Secretory component delays the conversion of secretory IgA into antigen-binding competent F(ab')2: a possible implication for mucosal defense.

Secretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at approximately 10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the alpha-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the kappa light chain was not digested at all, suggesting that the F(ab')2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the alpha-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab')2 is discussed in terms of mucosal immune defenses.

Integrin-mediated adhesion and soluble ligand binding stabilize COX-2 protein levels in endothelial cells by inducing expression and preventing degradation.

Cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin synthesis, is highly expressed during inflammation and cellular transformation and promotes tumor progression and angiogenesis. We have previously demonstrated that endothelial cell COX-2 is required for integrin alphaVbeta3-dependent activation of Rac-1 and Cdc-42 and for endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047; Dormond, O., Bezzi, M., Mariotti, A., and Ruegg, C. (2002) J. Biol. Chem. 277, 45838-45846). In this study, we addressed the question of whether integrin-mediated cell adhesion may regulate COX-2 expression in endothelial cells. We report that cell detachment from the substrate caused rapid degradation of COX-2 protein in human umbilical vein endothelial cells (HUVEC) independent of serum stimulation. This effect was prevented by broad inhibition of cellular proteinases and by neutralizing lysosomal activity but not by inhibiting the proteasome. HUVEC adhesion to laminin, collagen I, fibronectin, or vitronectin induced rapid COX-2 protein expression with peak levels reached within 2 h and increased COX-2-dependent prostaglandin E2 production. In contrast, nonspecific adhesion to poly-L-lysine was ineffective in inducing COX-2 expression. Furthermore, the addition of matrix proteins in solution promoted COX-2 protein expression in suspended or poly-L-lysine-attached HUVEC. Adhesion-induced COX-2 expression was strongly suppressed by pharmacological inhibition of c-Src, phosphatidylinositol 3-kinase, p38, extracellular-regulated kinase 1/2, and, to a lesser extent, protein kinase C and by the inhibition of mRNA or protein synthesis. In conclusion, this work demonstrates that integrin-mediated cell adhesion and soluble integrin ligands contribute to maintaining COX-2 steady-state levels in endothelial cells by the combined prevention of lysosomal-dependent degradation and the stimulation of mRNA synthesis involving multiple signaling pathways.

Glyphosate behavior in a Rhodic Oxisol under no-till and conventional agricultural systems

The behavior of glyphosate in a Rhodic Oxisol, collected from fields under no-till and conventional management systems in Ponta Grossa, Parana state (Brazil) was investigated. Both agricultural systems had been in production for 23 years. Glyphosate mineralization, soil-bound forms, sorption and desorption kinetics, sorption/desorption batch experiments, and soil glyphosate phythoavailability (to Panicum maximum) were determined. The mineralization experiment was set up in a completely randomized design with a 2 x 2 factorial scheme (two management systems and two 14C radiolabelled positions in the glyphosate), with five replicates. 14CO2 evolution was measured in 7-day intervals during 63 days. The glyphosate sorption kinetics was investigated in a batch experiment, employing a glyphosate concentration of 0.84 mg L-1. The equilibration solution was 0.01 mol L-1 CaCl2 and the equilibration times were 0, 10, 30, 60, 120, 240, and 360 min. Sorption/desorption of glyphosate was also investigated using equilibrium batch experiments. Five different concentrations of the herbicide were used for sorption (0.42, 0.84, 1.68, 3.36, and 6.72 mg L-1) and one concentration for desorption. Glyphosate phytoavailability was analyzed in a 2 x 5 factorial scheme with two management systems and five glyphosate concentrations added to soil (0, 4.2, 8.4, 42.0, and 210.0 µg g-1) in a completely randomized design. Phytotoxicity symptoms in P. maximum were evaluated for different periods. The soil under both management systems showed high glyphosate sorption, which impeded its desorption and impaired the mineralization in the soil solution. Practically the total amount of the applied glyphosate was quickly sorbed (over 90 % sorbed within 10 min). Glyphosate bound to residues did not have adverse effects on P. maximum growth. The mineralization of glyphosate was faster under no-till and aminomethylphosphonic acid was the main glyphosate metabolite.

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

Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas.

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.

Fine-Tuning Translation Kinetics Selection as the Driving Force of Codon Usage Bias in the Hepatitis A Virus Capsid

Hepatitis A virus (HAV), the prototype of genus Hepatovirus, has several unique biological characteristics that distinguish it from other members of the Picornaviridae family. Among these, the need for an intact eIF4G factor for the initiation of translation results in an inability to shut down host protein synthesis by a mechanism similar to that of other picornaviruses. Consequently, HAV must inefficiently compete for the cellular translational machinery and this may explain its poor growth in cell culture. In this context of virus/cell competition, HAV has strategically adopted a naturally highly deoptimized codon usage with respect to that of its cellular host. With the aim to optimize its codon usage the virus was adapted to propagate in cells with impaired protein synthesis, in order to make tRNA pools more available for the virus. A significant loss of fitness was the immediate response to the adaptation process that was, however, later on recovered and more associated to a re-deoptimization rather than to an optimization of the codon usage specifically in the capsid coding region. These results exclude translation selection and instead suggest fine-tuning translation kinetics selection as the underlying mechanism of the codon usage bias in this specific genome region. Additionally, the results provide clear evidence of the Red Queen dynamics of evolution since the virus has very much evolved to re-adapt its codon usage to the environmental cellular changing conditions in order to recover the original fitness.

Activation of Srk1 by the Mitogen-activated Protein Kinase Sty1/Spc1 Precedes Its Dissociation from the Kinase and Signals Its Degradation

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. The Schizosaccharomyces pombe SAPK Sty1/Spc1 orchestrates general changes in gene expression in response to diverse forms of cytotoxic stress. Here we show that Sty1/Spc1 is bound to its target, the Srk1 kinase, when the signaling pathway is inactive. In response to stress, Sty1/Spc1 phosphorylates Srk1 at threonine 463 of the regulatory domain, inducing both activation of Srk1 kinase, which negatively regulates cell cycle progression by inhibiting Cdc25, and dissociation of Srk1 from the SAPK, which leads to Srk1 degradation by the proteasome.

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

Kinetics of K release from soils of Brazilian coffee regions: effect of organic acids

Kinetic studies on soil potassium release can contribute to a better understanding of K availability to plants. This study was conducted to evaluate K release rates from the whole soil, clay, silt, and sand fractions of B-horizon samples of a basalt-derived Oxisol and a sienite-derived Ultisol, both representative soils from coffee regions of Minas Gerais State, Brazil. Potassium was extracted from each fraction after eight different shaking time periods (0-665 h) with either 0.001 mol L-1 citrate or oxalate at a 1:10 solid:solution ratio. First-order, Elovich, zero-order, and parabolic diffusion equations were used to parameterize the time dependence of K release. For the Oxisol, the first-order equation fitted best to the experimental data of K release, with similar rates for all fractions and independent of the presence of citrate or oxalate in the extractant solution. For all studied Ultisol fractions, in which K release rates increased when extractions were performed with citrate solution, the Elovich model described K release kinetics most adequately. The highest potassium release rate of the Ultisol silt fraction was probably due to the transference of "non-exchangeable" K to the extractant solution, whereas in the Oxisol exchangeable potassium represented the main K source in all studied fractions.

Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors

Plant growth and development are particularly sensitive to changes in the light environment and especially to vegetational shading. The shade-avoidance response is mainly controlled by the phytochrome photoreceptors. In Arabidopsis, recent studies have identified several related bHLH class transcription factors (PIF, for phytochrome-interacting factors) as important components in phytochrome signaling. In addition to a related bHLH domain, most of the PIFs contain an active phytochrome binding (APB) domain that mediates their interaction with light-activated phytochrome B (phyB). Here we show that PIF4 and PIF5 act early in the phytochrome signaling pathways to promote the shade-avoidance response. PIF4 and PIF5 accumulate to high levels in the dark, are selectively degraded in response to red light, and remain at high levels under shade-mimicking conditions. Degradation of these transcription factors is preceded by phosphorylation, requires the APB domain and is sensitive to inhibitors of the proteasome, suggesting that PIF4 and PIF5 are degraded upon interaction with light-activated phyB. Our data suggest that, in dense vegetation, which is rich in far-red light, shade avoidance is triggered, at least partially, as a consequence of reduced phytochrome-mediated degradation of transcription factors such as PIF4 and PIF5. Consistent with this idea, the constitutive shade-avoidance phenotype of phyB mutants partially reverts in the absence of PIF4 and PIF5

Green manure in coffee systems in the region of Zona da Mata, Minas Gerais: characteristics and kinetics of carbon and nitrogen mineralization

The use of green manure may contribute to reduce soil erosion and increase the soil organic matter content and N availability in coffee plantations in the Zona da Mata, State of Minas Gerais, in Southeastern Brazil. The potential of four legumes (A. pintoi, C. mucunoides, S. aterrimum and S. guianensis) to produce above-ground biomass, accumulate nutrients and mineralize N was studied in two coffee plantations of subsistence farmers under different climate conditions. The biomass production of C. mucunoides was influenced by the shade of the coffee plantation. C. mucunoides tended to mineralize more N than the other legumes due to the low polyphenol content and polyphenol/N ratio. In the first year, the crop establishment of A. pintoi in the area took longer than of the other legumes, resulting in lower biomass production and N2 fixation. In the long term, cellulose was the main factor controlling N mineralization. The biochemical characteristics, nutrient accumulation and biomass production of the legumes were greatly influenced by the altitude and position of the area relative to the sun.

Pitfalls in cefepime titration from human plasma: plasma- and temperature-related drug degradation in vitro.

While developing a high-pressure liquid chromatography assay for cefepime in plasma, we observed significant drug degradation at 20 and 37 degrees C but not at 4 degrees C. This plasma-related degradation persisted after protein removal. This warrants caution regarding cefepime assays for pharmacokinetic and pharmacodynamic studies of cefepime in vitro and in vivo.

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.