Dicarboxylic amino acids and glycine-betaine regulate chaperone-mediated protein-disaggregation under stress.

Active protein-disaggregation by a chaperone network composed of ClpB and DnaK + DnaJ + GrpE is essential for the recovery of stress-induced protein aggregates in vitro and in Escherichia coli cells. K-glutamate and glycine-betaine (betaine) naturally accumulate in salt-stressed cells. In addition to providing thermo-protection to native proteins, we found that these osmolytes can strongly and specifically activate ClpB, resulting in an increased efficiency of chaperone-mediated protein disaggregation. Moreover, factors that inhibited the chaperone network by impairing the stability of the ClpB oligomer, such as natural polyamines, dilution, or high salt, were efficiently counteracted by K-glutamate or betaine. The combined protective, counter-negative and net activatory effects of K-glutamate and betaine, allowed protein disaggregation and refolding under heat-shock temperatures that otherwise cause protein aggregation in vitro and in the cell. Mesophilic organisms may thus benefit from a thermotolerant osmolyte-activated chaperone mechanism that can actively rescue protein aggregates, correctly refold and maintain them in a native state under heat-shock conditions.

Effects of prior repeated-sprints with different recovery duration on oxygen uptake kinetics during subsequent heavy-exercise

Introduction: Prior repeated-sprints (6) has become an interesting method to resolve the debate surrounding the principal factors that limits the oxygen uptake (V'O2) kinetics at the onset of exercise [i.e., muscle O2 delivery (5) or metabolic inertia (3)]. The aim of this study was to compare the effects of two repeated-sprints sets of 6x6s separated by different recovery duration between the sprints on V'O2 and muscular de-oxygenation [HHb] kinetics during a subsequent heavy-intensity exercise. Methods: 10 male subjects performed a 6-min constant-load cycling test (T50) at intensity corresponding to half of the difference between V'O2max and the ventilatory threshold. Then, they performed two repeated-sprints sets of 6x6s all-out separated by different recovery duration between the sprints (S1:30s and S2:3min) followed, after 7-min-recovery, by the T50 (S1T50 and S2T50, respectively). V'O2, [HHb] of the vastus lateralis (VL) and surface electromyography activity [i.e., root-mean-square (RMS) and the median frequency of the power density spectrum (MDF)] from VL and vastus medialis (VM) were recorded throughout T50. Models using a bi-exponential function for the overall T50 and a mono-exponential for the first 90s of T50 were used to define V'O2 and [HHb] kinetics respectively. Results: V'O2 mean value was higher in S1 (2.9±0.3l.min-1) than in S2 (1.2±0.3l.min-1); (p<0.001). The peripheral blood flow was increased after sprints as attested by a higher basal heart rate (HRbaseline) (S1T50: +22%; S2T50: +17%; p≤0.008). Time delay [HHb] was shorter for S1T50 and S2T50 than for T50 (-22% for both; p≤0.007) whereas the mean response time of V'O2 was accelerated only after S1 (S1T50: 32.3±2.5s; S2T50: 34.4±2.6s; T50: 35.7±5.4s; p=0.031). There were no significant differences in RMS between the three conditions (p>0.05). MDF of VM was higher during the first 3-min in S1T50 than in T50 (+6%; p≤0.05). Conclusion: The study show that V'O2 kinetics was speeded by prior repeated-sprints with a short (30s) but not a long (3min) inter-sprints-recovery even though the [HHb] kinetics was accelerated and the peripheral blood flow was enhanced after both sprints. S1, inducing a greater PCr depletion (1) and change in the pattern of the fibres recruitment (increase in MDF) compared with S2, may decrease metabolic inertia (2), stimulate the oxidative phosphorylation activation (4) and accelerate V'O2 kinetics at the beginning of the subsequent high-intensity exercise.

Destino de adubos nitrogenados marcados com 15N em amostras de dois solos da Amazônia central

Foi estudado, em condições de casa de vegetação, em Nancy (França), em 1992, o destino de duas formas de fertilizantes nitrogenados, marcados com 15N, sulfato de amônio e uréia, em amostras do horizonte A de dois principais solos da Amazônia Central, classificados como latossolo amarelo e podzólico vermelho-amarelo. A planta teste foi o "rye-grass" da Itália (Lolium multiflorum L.). Em ambos os solos, a uréia foi mais bem utilizada do que o sulfato de amônio. Entre 60 e 70% do N aplicado como uréia foi absorvido pela planta, enquanto, com a aplicação de sulfato de amônio, esses valores variaram entre 44 e 49%. O balanço do 15N no final do ciclo da cultura mostrou que a imobilização do N nos dois solos foi maior na presença de uréia que na de sulfato de amônio. As perdas, estimadas por diferença, foram mais elevadas no tratamento com sulfato de amônio. Considerando que perdas por lixiviação foram praticamente nulas com a técnica de cultivo utilizada, elas devem ter ocorrido essencialmente por via gasosa.

A wearable system for multi-segment foot kinetics measurement.

This study aims to design a wearable system for kinetics measurement of multi-segment foot joints in long-distance walking and to investigate its suitability for clinical evaluations. The wearable system consisted of inertial sensors (3D gyroscopes and 3D accelerometers) on toes, forefoot, hindfoot, and shank, and a plantar pressure insole. After calibration in a laboratory, 10 healthy elderly subjects and 12 patients with ankle osteoarthritis walked 50m twice wearing this system. Using inverse dynamics, 3D forces, moments, and power were calculated in the joint sections among toes, forefoot, hindfoot, and shank. Compared to those we previously estimated for a one-segment foot model, the sagittal and transverse moments and power in the ankle joint, as measured via multi-segment foot model, showed a normalized RMS difference of less than 11%, 14%, and 13%, respectively, for healthy subjects, and 13%, 15%, and 14%, for patients. Similar to our previous study, the coronal moments were not analyzed. Maxima-minima values of anterior-posterior and vertical force, sagittal moment, and power in shank-hindfoot and hindfoot-forefoot joints were significantly different between patients and healthy subjects. Except for power, the inter-subject repeatability of these parameters was CMC>0.90 for healthy subjects and CMC>0.70 for patients. Repeatability of these parameters was lower for the forefoot-toes joint. The proposed measurement system estimated multi-segment foot joints kinetics with acceptable repeatability but showed difference, compared to those previously estimated for the one-segment foot model. These parameters also could distinguish patients from healthy subjects. Thus, this system is suggested for outcome evaluations of foot treatments.

Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness.

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ∼1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ∼1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.

Effects of aerobic fitness on oxygen uptake kinetics in heavy intensity swimming.

This study aimed to characterise both the [Formula: see text] kinetics within constant heavy-intensity swimming exercise, and to assess the relationships between [Formula: see text] kinetics and other parameters of aerobic fitness, in well-trained swimmers. On separate days, 21 male swimmers completed: (1) an incremental swimming test to determine their maximal oxygen uptake [Formula: see text], first ventilatory threshold (VT), and the velocity associated with [Formula: see text] [Formula: see text] and (2) two square-wave transitions from rest to heavy-intensity exercise, to determine their [Formula: see text] kinetics. All the tests involved breath-by-breath analysis of freestyle swimming using a swimming snorkel. [Formula: see text] kinetics was modelled with two exponential functions. The mean values for the incremental test were 56.0 ± 6.0 ml min(-1) kg(-1), 1.45 ± 0.08 m s(-1); and 42.1 ± 5.7 ml min(-1) kg(-1) for [Formula: see text], [Formula: see text] and VT, respectively. For the square-wave transition, the time constant of the primary phase (τ(p)) averaged 17.3 ± 5.4 s and the relevant slow component (A'(sc)) averaged 4.8 ± 2.9 ml min(-1) kg(-1) [representing 8.9% of the end-exercise [Formula: see text] (%A'(sc))]. τ(p) was correlated with [Formula: see text] (r = -0.55, P = 0.01), but not with either [Formula: see text] (r = 0.05, ns) or VT (r = 0.14, ns). The %A'(sc) did not correlate with either [Formula: see text] (r = -0.14, ns) or [Formula: see text] (r = 0.06, ns), but was inversely related with VT (r = -0.61, P < 0.01). This study was the first to describe the [Formula: see text] kinetics in heavy-intensity swimming using specific swimming exercise and appropriate methods. As has been demonstrated in cycling, faster [Formula: see text] kinetics allow higher aerobic power outputs to be attained. The slow component seems to be reduced in swimmers with higher ventilatory thresholds.

Eficiência da fertilização nitrogenada com uréia (15N) em Brachiaria brizantha cv. Marandu associada ao parcelamento de superfosfato simples e cloreto de potássio

Em pastagens, a avaliação da eficiência do processo de fertilização com N-uréia é importante em virtude das altas doses utilizadas. Vários trabalhos relatam que a aplicação de uréia associada com cloreto de potássio (KCl) e superfosfato simples (SFS) pode melhorar a sua eficiência. Na recuperação de pastagens degradadas ou em sistemas de exploração intensiva, as quantidades de KCl e SFS empregadas como fertilização corretiva são altas e aplicadas em novembro, no início da estação das águas. O parcelamento do KCl e SFS associado às coberturas nitrogenadas com uréia seria de fácil adoção sem custos adicionais. Com o objetivo de avaliar este manejo, foi desenvolvido um experimento em que se realizou o balanço anual da aplicação de 15N da uréia quando o SFS e, ou, o KCl foram parcelados, associados à aplicação da uréia em cinco coberturas, ou quando ambos foram aplicados de uma só vez, no mês de novembro. A maior produção de forragem ocorreu quando o SFS foi parcelado, seguido pelo parcelamento do KCl, da aplicação única de ambos e, finalmente, do parcelamento dos dois. A recuperação do N-uréia na parte aérea e no sistema solo-pastagem não diferiu com os tratamentos. Entretanto, a recuperação na coroa da planta e no sistema radicular diferiu entre os tratamentos e mostrou-se positivamente correlacionada com a produção de forragem. A recuperação de fertilizante na liteira foi favorecida pelo parcelamento do KCl. Apesar de não ter sido verificado aumento na recuperação total do sistema entre os tratamentos, o parcelamento do KCl e do SFS tem sido indicado por melhorar a recuperação do N-uréia em várias estruturas da planta, refletindo em maior produção da forrageira.

Recuperação do nitrogênio (15N) da uréia e da palhada por soqueira de cana-de-açúcar (Saccharum spp.)

Desenvolveu-se um experimento com vistas em avaliar a utilização do nitrogênio mineralizado da palhada (15N) e do nitrogênio da uréia (15N) aplicada em soqueira de cana-de-açúcar. O experimento foi desenvolvido em campo, num Argissolo Vermelho-Amarelo (Paleudalf), no município de Piracicaba (SP), de outubro de 1997 a agosto de 1998, e constou de quatro tratamentos: (T1) mistura de vinhaça e uréia aplicada em área total sobre o solo coberto com palhada-15N; (T2) mistura de vinhaça e uréia-15N aplicada em área total sobre o solo coberto com palhada; (T3) mistura de vinhaça e uréia-15N aplicada em área total sobre o solo sem a palhada; (T4) uréia-15N enterrada em sulcos nos dois lados das linhas da cana, com prévia aplicação de vinhaça sobre o solo sem cobertura de palhada. O delineamento experimental foi o de blocos casualizados, com quatro repetições. Comparações de componentes de produtividade da cultura, da acumulação de nitrogênio pela parte aérea e da utilização do nitrogênio da uréia-15N e do mineralizado da palhada-15N, foram realizadas entre os tratamentos. O desenvolvimento vegetal deu-se em um ciclo de 315 dias e foi semelhante nas condições com ou sem palhada de cana-de-açúcar. Do nitrogênio total acumulado na parte aérea da soqueira de cana-de-açúcar, 10 a 16 % foi absorvido do fertilizante e, em média, 4 % do N mineralizado da palhada. A eficiência de utilização do nitrogênio da uréia pela soqueira de cana-de-açúcar foi em média de 17 %, não havendo diferenças entre os tratamentos, e o da palhada foi em média de 8 %. O nitrogênio da palhada foi disponibilizado para a planta no final do ciclo da cultura.

Effect of glycine and alanine supplementation on development of cattle embryo cultured in CR1aa medium with or without cumulus cells

Selostus: Glysiinin ja alaniinin vaikutus CR1aa-liuoksessa viljeltyyn kumulussolullisen ja -soluttoman naudanalkion kehitykseen

Fat oxidation kinetics during exercise in obese individuals

Introduction An impaired ability to oxidize fat may be a factor in the obesity's aetiology (3). Moreover, the exercise intensity (Fatmax) eliciting the maximal fat oxidation rate (MFO) was lower in obese (O) compared with lean (L) individuals (4). However, difference in fat oxidation rate (FOR) during exercise between O and L remains equivocal and little is known about FORs during high intensities (>60% ) in O compared with L. This study aimed to characterize fat oxidation kinetics over a large range of intensities in L and O. Methods 12 healthy L [body mass index (BMI): 22.8±0.4] and 16 healthy O men (BMI: 38.9±1.4) performed submaximal incremental test (Incr) to determine whole-body fat oxidation kinetics using indirect calorimetry. After a 15-min resting period (Rest) and 10-min warm-up at 20% of maximal power output (MPO, determined by a maximal incremental test), the power output was increased by 7.5% MPO every 6-min until respiratory exchange ratio reached 1.0. Venous lactate and glucose and plasma concentration of epinephrine (E), norepinephrine (NE), insulin and non-esterified fatty acid (NEFA) were assessed at each step. A mathematical model (SIN) (1), including three variables (dilatation, symmetry, translation), was used to characterize fat oxidation (normalized by fat-free mass) kinetics and to determine Fatmax and MFO. Results FOR at Rest and MFO were not significantly different between groups (p≥0.1). FORs were similar from 20-60% (p≥0.1) and significantly lower from 65-85% in O than in L (p≤0.04). Fatmax was significantly lower in O than in L (46.5±2.5 vs 56.7±1.9 % respectively; p=0.005). Fat oxidation kinetics was characterized by similar translation (p=0.2), significantly lower dilatation (p=0.001) and tended to a left-shift symmetry in O compared with L (p=0.09). Plasma E, insulin and NEFA were significantly higher in L compared to O (p≤0.04). There were no significant differences in glucose, lactate and plasma NE between groups (p≥0.2). Conclusion The study showed that O presented a lower Fatmax and a lower reliance on fat oxidation at high, but not at moderate, intensities. This may be linked to a: i) higher levels of insulin and lower E concentrations in O, which may induce blunted lipolysis; ii) higher percentage of type II and a lower percentage of type I fibres (5), and iii) decreased mitochondrial content (2), which may reduce FORs at high intensities and Fatmax. These findings may have implications for an appropriate exercise intensity prescription for optimize fat oxidation in O. References 1. Cheneviere et al. Med Sci Sports Exerc. 2009 2. Holloway et al. Am J Clin Nutr. 2009 3. Kelley et al. Am J Physiol. 1999 4. Perez-Martin et al. Diabetes Metab. 2001 5. Tanner et al. Am J Physiol Endocrinol Metab. 2002

Eficiência e competitividade de variantes espontâneos isolados de estirpes de Bradyrhizobium spp recomendadas para a cultura da soja (Glycine max)

O cultivo sucessivo de soja inoculada numa mesma área proporcionou a adaptação de uma população de rizóbios, que podem não ser tão eficientes quanto à capacidade de fixação de N2, mas apresentam alta competitividade, dificultando a introdução de novas estirpes mais eficientes. Com a finalidade de avaliar o desempenho simbiótico (eficiência e competitividade) de variantes espontâneos isolados de estirpes de B. japonicum (SEMIA 5079 e SEMIA 5080) e B. elkanii (SEMIA 587 e SEMIA 5019), realizou-se um experimento em casa de vegetação onde os variantes foram inoculados isoladamente e em diferentes combinações entre os variantes e uma estirpe comprovadamente mais competitiva (SEMIA 587 ou SEMIA 5019) a partir da adição de inóculos mistos (1/1; v/v) no cultivar de soja BR-16. Por meio da avaliação das variáveis analisadas (nodulação, produção de matéria de seca da parte aérea, N total acumulado na parte aérea e ocupação nodular), foi possível constatar que o determinante da maior eficiência em tratamentos co-inoculados não foi a ocupação nodular de determinada estirpe ou variante presente no inóculo, mas, sim, o tipo de interação (sinérgica ou antagônica) predominante no tratamento co-inoculado e que é possível selecionar variantes eficientes e competitivos para a cultura da soja a partir de estirpes parentais que já apresentam características desejáveis para utilização em inoculantes comerciais.

Feasibility of in vivo 15N MRS detection of hyperpolarized 15N labeled choline in rats.

The increase of total choline in tumors has become an important biomarker in cancer diagnosis. Choline and choline metabolites can be measured in vivo and in vitro using multinuclear MRS. Recent in vivo(13)C MRS studies using labeled substrates enhanced via dynamic nuclear polarization demonstrated the tremendous potential of hyperpolarization for real-time metabolic studies. The present study demonstrates the feasibility of detecting hyperpolarized (15)N labeled choline in vivo in a rat head at 9.4 T. We furthermore report the in vitro (172 +/- 16 s) and in vivo (126 +/- 15 s) longitudinal relaxation times. We conclude that with appropriate infusion protocols it is feasible to detect hyperpolarized (15)N labeled choline in live animals.