Cytoxicity and Apoptotic Mechanism of Ruthenium(II) Amino Acid Complexes in Sarcoma-180 Tumor Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Apparent protein and energy digestibility and amino acid availability of corn and co-products in extruded diets for nile tilapia, oreochromis niloticus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Learning HMMs for nucleotide sequences from amino acid alignments

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Site and extent of amino acid digestion in dairy cattle fed with corn and its byproducts

The study was conducted to evaluated the site and extent of dry matter (DM), crude protein (CP), methionine (Met), lysine (Lys), and threonine (Thr) digestion of corn and byproducts obtained from corn germ mixed with different amounts of extruded or non-extruded ether extract (EE) in dairy cattle. Treatments consisted in eight types of feed and two processing in a 4 x 2 factorial design. There were four feeds: corn grain cracked (Corn), corn germ meal with 1% EE (CG1), corn germ meal with 7% EE (CG7), and corn germ meal with 10% EE (CG10). The feeds were processed in one of two ways: extruded (Ex) and not extruded. In situ techniques were used to determine DM, CP, Met, Lys, and Thr partial and total tract digestion. A basic diet was compounded of corn germ meal, soybean meal and coastcross hay in a 70: 30 roughage to concentrate ratio. There was no interaction (P>0.05) between feeds and processing method. Extrusion improved (P<0.05) total tract digestibility of corn DM but not CP. Intestinal digestibility was similar (P>0.05) for corn and corn germ meal mixed with 7 and 10% EE, regardless of EE processing method. The CP total tract digestibility of corn germ meal with 1% non-extruded EE was 16.62% higher (P<0.05) than that of the extruded form. The best total CP digestibility was obtained for corn germ meal with 7% EE, independently of the processing method. The effects of EE processing method on partial and total digestibility differed between amino acid. Corn and corn byproduct extrusion may improve dry matter digestibility, but do not necessarily influence crude protein digestion. Ruminal and intestinal digestibility of Met, Lys, and Thr depends on both feed type and processing method. Therefore, amino acid availability should be considered individually.

Modeling amino acid requirements of poultry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identities among actin-encoding cDNAs of the Nile tilapia (Oreochromis niloticus) and other eukaryote species revealed by nucleotide and amino acid sequence analyses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

l-amino acid oxidase from Bothrops marajoensis causes nephrotoxicity in isolated perfused kidney and cytotoxicity in MDCK renal cells

Renal alterations caused by Bothrops venom and its compounds are studied to understand these effects and provide the best treatment. Previously, we studied the renal effect of the whole venom of Bothrops marajoensis and its phospholipase A2 (PLA2), but these effects could not to be attributed to PLA2. To continue the study, we report in this short communication the effects of l-amino acid oxidase from B. marajoensis venom (LAAOBm) on renal function parameter alterations observed in the same model of isolated perfused kidney, as well as the cytotoxic effect on renal cells. LAAOBm caused a decrease in PP, RVR, UF, GFR, %TNa(+) and %TCl(-), very similar to the effects of whole venom using the same model. We also demonstrated its cytotoxicity in MDCK cells with IC50 of 2.5 μg/mL and late apoptotic involvement demonstrated by flow cytometry assays. In conclusion, we suggested that LAAOBm is a nephrotoxic compound of B. marajoensis venom.

Percolated non-Newtonian flow for silicone obtained from exfoliated bioinorganic layered double hydroxide intercalated with amino acid

A simple and scalable procedure was used to obtain thin, stable, homogeneous, and easy-to-handle films composed of silicone derived from dimethicones containing dispersed hydrotalcite-type materials previously organo-modified with amino acids. The absence of the typical X-ray pattern of the bioinorganic LDH filler suggested an exfoliation process that was further indirectly evidenced by a drastic change in the rheological behavior, which turned from a quasi-Newtonian behavior for the silicone free of LDH filler to an extensive developed gel-like structure for the nanocomposite derivatives. Visualized by the shear-thinning exponent of the complex viscosity in the low-frequency range, the percolation threshold was evident for filler loading as low as <5 w/W%, suggesting the presence of a largely developed interface between the filler and the polymer. The increase of more than one order of magnitude in viscosity was explained by the rather strong attrition phenomenon between the tethered amino acid anions and the silicone chains. UVB radiation absorption profiles make such bioinorganic polymer nanocomposites potentially applicable in skin protection. Thermo-gravimetric analysis revealed significant improvement in the thermal stability, especially in the final step of the polymer combustion, thus underlining the role of the hybrid material as a thermal retardant agent. (C) 2011 Elsevier B.V. All rights reserved.

Is the poly (methylene blue)-modified glassy carbon electrode an adequate electrode for the simple detection of thiols and amino acid-based molecules?

This paper describes the preparation, characterization, and use of poly (methylene blue) (PMB)-modified glassy carbon electrodes (GCE) (GCE-PMB) in the detection of the thiols L-cysteine (L-CySH) and N-acetyl cysteine (Acy), and the herbicide glyphosate (GLYP) in pH 5.3 aqueous solution. The polymer film prepared by electropolymerization showed different characteristics such as robustness, stability, and redox properties satisfactorily. The surface coverage concentration (Gamma) of PMB was found to be 7.90 x 10(-9) - mol cm(-2). Moreover, we observed strong adhesion of the polymer film to the electrode surface. The results using GCE-PMB as a sensor indicated that this modified electrode exhibited electrocatalytic activity toward the detection of thiols and glyphosate in 0.1 mol L-1 KO (pH 5.3). Meanwhile, strong adsorption of the analytes on the GCE-PMB electrodes was also observed. Otherwise, using a low concentration (1 x 10(-4) mol L-1) of L-cysteine and N-acetyl cysteine and 8.9 x 10(-6) mol L-1 of glyphosate, separately, it was possible to observe a well-defined electrochemical response, thus providing an opportunity to further understand the applicability of PMB as a sensor for amino acid-based molecules. (C) 2012 Elsevier B.V. All rights reserved.

Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom

The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (K-m of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 mu g of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC50: 22.7 mu g/mL) and on MCF-7 cell line (breast adenocarcinoma, IC50:1.41 mu g/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC50: 2.22 mu g/nnL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions. (C) 2012 Elsevier Ltd. All rights reserved.

The 2:1 cycloadducts from [3+2] 1,3-dipolar cycloaddition of nitrile oxide and vinylacetic acid. Synthesis and liquid crystal behaviour

Four liquid crystals (LC) 3,7a-bis(4-alkyloxyphenyl)-7,7a-dihydro-6H-isoxazolo[2,3-d][1,2,4]oxadiazol-6-yl)acetic acid (7a-d) were synthesised and the mesomorphic behaviour reported. The LCs were characterised as 2: 1 bisadducts, which were obtained from a double [3+2] 1,3-dipolar cycloaddition. In the first step, the cycloaddition of 4-alkyloxyphenylnitrile oxide (4a-d) and vinylacetic acid (5) gave the initial unobserved 1:1 cycloadducts 2-[3-(4-alkyloxyphenyl)-4,5-dihydroisoxazol-5-yl]acetic acid (6a-d). In the second step, the addition of a second equivalent of 4 to 6 yielded the 2: 1 bisadducts 7a-d without any traces of 6. All compounds 7a-d were unstable during the transition from the mesophase to the isotropic state upon first heating as evidenced by the large peaks in the differential scanning calorimetry traces. Due to the chemical instability of the compounds upon heating, the transition temperature related to the smectic C to smectic A transitions was acquired by means of an image processing method. X-Ray diffraction experiments were also used to analyse the liquid-crystalline phases. A theoretical calculation was performed using density functional theory (DFT) methods at the PBE1PBE/6-311+G(2d,p) level (with solvent effect) in order to get information about the energetic profile of the 2: 1 cycloaddition. DFT studies revealed that the cycloaddition process is controlled by the HOMO(dipolarophile) - LUMO(1,3-dipole), and that the double [3+2] 1,3-dipolar cycloaddition reaction is quite possible.

Effects of microbial inoculants and amino acid production by-product on fermentation and chemical composition of sugarcane silages

The objective of this study was to evaluate the chemical composition, fermentation patterns and aerobic stability of sugarcane silages with addition of amino acid production (monosodium glutamate) by-product (APB) and microbial inoculants. Mature sugarcane was chopped and ensiled in laboratory silos (n = 4/treatment) without additives (control) and with APB (10 g/kg), Pioneer 1174® (PIO, 1.0 mg/kg, Lactobacillus plantarum + Streptoccoccus faecium, Pioneer), Lalsil Cana (2.0 mg/kg, Lactobacillus buchineri, Lallemand) or Mercosil Maís 11C33® (1.0 mg/kg, Lactobacillus buchineri + Lactobacillus plantarum + Streptoccoccus faecium, Timac Agro). Fresh silage and silage liquor samples were obtained to assess pH, chemical composition and organic acid concentrations. Silage temperature was recorded throughout seven days to evaluate aerobic stability. The addition of APB decreased lactic acid levels, increased pH and N-NH3 and did not alter ethanol, acetic and butyric acids concentrations or dry matter (DM) losses. Microbial inoculants enhanced acetic acid levels, although only Pioneer 1174® and Mercosil Maís 11C33® lowered ethanol, butyric acid and DM losses. The addition of APB increased CP content and did not modify DM, soluble carbohydrates contents or in vitro dry matter digestibility. Additives did not alter silage maximum temperature or temperature increasing rate; however, Pioneer 1174® and Mercosil Maís 11C33® increased the time elapsed to reach maximum temperature. Monosodium glutamate production by-product does not alter fermentation patterns or aerobic stability of sugarcane silages, whereas homofermentative bacteria can provide silages of good quality.

Ileal amino acid digestibility in canola meals from yellow- and black-seeded Brassica napus and Brassica juncea fed to growing pigs

Twelve ileal cannulated pigs (30.9 ± 2.7 kg) were used to determine the apparent (AID) and standardized (SID) ileal digestibility of protein and AA in canola meals (CM) derived from black- (BNB) and yellow-seeded (BNY) Brassica napus canola and yellow-seeded Brassica juncea (BJY). The meals were produced using either the conventional pre-press solvent extraction process (regular meal) or a new, vacuum-assisted cold process of meal de-solventization (white flakes) to provide 6 different meals. Six cornstarch-based diets containing 35% canola meal as the sole source of protein in a 3 (variety) × 2 (processing) factorial arrangement were randomly allotted to pigs in a 6 × 7 incomplete Latin square design to have 6 replicates per diet. A 5% casein diet was fed to estimate endogenous AA losses. Canola variety and processing method interacted for the AID of DM (P = 0.048), N (P = 0.010), and all AA (P < 0.05), except for Arg, Lys, Phe, Asp, Glu, and Pro. Canola variety affected or tended to affect the AID of most AA but had no effect on the AID of Lys, Met, Val, Cys, and Pro, whereas processing method had an effect on only Lys and Asp and tended to affect the AID of Thr, Gly and Ser. The effects of canola variety, processing method, and their interaction on the SID values for N and AA followed a similar pattern as for AID values. For the white flakes, SID of N in BJY (74.2%) was lower than in BNY and BNB, whose values averaged 78.5%; however, among the regular meals, BJY had a greater SID value for N than BNY and BNB (variety × processing, P = 0.015). For the white flakes, the SID of Ile (86.4%), Leu (87.6%), Lys (88.9%), Thr (87.6%) and Val (84.2%) in BNB were greater than BNY and BJY. Opposite results were observed for the regular processing, with SID of Lys (84.1%), Met (89.5%), Thr (84.1%), and Val (83.6%) being greater in BJY, followed by BNB and BNY(variety × processing, P < 0.057). The SID of Met was greatest for the white flakes (90.2%) but least for the regular processing (83.0%) in BNY (variety × processing, P < 0.057). It was concluded that the AID and SID of N and AA of the CM tested varied according to canola variety and the processing method used. Overall, the SID values for Ile, Leu, Lys, Met, Thr, and Val averaged across CM types and processing methods were 81.8, 82.6, 83.4, 85.9, 80.8, and 78.4%, respectively.

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

Parallel amino acid replacements in the rhodopsins of the rockfishes (Sebastes spp.) associated with shifts in habitat depth

Among various groups of fishes, a shift in peak wavelength sensitivity has been correlated with changes in their photic environments. The genus Sebastes is a radiation of marine fish species that inhabit a wide range of depths from intertidal to over 600 m. We examined 32 species of Sebastes for evidence of adaptive amino acid substitution at the rhodopsin gene. Fourteen amino acid positions were variable among these species. Maximum likelihood analyses identify several of these to be targets of positive selection. None of these correspond to previously identified critical amino acid sites, yet they may in fact be functionally important. The occurrence of independent parallel changes at certain amino acid positions reinforces this idea. Reconstruction of habitat depths of ancestral nodes in the phylogeny suggests that shallow habitats have been colonized independently in different lineages. The evolution of rhodopsin appears to be associated with changes in depth, with accelerated evolution in lineages that have had large changes in depth.