4.04 kGy/h (3 kGy). Batches of irradiated and control samples were evaluated during 11 d of refrigerated (2 +/- 1 degrees C) storage for the following parameters: total psychrotrophic bacteria count, thiobarbituric acid reactive substances (TBARS), evaluation of objective color (L*, a*, and b*) and a sensory evaluation (irradiated odor, oxidized odor, pink and brown colors). No statistical difference (P > 0.05) was found amongst the TBARS values obtained for the MDCM samples irradiated with dose rates of 0.32 and 4.04 kGy/h. There was a significant increase (P < 0.05) in the psychrotrophic bacterial count as from the 7th day of refrigerated storage, for the MDCM samples irradiated at the dose rate of 4.04 kGy/h. With respect to the attribute of oxidized odor, the samples irradiated with a dose rate of 0.32 kGy/h showed a stronger intensity and were significantly different (P < 0.05) from the sample irradiated with a dose rate of 4.04 kGy/h on days 0 and 2 of refrigerated storage. Irradiation with a dose rate of 4.04 kGy/h (3 kGy) was shown to be the best condition for the processing of MDCM according to the evaluation of all the variables, under the conditions of this study. Practical Application The results obtained for the application of different dose rates of ionizing radiation to mechanically deboned chicken meat will provide the food industry with information concerning the definition of the best processing conditions to maximize the sensory and food quality.

Determination of the median lethal concentration (LC(50)) of mycoinsecticides for the control of Ceratitis capitata (Diptera: Tephritidae)

The aim of this study was to determine the median lethal concentration (LC(50)) of the commercial products Boveril WP (R) (Beauveria bassiana) and Metarril WP (R) (Metarhizium anisopliae) on the larvae and pupae of the fruit Ceratitis capitata. Insects used in this study came from a laboratory colony. The evaluated product concentrations were 10.00, 15.00, 20.00 and 25.00 g/L of water, which correspond, respectively, to 5.00x10(9), 7.50x10(9), 10.00x10(9) and 12.50x10(9) viable conidia/L of water for the two products, and in the control only water was applied. Third instar larvae and pupae of C. capitata were used in this study. Results showed an overall mortality of larvae with all conidial concentrations of M. anisopliae. The LC(50) values for larvae were 2.99 and 2.97 g/L for Boveril (R) and Metarril (R), respectively, while for pupae they were 3.12 and 4.74 g/L for Boveril (R) and Metarril (R), respectively. The high pathogenicity demonstrated by lower conidial concentrations of the tested products may mean greater efficiency from both economic and environmental points of view.

Influence of magnesium status and magnesium intake on the blood glucose control in patients with type 2 diabetes

Background & aims: This study was undertaken to assess magnesium intake and magnesium status in patients with type 2 diabetes, and to identify the parameters that best predict alterations in fasting glucose and plasma magnesium. Methods: A cross-sectional study was carried out in patients with type 2 diabetes (n = 51; 53.6 +/- 10.5 y) selected within the inclusion factors, at the University Hospital Onofre Lopes. Magnesium intake was assessed by three 24-h recalls. Urine, plasma and erythrocytes magnesium, fasting and 2-h postprandial glucose, HbA1, microalbuminuria, proteinuria, and serum and urine creatinine were measured. Results: Mean magnesium intake (9.37 +/- 1.76 mmol/d), urine magnesium (2.80 +/- 1.51 mmol/d), plasma magnesium (0.71 +/- 0.08 mmol/L) and erythrocyte magnesium (1.92 +/- 0.23 mmol/L) levels were low. Seventy-seven percent of participants presented one or more magnesium status parameters below the cut-off points of 3.00 mmol/L for urine, 0.75 mmol/L for plasma and 1.65 mmol/L for erythrocytes. Subjects presented poor blood glucose control with fasting glucose of 8.1 +/- 3.7 mmol/L, 2-h postprandial glucose of 11.1 +/- 5.1 mmol/L, and HbA1 of 11.4 +/- 3.0%. The parameters that influenced fasting glucose were urine, plasma and dietary magnesium, while plasma magnesium was influenced by creatinine clearance. Conclusions: Magnesium status was influenced by kidney depuration and was altered in patients with type 2 diabetes, and magnesium showed to play an important role in blood glucose control. (C) 2011 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Consumer`s evaluation of the effects of gamma irradiation and natural antioxidants on general acceptance of frozen beef burger

The effect of addition of rosemary and oregano extracts on the sensory quality of irradiated beef burger was investigated. Batches of beef burgers were prepared with 400 ppm of rosemary or oregano extract and a group prepared with 200 ppm of synthetic butyl-hydroxytoluene (BHT)/butyl-hydroxy-anisol (BHA) was used as a control. Half of each formulation was irradiated at the maximum dose allowed for frozen meat (7 kGy). Samples were kept under frozen conditions (-20 degrees C) during the whole storage period, including during irradiation. Two analyses were performed after 20 and 90 days to verify the influence of the addition of the different types of antioxidants and the effect of irradiation and storage time on the acceptance of the product. Thirty-three and thirty-four untrained panelists were invited to participate in the first and second test, respectively. A structured hedonic scale ranging from 1 to 9 points was used in both analyses. BHT/BHA formulation obtained the highest score (6.73) and regarding the natural antioxidants, oregano received better acceptance (6.36). Irradiated samples formulated with oregano received a lower score, 6.03 in the first test and 5.06 in the second one, compared to the non-irradiated sample (6.36 and 5.79). In the second test (90 days), the sample formulated with BHT/BHA and which was irradiated received a higher score (6.59) when compared to the non-irradiated one (5.85). In both tests, the irradiated samples formulated with rosemary extract obtained a better score compared to the non-irradiated one, the scores being 5.00-3.82 and 5.00-3.76 in the first and second test, respectively. Our results allowed us to conclude that the natural antioxidants, rosemary and oregano extracts, present a good alternative for replacing synthetic additives in food industries, and that the irradiation process, in some cases, may help to enhance the sensory quality of food. (C) 2008 Elsevier Ltd. All rights reserved.

Abnormal Neurovascular Control in Anabolic Androgenic Steroids Users

NUNES ALVES, M. J. N., M. R. DOS SANTOS, R. G. DIAS, C. A. AKIHO, M. C. LATERZA, M. U. P. B. RONDON, R. L. DE MORAES MOREAU, and C. E. NEGRAO. Abnormal Neurovascular Control in Anabolic Androgenic Steroids Users. Med. Sci. Sports Exerc., Vol. 42, No. 5, pp. 865-871, 2010. Purpose: Previous studies showed that anabolic androgenic steroids (AAS) increase vascular resistance and blood pressure (BP) in humans. In this study, we tested the hypotheses 1) that AAS users would have increased muscle sympathetic nerve activity (MSNA) and reduced forearm blood flow (FBF) compared with AAS nonusers and 2) that there would be an association between MSNA and 24-h BP. Methods: Twelve AAS users aged 31 +/- 2 yr (means +/- SE) and nine age-matched AAS nonusers aged 29 T 2 yr participated in the study. All individuals were involved in strength training for at least 2 yr. AAS was determined by urine test (chromatography-mass spectrometry). MSNA was directly measured by microneurography technique. FBF was measured by venous occlusion plethysmography. BP monitoring consisted of measures of BP for 24 h. Results: MSNA was significantly higher in AAS users than that in AAS nonusers (29 +/- 3 vs 20 +/- 1 bursts per minute, P = 0.01). FBF (1.92 +/- 0.17 vs 2.77 +/- 0.24 mL.min(-1).100 mL(-1), P = 0.01) and forearm vascular conductance (2.01 +/- 0.17 vs 2.86 +/- 0.31 U, P = 0.02) were significantly lower in AAS users than that in AAS nonusers. Systolic (131 +/- 4 vs 120 +/- 3 mm Hg, P = 0.001), diastolic (74 +/- 4 vs 68 +/- 3 mm Hg, P = 0.02), and mean BP (93 +/- 4 vs 86 +/- 3 mm Hg, P = 0.005) and heart rate (74 +/- 3 vs 68 +/- 3 bpm, P = 0.02) were significantly higher in AAS users when compared with AAS nonusers. Further analysis showed that there was a significant correlation between MSNA and 24-h mean BP (r = 0.75, P = 0.002). Conclusions: AAS increases MSNA and reduces muscle blood flow in young individuals. In addition, the increase in BP levels in AAS users is associated with augmented sympathetic outflow. These findings suggest that AAS increases the susceptibility for cardiovascular disease in humans.

Detection of the TLR4 1196C > T Polymorphism by Mismatched-Polymerase Chain Reaction Using Plasmid DNA as Internal Control in Restriction Fragment Length Polymorphism Assays

Background: Restriction fragment length polymorphism (RFLP) is a common molecular assay used for genotyping, and it requires validated quality control procedures to prevent mistyping caused by impaired endonuclease activity. We have evaluated the usefulness of a plasmid-based internal control in RFLP assays. Results: Blood samples were collected from 102 individuals with acute myocardial infarction (AMI) and 108 non-AMI individuals (controls) for DNA extraction and laboratory analyses. The 1196C> T polymorphism in the toll-like receptor 4 (TLR4) gene was amplified by mismatched-polymerase chain reaction (PCR). Amplicons and pBluescript II SK-plasmid were simultaneously digested with endonuclease HincII. Fragments were separated on 2% agarose gels. Plasmid was completely digested using up to 55.2 nmL/L DNA solutions and 1 mu L PCR product. Nevertheless, plasmid DNA with 41.4 nM or higher concentrations was incompletely digested in the presence of 7 mL PCR product. In standardized conditions, TLR4 1196C> T variant was accurately genotyped. TLR4 1196T allele frequency was similar between AMI (3.1%) and controls (2.0%, p = 0.948). TLR4 SNP was not associated with AMI in this sample population. In conclusion, the plasmid-based control is a useful approach to prevent mistyping in RFLP assays, and it is validate for genetic association studies such as TLR4 1196C> T.

Apoptosis induction by 4-nerolidylcatechol in melanoma cell lines

Pothomorphe umbellata, a native Brazilian plant, is popularly known to be effective in the treatment of skin lesions. This benefit is attributed to 4-nerolidylcatechol (4-NC) a compound extracted from P. umbellata. Since melanomas show prominent resistance to apoptosis and exhibit extreme chemoresistance to multiple forms of therapy, novel compounds addressing induction of cell death are worth investigating. Here, we evaluated effects on cell cycle progression and possible cytotoxic activity of 4-NC in melanoma cell lines as well as human dermal fibroblasts. Inhibitory effects on cell invasion and MMP activity were also investigated. 4-NC showed cytotoxic activity for all melanoma cell lilies tested (IC(50) = 20-40 mu M, 24 h for tumoral cell lines: IC(50) = 50 mu M for fibroblast cell line) associated with its capacity to induce apoptosis. Furthermore, this is the first time that 4-NC is described as an inhibitor of cell invasiveness, due mainly to a G I cell cycle arrest and inhibition of MMP-2 activity in melanoma cell lines. (C) 2008 Elsevier Ltd. All rights reserved.

Influence of ammonium sulphate feeding time on fed-batch Arthrospira (Spirulina) platensis cultivation and biomass composition with and without pH control

Previous work demonstrated that a mixture of NH(4)Cl and KNO(3) as nitrogen source was beneficial to fed-batch Arthrospira (Spirulina) platensis cultivation, in terms of either lower costs or higher cell concentration. On the basis of those results, this study focused on the use of a cheaper nitrogen source mixture, namely (NH(4))(2)SO(4) plus NaNO(3), varying the ammonium feeding time (T = 7-15 days), either controlling the pH by CO(2) addition or not. A. platensis was cultivated in mini-tanks at 30 degrees C, 156 mu mol photons m(-2) s(-1), and starting cell concentration of 400 mg L(-1), on a modified Schlosser medium. T = 13 days under pH control were selected as optimum conditions, ensuring the best results in terms of biomass production (maximum cell concentration of 2911 mg L(-1), cell productivity of 179 mg L(-1) d(-1) and specific growth rate of 0.77 d(-1)) and satisfactory protein and lipid contents (around 30% each). (C) 2011 Elsevier Ltd. All rights reserved.

Sterilization of Medical Devices by Ethylene Oxide, Determination of the Dissipation of Residues, and Use of Green Fluorescent Protein as an Indicator of Process Control

Ethylene oxide (EO) is used to sterilize Oxygenator and Tubing applied to heart surgery. Residual levels of EO and its derivatives, ethylene chlorohydrin (ECH) and ethylene glycol (EG), may be hazardous to the patients. Therefore, it must be removed by the aeration process. This study aimed to estimate the minimum aeration time for these devices to attain safe limits for use (avoiding excessive aeration time) and to evaluate the Green Fluorescent Protein (GFP) as a biosensor capable of best indicating the distribution and penetration of EO gas throughout the sterilization chamber. Sterilization cycles of 2, 4, and 8 h were monitored by Bacillus atrophaeus ATCC 9372 as a biological indicator (131) and by the GFP. Residual levels of EO, ECH, and EG were determined by gas chromatography (GC), and the residual dissipation was studied. Safe limits were reached right after the sterilization process for Oxygenator and after 204 h of aeration for Tubing. In the 2 h cycle, the GFP concentration decreased from 4.8 (+/- 3.2)% to 7.5 (+/- 2.5)%. For the 4 h cycle, the GFP concentration decreased from 17.4 (+/- 3.0)% to 21.5 (+/- 6.8)%, and in the 8 h cycle, it decreased from 22.5 (+/- 3.2)% to 23.9 (+/- 3.9)%. This finding showed the potentiality for GFP applications as an EO biosensor. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 9113: 626-630, 2009

Ultra-Fast Gradient LC Method for Omeprazole Analysis Using a Monolithic Column: Assay Development, Validation, and Application to the Quality Control of Omeprazole Enteric-Coated Pellets

A method was optimized for the analysis of omeprazole (OMZ) by ultra-high speed LC with diode array detection using a monolithic Chromolith Fast Gradient RP 18 endcapped column (50 x 2.0 mm id). The analyses were performed at 30 degrees C using a mobile phase consisting of 0.15% (v/v) trifluoroacetic acid (TFA) in water (solvent A) and 0.15% (v/v) TFA in acetonitrile (solvent B) under a linear gradient of 5 to 90% B in 1 min at a flow rate of 1.0 mL/min and detection at 220 nm. Under these conditions, OMZ retention time was approximately 0.74 min. Validation parameters, such as selectivity, linearity, precision, accuracy, and robustness, showed results within the acceptable criteria. The method developed was successfully applied to OMZ enteric-coated pellets, showing that this assay can be used in the pharmaceutical industry for routine QC analysis. Moreover, the analytical conditions established allow for the simultaneous analysis of OMZ metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in the same run, showing that this method can be extended to other matrixes with adequate procedures for sample preparation.

Antinociceptive and anti-inflammatory properties of 7-hydroxycoumarin in experimental animal models: potential therapeutic for the control of inflammatory chronic pain

Objectives In the present study we investigated the anti nociceptive, anti-inflammatory and antipyretic effects of 7-hydroxycoumarin (7-HC) in animal models. Methods The effects of oral 7-HC were tested against acetic acid-induced writhing, formalin test, tail flick test, complete Freund`s adjuvant (CFA)-induced hypemociception, carrageenan-induced paw oedema, lipopolysaccharide-induced fever and the rota rod test. Key findings 7-HC (3-60 mg/kg) produced a dose-related antinociception against acetic acid-induced writhing in mice and in the formalin test. In contrast, treatment with 7-HC did not prevent thermal nociception in the tail flick test. A single treatment with 7-HC, 60 mg/kg, produced a long-lasting antinociceptive effect against CFA-induced hypernociception, a chronic inflammatory pain stimulus. Notably, at 60 mg/kg per day over 4 days the administration of 7-HC produced a continuous antinociceptive effect against CFA-induced hypernociception. 7-HC (30-120 mg/kg) produced anti-inflammatory and antipyretic effects against carrageenan-induced inflammation and lipopolysaccharide-induced fever, respectively. Moreover, 7-HC was found to be safe with respect to ulcer induction. In the rota rod test, 7-HC-treated mice did not show any motor performance alterations. Conclusions The prolonged antinociceptive and anti-inflammatory effects of 7-HC, in association with its low ulcerogenic activity, indicate that this molecule might be a good candidate for development of new drugs for the control of chronic inflammatory pain and fever.