Comparative Study of Chemical Approaches to the Solid-Phase Synthesis of a Tumor-Seeking α-MSH Analogue

The synthesis of a cyclic melanocortin analogue (H-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂), where the Boc-protected derivative of a metal-chelating pyrazolyl ligand (pz) was inserted as N-terminal residue, was addressed by several different Fmoc/tBu and Boc/Bzl solid-phase strategies. On-resin cyclization was achieved immediately following incorporation of Asp, by condensation of the Asp side chain carboxyl with the Lys side chain primary amine after selective and simultaneous removal of side chain protecting groups. The success of the synthesis was highly dependent on the chemical strategy employed, with Boc/Bzl chemistry giving the best results. On the light of our findings, Fmoc/tBu strategies are not advantageous for the solid-phase synthesis of this particular type of lactam-bridged peptides. Last, but not least, the target peptide was recently found to have promising tumor-seeking properties (J Biol Inorg Chem 13:449–459, 2008).     

Metformin promotes isolated rat liver mitochondria impairment

Metformin, a drug widely used in the treatment of type 2 diabetes, has recently received attention due to the new and contrasting findings regarding its effects on mitochondrial function. In the present study, we evaluated the effect of metformin in isolated rat liver mitochondria status. We observed that metformin concentrations ≥8 mM induce an impairment of the respiratory chain characterized by a decrease in RCR and state 3 respiration. However, only metformin concentrations ≥10 mM affect the oxidative phosphorylation system by decreasing the mitochondrial transmembrane potential and increasing the repolarization lag phase. Moreover, our results show that metformin does not prevent H₂O₂ production, neither protects against lipid peroxidation induced by the pro-oxidant pair ADP/Fe²⁺. In addition, we observed that metformin exacerbates Ca²⁺-induced permeability transition pore opening by decreasing the capacity of mitochondria to accumulate Ca²⁺ and increasing the oxidation of thiol groups. Taken together, our results show that metformin can promote liver mitochondria injury predisposing to cell death. Cristina Carvalho and Sónia Correia contributed equally to this work.     

Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1

Molecular cues, such as netrin 1, guide axons by influencing growth cone motility. Rho GTPases are a family of intracellular proteins that regulate the cytoskeleton, substrate adhesion and vesicle trafficking. Activation of the RhoA subfamily of Rho GTPases is essential for chemorepellent axon guidance; however, their role during axonal chemoattraction is unclear. Here, we show that netrin 1, through its receptor DCC, inhibits RhoA in embryonic spinal commissural neurons. To determine whether netrin 1-mediated chemoattraction requires Rho function, we inhibited Rho signaling and assayed axon outgrowth and turning towards netrin 1. Additionally, we examined two important mechanisms that influence the guidance of axons to netrin 1: substrate adhesion and transport of the netrin receptor DCC to the plasma membrane. We found that inhibiting Rho signaling increased plasma membrane DCC and adhesion to substrate-bound netrin 1, and also enhanced netrin 1-mediated axon outgrowth and chemoattractive axon turning. Conversely, overexpression of RhoA or constitutively active RhoA inhibited axonal responses to netrin 1. These findings provide evidence that Rho signaling reduces axonal chemoattraction to netrin 1 by limiting the amount of plasma membrane DCC at the growth cone, and suggest that netrin 1-mediated inhibition of RhoA activates a positive-feedback mechanism that facilitates chemoattraction to netrin 1. Notably, these findings also have relevance for CNS regeneration research. Inhibiting RhoA promotes axon regeneration by disrupting inhibitory responses to myelin and the glial scar. By contrast, we demonstrate that axon chemoattraction to netrin 1 is not only maintained but enhanced, suggesting that this might facilitate directing regenerating axons to appropriate targets.     

Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site affects phosphate activation.

To identify those residues involved in fructose 6-phosphate binding to the kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase site-directed mutations were engineered at Lys194, Arg195, Arg230, and Arg238. The mutant enzymes were purified to homogeneity by anion exchange and Blue-Sepharose chromatography and/or substrate elution from phosphocellulose columns. Circular dichroism experiments demonstrated that all of the single amino acid mutations had no effect on the secondary structure of the protein. In addition, when fructose-2,6-bisphosphatase activity was measured, all mutants had Km values for fructose 2,6-bisphosphate, Ki values for fructose 6-phosphate, and maximal velocities similar to that of the wild-type enzyme. Mutation of Arg195----Ala, or His, had little or no effect on the maximal velocity of the kinase but increased the Km for fructose 6-phosphate greater than 3,000-fold. Furthermore, the Ka for phosphate for Arg195Ala was increased 100-fold compared with the wild-type enzyme. Mutation of Lys194----Ala had no effect on maximal velocity or the Km for fructose 6-phosphate. Mutation of either Arg230 or Arg238----Ala increased the maximal velocity and the Km for fructose-6 phosphate of the kinase by 2-3-fold but had no effect on fructose-2,6-bisphosphatase. However, the Km values for ATP of the Arg230Ala and Arg238Ala mutants were 30-40-fold higher than that for the wild-type enzyme. Mutation of Gly48----Ala resulted in a form with no kinase activity, but fructose-2,6-bisphosphatase activity was identical to that of the wild-type enzyme. The results indicate that: 1) Arg195 is a critical residue for the binding of fructose 6-phosphate to the 6-phospho-fructo-2-kinase domain, and that interaction of the sugar phosphate with Arg195 is highly specific since mutation of the adjacent Lys194----Ala had no effect on fructose 6-phosphate binding; 2) Arg195 also play an important role in the binding of inorganic phosphate; and 3) Gly48 is an important residue in the nucleotide binding fold of 6-phosphofructo-2-kinase and that both Arg230 and Arg238 are also involved in ATP binding; and 4) the bifunctional enzyme has two separate and independent fructose 6-phosphate binding sites.     

Targeted integration of foreign genes into repetitive sequences of the Brevibacterium lactofermentum chromosome

Abstract The nucleotide sequence of a gene coding for a 37 kDa subunit of a cytosolic malate dehydrogenase of Trichomonas vaginalis was established. The sequences of a gDNA clone and a cDNA clone, which lacked seven amino-terminal codons, were identical, indicating an absence of introns from the gene. Cell fractionation combined with sequencing of peptide fragments of the purified enzyme showed that the gene codes for an expressed cytosolic enzyme. The derived amino acid sequence was closely related to cytosolic malate dehydrogenases from animals and plants and from the eubacteria Thermus aquaticus and Mycobacterium leprae and was more distant from the enzyme of mitochondria and from Escherichia coli and certain other eubacteria. In phylogenetic reconstructions this enzyme shared a most recent common ancestor with other cytosolic enzymes.     

Seed priming with iron and zinc in bread wheat: effects in germination,mitosis and grain yield

Currently, the biofortification of crops like wheat with micronutrients such as iron (Fe) and zinc (Zn) is extremely important due to the deficiencies of these micronutrients in the human diet and in soils. Agronomic biofortification with Fe and Zn can be done through different exogenous strategies such as soil application, foliar spraying, and seed priming. However, the excess of these micronutrients can be detrimental to the plants. Therefore, in the last decade, a high number of studies focused on the evaluation of their phytotoxic effects to define the best strategies for biofortification of bread wheat. In this study, we investigated the effects of seed priming with different dosages (1 mg L^?1 to 8 mg L^?1) of Fe and/or Zn in germination, mitosis and yield of bread wheat cv. ‘Jordão’ when compared with control. Overall, our results showed that: micronutrient dosages higher than 4 mg L^?1 negatively affect the germination; Fe and/or Zn concentrations higher than 2 mg L^?1 significantly decrease the mitotic index and increase the percentage of dividing cells with anomalies; treatments performed with 8 mg L^?1 of Fe and/or 8 mg L^?1 Zn caused negative effects in germination, mitosis and grain yield. Moreover, seed priming with 2 mg L^?1 Fe?+?2 mg L^?1 Zn has been shown to be non-cytotoxic, ensuring a high rate of germination (80%) and normal dividing cells (90%) as well as improving tillering and grain yield. This work revealed that seed priming with Fe and Zn micronutrients constitutes a useful and alternative approach for the agronomic biofortification of bread wheat.     

Metabolic fingerprinting analysis of oil palm reveals a set of differentially expressed metabolites in fatal yellowing symptomatic and non-symptomatic plants

Introduction

Oil palm (E. guineensis), the most consumed vegetable oil in the world, is affected by fatal yellowing (FY), a condition that can lead to the plant’s death. Although studies have been performed since the 1980s, including investigations of biotic and abiotic factors, FY’s cause remains unknown and efforts in researches are still necessary.

Objectives

This work aims to investigate the metabolic expression in plants affected by FY using an untargeted metabolomics approach.

Method

Metabolic fingerprinting analysis of oil palm leaves was performed using ultra high liquid chromatography–electrospray ionization–mass spectrometry (UHPLC–ESI–MS). Chemometric analysis, using principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA), was applied to data analysis. Metabolites identification was performed by high resolution mass spectrometry (HRMS), MS/MS experiments and comparison with databases and literature.

Results

Metabolomics analysis based on MS detected more than 50 metabolites in oil palm leaf samples. PCA and PLS-DS analysis provided group segregation and classification of symptomatic and non-symptomatic FY samples, with a great external validation of the results. Nine differentially expressed metabolites were identified as glycerophosphorylcholine, arginine, asparagine, apigenin 6,8-di-C-hexose, tyramine, chlorophyllide, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, proline and malvidin 3-glucoside-5-(6″-malonylglucoside). Metabolic pathways and biological importance of those metabolites were assigned.

Conclusion

Nine metabolites were detected in a higher concentration in non-symptomatic FY plants. Seven are related to stress factors i.e. plant defense and nutrient absorption, which can be affected by the metabolic depression of these compounds. Two of those metabolites (glycerophosphorylcholine and 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine) are presented as potential biomarkers, since they have no known direct relation to plant stress.