Conservation of dual-targeted proteins in Arabidopsis and rice points to a similar pattern of gene-family evolution

Gene duplication followed by acquisition of specific targeting information and dual targeting were evolutionary strategies enabling organelles to cope with overlapping functions. We examined the evolutionary trend of dual-targeted single-gene products in Arabidopsis and rice genomes. The number of paralogous proteins encoded by gene families and the dual-targeted orthologous proteins were analysed. The number of dual-targeted proteins and the corresponding gene-family sizes were similar in Arabidopsis and rice irrespective of genome sizes. We show that dual targeting of methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase, and tyrosyl-tRNA synthetase was maintained despite occurrence of whole-genome duplications in Arabidopsis and rice as well as a polyploidization followed by a diploidization event (gene loss) in the latter.

Glutamine in vitro supplementation partly reverses impaired macrophage function resulting from early weaning in mice

Objective: Glutamine is one of the most abundant amino acids found in maternal milk, and its concentration increases throughout lactation. Because glutamine is essential for macrophage functionality, it is hereby suggested that early weaning in conjunction with the absence of glutamine consumption impairs the functioning of macrophages, which could in turn be reversed with an in vitro supplementation with glutamine. Methods: Swiss Webster mice were early weaned at 14 d of age (EW group) or at 21 d of age (control group, n = 8 per group). The EW group was fed a glutamine-free diet from days 14 to 21 of life. Results: Mice in the EW group presented a significant decrease in plasma and muscle concentrations of glutamine and an increase in the activity of glutamine synthetase in the muscle. Peritoneal macrophages obtained from animals in the EW group presented a significant increase in the production of interleukin (IL)-10 and a significant decrease in the synthesis of IL-1 beta, IL-6, tumor necrosis factor-a, nitric oxide, and hydrogen peroxide and in their ability to adhere, spread, phagocytize, and kill fungi. Glutamine in vitro supplementation reversed the decrease in IL-6, nitric oxide, and hydrogen peroxide synthesis and the decrease in the capacity to adhere, spread, and phagocytize in animals of the EW group. Conclusion: These new. data may imply that a lack of glutamine intake in early weaned mice hampers the functioning of peritoneal macrophages. (C) 2008 Elsevier Inc. All rights reserved.

Dietary glutamine supplementation increases the activity of peritoneal macrophages and hemopoiesis in early-weaned mice inoculated with Mycobacterium bovis bacillus Calmette-Guerin

Infants who are breast-fed have been shown to have a lower incidence of certain infectious diseases compared with formula-fed infants. Glutamine is one of the most abundant amino acids found in maternal milk and it is essential for the function of immune system cells such as macrophages. The purpose of this study was to investigate the effect of glutamine supplementation on the function of peritoneal macrophages and on hemopoiesis in early-weaned mice inoculated with Mycobacterium bovis bacillus Calmette-Guerin (BCG). Mice were wearied at 14 d of age and distributed to 2 groups and fed either a glutamine-free diet (n = 16) or a glutamine-supplemented diet (+Gln (n = 16). Both diets were isonitrogenous (with addition of a mixture of nonessential amino acids) and isocaloric. At d 21, 2 subgroups of mice (n = 16) were intraperitoneally injected with BCG and all mice were killed at d 28. Plasma, muscle and liver glutamine concentrations and muscle glutamine synthetase activity were not affected by diet or inoculation with BCG. The +GIn diet led to increased leukocyte and lymphocyte counts in the peripheral blood (P < 0.05) and granulocyte and lymphocyte counts in the bone marrow and spleen (P < 0.05). The +GIn diet increased spreading and adhesion capacities, hydrogen peroxide, nitric oxide, and tumor necrosis factor-alpha (TNF alpha) syntheses and the phagocytic and fungicidal activity of peritoneal macrophages (P < 0.05). The interaction between the +GIn diet and BCG inoculation increased the area under the curve of interleukin (IL)-1 beta and TNF alpha syntheses (P < 0.05). In conclusion, the intake of glutamine increases the function of peritoneal macrophages and hemopoiesis in early-weaned and BCG-inoculated mice. These data have important implications for the design of breast milk substitutes for human infants.

Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications. (C) 2010 Elsevier GmbH. All rights reserved.

Microcystin production by a freshwater spring cyanobacterium of the genus Fischerella

We investigated the production of a hepatotoxic, cyclic heptapeptide, microcystin, by a filamentous branched cyanobacterium belonging to the order Stigonematales, genus Fischerella. The freshwater Fischerella sp. strain CENA161 was isolated from spring water in a small concrete dam in Piracicaba, Sao Paulo State, Brazil, and identified by combining a morphological description with 16S rRNA gene sequencing and phylogenetic analysis. Microcystin (MCYST) analysis performed using an ELISA assay on cultured cells gave positive results. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis detected 33.6 mu g MCYST-LR per gram dry weight of cyanobacterial cells. Microcystin profile revealed by quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS) analysis confirmed the production of MCYST-LR. Furthermore, genomic DNA was analyzed by PCR for sequences similar to the ketosynthase (KS) domain of the type I polyketide synthase gene, which is involved in microcystin biosynthesis. This revealed the presence of a KS nucleotide fragment similar to the mcyD and ndaD genes of the microcystin and nodularin synthetase complexes. Phylogenetic analysis grouped the Fischerella KS sequence together with mcyD sequences of the three known microcystin synthetase operon (Microcystis, Planktothrix and Anabaena) and ndaD of the nodularin synthetase operon, with 100% bootstrap support. Our findings demonstrate that Fischerella sp. CENA161 produces MYCST-LR and for the first time identify a nucleotide sequence putatively involved in microcystin synthesis in this genus. (C) 2009 Elsevier Ltd. All rights reserved.

Mandibular appliance modulates condylar growth through integrins

Functional orthopedic therapy corrects growth discrepancies between the maxilla and mandible, possibly through postural changes in the musculature and modulation of the mandibular condylar cartilage growth. Using Wistar rats, we tested the hypothesis that chondrocytes respond to forces generated by a mandibular propulsor appliance by changes in gene expression, and that integrins are important mediators in this response. Immunohistochemical analyses demonstrated that the use of the appliance for different periods of time modulated the expression of fibronectin, alpha 5 and alpha v integrin subunits, as well as cell proliferation in the cartilage. In vitro, cyclic distension of condylar cartilage-derived cells increased fibronectin mRNA, as well as Insulinlike Growth Factor-I and II mRNA and cell proliferation. A peptide containing the Arginine-Glycine-Asparagine sequence (RGD), the main cell-binding sequence in fibronectin, blocked almost all these effects, confirming that force itself modulates the growth of the rat condylar cartilage, and that RGD-binding integrins participate in mechanotransduction.

Dynamics of biochemical and morphophysiological changes during zygotic embryogenesis in Acca sellowiana (Berg.) Burr.

Acca sellowiana (Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a domestication and breeding program. Biotechnological tools have been used to assist in this program. The establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis. Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines (PAs), IAA and ABA, in different stages of A. sellowiana zygotic embryogenesis. Starch is the predominant storage compound during zygotic embryo development. Increased synthesis of amino acids in the cotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages. IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this species as well as in other woody angiosperms.

Nitrogen Metabolism in leaves of a tank epiphytic bromeliad: Characterization of a spatial and functional division

The leaf is considered the most important vegetative organ of tank epiphytic bromeliads due to its ability to absorb and assimilate nutrients. However, little is known about the physiological characteristics of nutrient uptake and assimilation. In order to better understand the mechanisms utilized by some tank epiphytic bromeliads to optimize the nitrogen acquisition and assimilation, a study was proposed to verify the existence of a differential capacity to assimilate nitrogen in different leaf portions. The experiments were conducted using young plants of Vriesea gigantea. A nutrient solution containing NO(3)(-)/NH(4)(+) or urea as the sole nitrogen source was supplied to the tank of these plants and the activities of urease, nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (NADH-GDH) were quantified in apical and basal leaf portions after 1, 3, 6, 9, 12, 24 and 48 h. The endogenous ammonium and urea contents were also analyzed. Independent of the nitrogen sources utilized, NR and urease activities were higher in the basal portions of leaves in all the period analyzed. On the contrary. GS and GDH activities were higher in apical part. It was also observed that the endogenous ammonium and urea had the highest contents detected in the basal region. These results suggest that the basal portion was preferentially involved in nitrate reduction and urea hydrolysis, while the apical region could be the main area responsible for ammonium assimilation through the action of GS and GDH activities. Moreover, it was possible to infer that ammonium may be transported from the base, to the apex of the leaves. In conclusion, it was suggested that a spatial and functional division in nitrogen absorption and NH(4)(+) assimilation between basal and apical leaf areas exists, ensuring that the majority of nitrogen available inside the tank is quickly used by bromeliad`s leaves. (C) 2011 Elsevier GmbH. All rights reserved.

Polyamines, amino acids, IAA and ABA contents during Ocotea catharinensis seed germination

The aim of this work was to Study biochemical variations of IAA (indole-3-acetic acid), ABA (abscisic acid). PAs (polyamines) and amino acids at endogenous levels, during seed germination in Ocotea catharinensis. Seeds were germinated in a vermiculite substratum (100%), samples being collected after 15, 30 and 60 days. Total amino acid levels decreased during the first 15 days. Followed by all increment at the end of germination. Among amino acids, higher concentration was observed in asparagine, this being the predominant amino acid during the whole germination period. Total PAs (free + conjugated) content increased during the first 15 days, followed by a decrease and stabilization between 30 and 60 days of germination. Among the PAs, free putrescine levels rose during the first 15 days, followed by a drop and Stabilization up to 60 days of germination, while spermidine and spermine (spm) contents diminished during the period. Only spin was detected in a conjugated form, with increasing concentrations starting from 30 days on. IAA levels increased during tire first 15 days. followed by a decrease and stabilization until the end of germination (60 days), while ABA contents dwindled during the first 15 days, with similar Values until the end of germination.

Enhancement of the citrulline-nitric oxide cycle in astroglioma cells by the proline-rich peptide-10c from Bothrops jararaca venom

The biological activity of the proline rich decapeptde Bj PRO 10c a processing product of the C type natriuretic peptide precursor protein, expressed in the brain and the venom gland of the pit viper Bothrops jararaca, was originally attributed to the inhibition of the somatic angiotensm converting enzyme activity with subsequent ant hypertensive effect However recent results suggest broader biological activity may also be involved in the cardiovascular effects of this peptide Here we show that Bj PRO 10c enhances and sustains the generation of nitric made (NO) by regulating argininosuccinate synthase activity and thereby velocity of the citrulline NO cycle Bj PRO 10c-mediated effects not restricted to the cardiovascular system since NO production was also induced in cells of astroglial origin Bj PRO 10c was internalized by C6 astroglioma cells where it induces NO production and upregulation of the citrulline NO cycle cells in a dose dependent fashion In view of that, astroglial cells function as L arginine pool for NO production in neighboring neurons, we suggest a regulatory function for Bj PRO-10c on the metabolism of this gaseous neurotransmitter in the CNS Moreover, proliferation of astroglial cells was reduced in the presence of Bj PRO 10c however, cell death was not induced Since NO donors have been studied for the treatment of solid cancers Bj PRO 10c may serve as structural model for developing drugs to improve the effects of cancer therapy based on the peptide`s ability to augment NO production (C) 2010 Elsevier B V All rights reserved

EFFECT OF EXERCISE ON GLUTAMINE SYNTHESIS AND TRANSPORT IN SKELETAL MUSCLE FROM RATS

P>Reductions in plasma glutamine are observed after prolonged exercise. Three hypotheses can explain such a decrease: (i) high demand by the liver and kidney; (ii) impaired release from muscles; and (iii) decreased synthesis in skeletal muscle. The present study investigated the effects of exercise on glutamine synthesis and transport in rat skeletal muscle. Rats were divided into three groups: (i) sedentary (SED; n = 12); (ii) rats killed 1 h after the last exercise bout (EX-1; n = 15); and (iii) rats killed 24 h after the last exercise bout (EX-24; n = 15). Rats in the trained groups swam 1 h/day, 5 days/week for 6 weeks with a load equivalent to 5.5% of their bodyweight. Plasma glutamine and insulin were lower and corticosterone was higher in EX-1 compared with SED rats (P < 0.05 and P < 0.01, respectively). Twenty-four hours after exercise (EX-24), plasma glutamine was restored to levels seen in SED rats, whereas insulin levels were higher (P < 0.001) and costicosterone levels were lower (P < 0.01) than in EX-1. In the soleus, ammonia levels were lower in EX-1 than in SED rats (P < 0.001). After 24 h, glutamine, glutamate and ammonia levels were lower in EX-24 than in SED and EX-1 rats (P < 0.001). Soleus glutamine synthetase (GS) activity was increased in EX-1 and was decreased in EX-24 compared with SED rats (both P < 0.001). The decrease in plasma glutamine concentration in EX-1 is not mediated by GS or glutamine transport in skeletal muscle. However, 24 h after exercise, lower GS may contribute to the decrease in glutamine concentration in muscle.

Oxidative stress protection of Trypanosomes requires selenophosphate synthase

Selenoproteins are characterized by the incorporation of at least one amino acid selenocysteine (Sec-U) encoded by in-frame UGA stop codons. These proteins, as well as the components of the Sec synthesis pathway, are present in members of the bacteria, archaea and eukaryote domains. Although not a ubiquitous pathway in all organisms, it was also identified in several protozoa, including the Kinetoplastida. Genetic evidence has indicated that the pathway is non-essential to the survival of Trypanosoma growing in non-stressed conditions. By analyzing the effects of RNA interference of the Trypanosoma brucei selenophosphate synthetase SPS2, we found a requirement under sub-optimal growth conditions. The present work shows that SPS2 is involved in oxidative stress protection of the parasite and its absence severely hampers the parasite survival in the presence of an oxidizing environment that results in an apoptotic-like phenotype and cell death. (C) 2011 Elsevier B.V. All rights reserved.

Fluorimetric determination of intra- and extracellular free amino acids in the microalgae Tetraselmis gracilis (Prasinophyceae) using monolithic column in reversed phase mode

This paper describes the development and application of an RP HPLC method using a C(18) monolithic stationary phase for the separation and quantification of extra- and intracellular amino acids in a batch cultivation of the marine alga Tetraselmis gracilis. Fluorimetric detection was made after separation of the o-phthaldialdehyde 2-mercaptoethanol (OPA-2MCE) derivatives using a binary gradient elution. Separation of 19 amino acids was achieved with resolution >1.5 in about 39 min at a flow rate of 1.5 mL/min. RSD of analyses in seawater medium ranged from 0.36% for Orn (0.50 mu mol/L) to 12% for Ile (0.10 mu mol/L). The main constituents of the intracellular dissolved free amino acids (DFAAs) in the exponential growth phase were arginine (Arg), asparagine (Asn), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), glutamine (Gln), and leucine (Leu). The major amino acids excreted to the media were valine (Val), Ala, Ser, and Gly. The monolithic phase facilitates the analysis by shortening the separation time and saving solvents and instrumentation costs (indeed conventional HPLC instrumentation can be used, running at lower pressures than those ones used with packed particle columns).

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with ophthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C(18) stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L(-1) phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L-1 phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, SO:SO and 65:35. At a flow rate of 10 mu L s(-1) a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 mu L s(-1) such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 mu mol L(-1) for Tyr to 0.51 mu mol L(-1) for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%. (C) 2008 Elsevier B.V. All rights reserved.