Fluorescent peptide hormones: development of high affinity vasopressin analogues.

Highly potent and specific peptide hormone analogues with fluorescent reporter groups are current research goals. Until now, however, only moderately potent analogues have been described. We report here several types of vasopressin (VP) analogues with different fluorophores attached to the peptide. In a first series, fluorophores were attached to the free epsilon amino function of [des-amino1-lysine8]VP (dLVP), producing agonistic analogues. In a second series, reporter groups were added to the N-terminal of open-chain antagonist structures. The biological activities of these analogues were assessed by two different sets of experiments: 1) The measurement of their binding affinities towards the V1a-vasopressin receptor subtype from WRK1 cells or rat liver membrane preparations; 2) Their ability to stimulate the phospholipase C activity in WRK1 cells. As expected, a simple acylation of fluorophores to dLVP resulted in a considerable loss of affinity. If however, the Lys8 side chain was extended through double Schiff-base formation with glutaraldehyde-ethylenediamine followed by reduction to an aminoalkyl aminoalkylamine, single fluorophores could be added without loss of affinity compared to VP. The open-chain analogues, on the other hand, while displaying weak affinity, nevertheless exhibited pure antagonistic behavior.     

Effect of cell therapy with allogeneic osteoblasts on bone repair of rat calvaria defects

Background aims

Regenerative medicine strategies based on cell therapy are considered a promising approach to repair bone defects. The aims of this study were to evaluate the effect of subculturing on the osteogenic potential of osteoblasts derived from newborn rat calvaria and the effect of these osteoblasts on bone repair of rat calvaria defects.

Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution

Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [¹³C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [¹³C]glutamate and [¹⁵N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the ¹³C/¹⁵N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.Plants are sessile, unable to relocate when exposed to diverse environmental and seasonal stimuli, and hence must be able to respond rapidly to survive stress conditions. Flooding or waterlogging of the soil is a common environmental condition that can greatly affect crop production and quality by blocking the entry of oxygen into the soil so that roots and other belowground organs cannot maintain respiration. In recent decades, the number of extreme floodings has strongly increased, which is especially tragic because most arable land worldwide is located in regions that are threatened by regular flooding events (Voesenek and Bailey-Serres, 2015).In plant heterotrophic tissues, respiratory metabolism is composed of various pathways, including glycolysis, the mitochondrial tricarboxylic acid cycle, and the mitochondrial electron transport chain. Under normal conditions, the conversion of Glc to pyruvate in the cytosol involves an initial input of ATP and produces the reduced cofactor NADH. The reactions of the tricarboxylic acid cycle occur within the mitochondrial matrix and lead to the complete oxidation of pyruvate, moving electrons from organic acids to the oxidized redox cofactors NAD⁺ and FAD, forming the reducing equivalents NADH and FADH₂ and concomitantly releasing carbon dioxide (Tovar-Méndez et al., 2003; Millar et al., 2011). Finally, the reduced cofactors generated during glycolysis and the tricarboxylic acid cycle are subsequently oxidized by the mitochondrial electron transport chain to fuel ATP synthesis by a process known as oxidative phosphorylation (Fernie et al., 2004; Plaxton and Podesta, 2006). The tricarboxylic acid cycle turnover rate depends greatly on the rate of NADH reoxidation by the mitochondrial electron transport chain and on the cellular rate of ATP utilization (Plaxton and Podesta, 2006). Besides supporting ATP synthesis, the reactions of the tricarboxylic acid cycle also contribute to the production of key metabolic intermediates for use in many other fundamental biosynthetic processes elsewhere in the cell (Fernie et al., 2004; Sweetlove et al., 2010; van Dongen et al., 2011; Araújo et al., 2012). Nevertheless, the control and regulation of the carbon flux through the tricarboxylic acid cycle are still poorly understood in plants, and noncyclic modes have been described to operate under certain circumstances (Rocha et al., 2010; Sweetlove et al., 2010; Araújo et al., 2012).Upon hypoxia, respiratory energy (ATP) production via oxidative phosphorylation by the mitochondrial electron transport chain goes down. To compensate for this, the glycolytic flux increases and Glc is consumed faster in an attempt to produce ATP via the glycolytic pathway, a process known as the Pasteur effect. To survive short-term hypoxia during flooding or waterlogging, plants must generate sufficient ATP and regenerate NADP⁺ and NAD⁺, which are required for glycolysis (Narsai et al., 2011; van Dongen et al., 2011). In addition to the accumulation of ethanol and lactate in oxygen-deprived plant tissues, metabolites such as Ala, succinate, and γ-aminobutyric acid (GABA) have also been shown to accumulate (Sousa and Sodek, 2003; Kreuzwieser et al., 2009; van Dongen et al., 2009; Rocha et al., 2010; Zabalza et al., 2011), although hardly anything is known about the fate of these products of hypoxic metabolism. However, the relative abundance of these products of hypoxic metabolism varies between plant species, genotypes, and tissues and can change throughout the course of oxygen limitation stress as well (Narsai et al., 2011).A model describing metabolic changes during hypoxia has been described previously for waterlogged roots of the highly flood-tolerant model crop legume Lotus japonicus (Rocha et al., 2010): upon waterlogging, the rate of pyruvate production is enhanced due to the activation of glycolysis (Pasteur effect) and the concomitant production of ATP via substrate-level phosphorylation. At the same time, the fermentation pathway is activated with the accumulation of lactate via lactate dehydrogenase and ethanol via two subsequent reactions catalyzed by pyruvate decarboxylase and alcohol dehydrogenase (Tadege et al., 1999). The amount of pyruvate produced can be reduced via alanine aminotransferease (AlaAT), which catalyzes the reversible reaction interconverting pyruvate and Glu to Ala and 2-oxoglutarate (2OG). Concomitantly, 2OG was suggested to reenter the tricarboxylic acid cycle to be used to produce another ATP and also succinate, which accumulates in the cell (Rocha et al., 2010). This Ala pathway provides a means for the role of Ala accumulation during hypoxia via reorganization of the tricarboxylic acid cycle. Furthermore, given that the use of this strategy prevents pyruvate accumulation, the continued operation of glycolysis during waterlogging can occur.It should be noted, however, that measurements of metabolite levels alone do not provide information about the actual activity of the metabolic pathways involved. Furthermore, the previous studies did not reveal which enzymes of the tricarboxylic acid cycle change their activity that leads to reorganization of the tricarboxylic acid cycle. To overcome this, analysis of metabolism using isotope-labeled substrates has proven to be essential for understanding the control and regulation of metabolic networks, and it has often been observed that significant changes in carbon flow are sometimes associated with only small adjustments in metabolite abundance (Schwender et al., 2004; Ratcliffe and Shachar-Hill, 2006). Metabolomics studies that require extensive metabolite labeling utilize uniformly labeled stable isotope tracers. Alternatively, detailed analysis of central carbon metabolism can make use of positional labeling as well. Following the extraction of labeled metabolites, the ¹³C label redistribution is measured usually with NMR or gas chromatography-mass spectrometry methods (Jorge et al., 2015). Schwender and Ohlrogge (2002) used both labeling approaches to investigate embryo development in Brassica napus seeds. While uniformly labeled [¹³C₆]Glc and [¹³C₁₂]Suc were applied to determine the metabolic flux through the major pathways of carbon metabolism, positionally labeled [1,2-¹³C]Glc was used to specifically outline the glycolytic/oxidative pentose phosphate pathway network during embryo development (Schwender and Ohlrogge, 2002). Gas chromatography-mass spectrometry analysis was used in this study to evaluate the ¹³C enrichment and isotopomer composition. In earlier studies of hypoxic metabolism, positionally labeled [1-¹³C]Glc was used to specifically investigate energy metabolism and pH regulation in hypoxic maize (Zea mays) root tips (Roberts et al., 1992; Edwards et al., 1998).In this study, we performed stable isotope labeling experiments using wild-type soybean (Glycine max) roots in order to better understand the dynamics of metabolism in operation in plant cells under hypoxic conditions. For this, we used fully labeled ¹³C and ¹⁵N tracers rather than positional labeling, as this allowed us to cover a broad view of the central carbon and nitrogen metabolic network. The labeling pattern of metabolites was subsequently measured with gas chromatography-time of flight-mass spectrometry (GC-TOF-MS). Our studies confirm the activity of Ala metabolism while revealing the parallel activity of the GABA shunt. The results provide evidence that the bifurcation of the tricarboxylic acid cycle results from the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), also known as complex II of the mitochondrial electron transport chain (mETC).     

An in vitro growth inhibition test for measuring the potency of Leptospira spp. Sejroe group vaccine in buffaloes

Leptospira spp. serovars Hardjo and Wollfi from Sejroe serogroup have been detected in livestock in Brazil, where the main control procedures rely on vaccination. The potency of two commercial vaccines available in this country was monitored by microagglutination test-MAT and in vitro growth inhibition test-GIT in serum samples from 33 female buffaloes divided into: G1-unvaccinated control; G2-vaccinated with Leptobac-6^® containing serovars Hardjo and Wolffi and G3-vaccinated with Triangle-9^® containing serovar Hardjo. G2 and G3 animals were vaccinated on day zero, and received a booster and two revaccinations on days 30, 210 and 390 and G1 animals received phosphate buffered saline. Serum samples were collected at 15-day intervals between days 0 and 60; and at 30-day intervals between days 60 and 540 and were tested by MAT and GIT with serovars Hardjo and Wolffi. G1 remained negative throughout the experiment. Both vaccines were able to induce agglutinating and growth inhibition antibodies. Six months after the last revaccination, all animals tested negative by MAT, but still were positive by GIT until the end of experimental period. GIT could be a good tool to evaluate the potency and to monitor antibodies responses of vaccines of Sejroe group serovars.     

The role of anticipatory postural adjustments in compensatory control of posture: 2. Biomechanical analysis

The central nervous system (CNS) utilizes anticipatory (APAs) and compensatory (CPAs) postural adjustments to maintain equilibrium while standing. It is known that these postural adjustments involve displacements of the center of mass (COM) and center of pressure (COP). The purpose of the study was to investigate the relationship between APAs and CPAs from a kinetic and kinematic perspective. Eight subjects were exposed to external predictable and unpredictable perturbations induced at the shoulder level while standing. Kinematic and kinetic data were recorded and analyzed during the time duration typical for anticipatory and compensatory postural adjustments. When the perturbations were unpredictable, the COM and COP displacements were larger compared to predictable conditions with APAs. Thus, the peak of COM displacement, after the pendulum impact, in the posterior direction reached 28 ± 9.6 mm in the unpredictable conditions with no APAs whereas it was 1.6 times smaller, reaching 17 ± 5.5 mm during predictable perturbations. Similarly, after the impact, the peak of COP displacement in the posterior direction was 60 ± 14 mm for unpredictable conditions and 28 ± 3.6 mm for predictable conditions. Finally, the times of the peak COM and COP displacements were similar in the predictable and unpredictable conditions. This outcome provides additional knowledge about how body balance is controlled in presence and in absence of information about the forthcoming perturbation. Moreover, it suggests that control of posture could be enhanced by better utilization of APAs and such an approach could be considered as a valuable modality in the rehabilitation of individuals with balance impairment.     

Activation of membrane phospholipase C by vasopressin. A requirement for guanyl nucleotides

Vasopressin stimulates the liberation of labelled inositol phosphate in partially purified plasma membranes prepared from myo-[3H]inositol prelabelled WRK1 cells. This stimulatory effect was very rapid (165% stimulation of inositol trisphosphate accumulation after a 10 s incubation period in the presence of 1 microM vasopressin), concentration dependent (EC50 = 12 nM) and was abolished by an antagonist of the vasopressor response to vasopressin. GTP, even at high concentrations (0.1 mM), did not increase inositol phosphate release: it was found to be absolutely necessary for hormonal stimulation of phospholipase C activity. Non-hydrolysable analogues of GTP may also stimulate this enzyme activity.     

Production of biosurfactant by a genetically-modified strain ofBacillus subtilis expressing green fluorescent protein

Biosurfactant production was investigated using two strains ofBacillus subtilis, being one a reference strain (B. subtilis 1012) and the other a genetically-modified strain (B. subtilis W1012) made able to produce the green fluorescent protein (GFP). A new method based on oil displacement technique was set up to measure the biosurfactant level in the medium. Although the tested microorganisms showed similar results in terms of cell growth parameters, the recombinant strain, besides expressing GFP, exhibited an average yield of extracellular surfactant on biomass (Y _{B/X, av} =239 mg_B g_x ^?1) more than twice that of the reference strain. The ability of the genetically-modified strain to simultaneously overproduce biosurfactant and GFP even at low cell concentration makes it an interesting candidate for possible use as a biological index-finger to monitor cell viability in bioremediation and oil recovery operations.