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Proper physiological function of mammalian airways requires the differentiation of basal stem cells into secretory or multiciliated cells, among others. In addition, the self-renewal ability of these basal stem cells is crucial for developing a quick response to toxic agents in order to re-establish the epithelial barrier function of the airways. Although these epithelial missions are vital, little is known about those mechanism controlling airway epithelial regeneration in health and disease. p53 has been recently proposed as the guardian of homeostasis, promoting differentiation programs, and antagonizing a de-differentiation program. Here, we exploit mouse and human tracheal epithelial cell culture models to study the role of MDM2-p53 signaling in self-renewal and differentiation in the airway epithelium. We show that p53 protein regulation by MDM2 is crucial for basal stem cell differentiation and to keep proper cell proliferation. Therefore, we suggest that MDM2/p53 interaction modulation is a potential target to control regeneration of the mammalian airway epithelia without massively affecting the epithelium integrity and differentiation potential.

Tight-junction (TJ) proteins are essential for establishing the barrier function between neighbor epithelial cells, but also for recognition of pathogens or cell migration. Establishing the expression pattern and localization of different TJ proteins will help to understand the development and physiology of the airway. Here we identify that the junctional adhesion molecule 3 (Jam3) expression is restricted to multiciliated cells (MCCs) in the airway epithelium. In vitro, Jam3 expression varies along airway basal stem cell (BSC) differentiation and upon DAPT treatment or IL6 exposure. However, Jam3 is not required for BSC differentiation to specific cell types. In addition, we found that MCC lacking Jam3 display normal cilia morphology and cilia beating frequency with a delay in BB assembly/positioning in MCCs during differentiation. Remarkably, Jam3 in MCC is mostly localized to subapical organelles, which are negative for the apical recycling endosome marker Rab11 and positive for EEA1. Our data show that Jam3 expression is connected to mature MCC in the airway epithelium and suggest a Jam3 role unrelated to its known barrier function.

Growing evidence suggests that Diabetes Mellitus increases the risk of developing Alzheimer's disease. It is well known that hyperglycemia, a key feature of Diabetes Mellitus, may induce plasma osmolarity disturbances. Both hyperglycemia and hyperosmolarity promote the altered post-translational regulation of microtubule-associated protein Tau. Interestingly, abnormal hyperphosphorylation and cleavage of Tau have been proven to lead to the genesis of filamentous structures referred to as neurofibrillary tangles, the main pathological hallmark of Alzheimer's disease. We have previously described that hyperosmotic stress induced by sorbitol promotes Tau proteolysis and apoptosis in SH-SY5Y cells via caspase-3 activation. In order to gain insights into the regulatory mechanisms of such processes, in this work we explored the intracellular signaling pathways that regulate these events. We found that sorbitol treatment significantly enhanced the activation of conventional families of MAPK in SH-SY5Y cells. Tau proteolysis was completely prevented by JNK inhibition but not affected by either ERK1/2 or p38 MAPK blockade. Moreover, inhibition of JNK, but not ERK1/2 or p38 MAPK, efficiently prevented sorbitol-induced apoptosis and caspase-3 activation. In summary, we provide evidence that JNK signaling pathway is an upstream regulator of hyperosmotic stress-induced Tau cleavage and apoptosis in SH-SY5Y through the control of caspase-3 activation.

Microbially induced carbonate precipitation (MICP), or biocementation, consists in using microorganisms living in the soil to produce calcium carbonate (biocement). This mineral bonds the grains and therefore improves the soil hydro-mechanical properties. When using this technique, one of the challenges is to ensure homogeneous treatment in the entire volume. In this study, an experimental device was developed to apply this treatment in cylindrical soil samples with 7.2 cm diameter and 12 cm height. Two distinct sample preparation techniques were tested: (i) pre-mixing the soil with bacteria, and then inject the feeding solution; (ii) inject bacteria followed by injecting the feeding solution. In both, the injection conditions varied in two distinct ways: (i) infiltration column, from the top and (ii) injecting through a perforated central tube. The homogeneity of the biocement in the volume was evaluated using X-ray and SEM images from small samples taken from different locations in the specimens and analysing different parameters. Mercury intrusion porosimetry (MIP) and CaCO3 dissolution tests revealed uneven distribution of CaCO3 content between the top and bottom sections, as well as along radial direction. The most homogeneous samples were found when bacteria were premixed with the soil before injecting the feeding solution. Unconfined compression tests (UCS) were also performed in samples with and without treatment. The treatment increased stiffness and strength significantly and soil rupture occurred mostly near the bottom, where the lowest CaCO3 contents were detected.

Organelle positioning as many other morphological parameters in a cell is not random. Centriole positioning as centrosomes or ciliary basal bodies is not an exception to this rule in cell biology. Indeed, centriole positioning is a tightly regulated process that occurs during development, and it is critical for many organs to function properly, not just during development but also in the adulthood. In this book chapter, we overview our knowledge on centriole positioning in different and highly specialized animal cells like photoreceptor or ependymal cells. We will also discuss recent advances in the discovery of molecular pathways involved in this process, mostly related to the cytoskeleton and the cell polarity pathways. And finally, we present quantitative methods that have been used to assess centriole positioning in different cell types although mostly in epithelial cells.

Tau is a microtubule-associated protein implicated in the pathogenesis of Alzheimer's disease and other related tauopathies. In this subset of neurodegenerative disorders, Tau auto-assembles into insoluble fibrils that accumulate in neurons as paired helical filaments (PHFs), promoting cellular dysfunction and cytotoxic effects. Growing evidence suggests that abnormal post-translational regulation, mainly hyperphosphorylation and aberrant cleavage, drives Tau to this pathological state. In this work we show that sorbitol-induced hyperosmotic stress promotes Tau proteolysis in SH-SY5Y neuroblastoma cells. The appearance of cleaved Tau was preceded by the activation of μ-calpain, the proteasome system and caspase-3. Tau proteolysis was completely prevented by caspase-3 inhibition but unaffected by neither the proteasome system nor μ-calpain activity blockade. Concomitantly, hyperosmotic stress induced apoptosis in SH-SY5Y cells, which was efficiently avoided by the inhibition of caspase-3 activity. Altogether, our results provide the first evidence that Tau protein is susceptible to caspase-3 proteolysis under hyperosmotic stress and suggest a positive relationship between Tau proteolysis and apoptosis in SH-SY5Y cells. J. Cell. Biochem. 117: 2781-2790, 2016. © 2016 Wiley Periodicals, Inc.

Organelle positioning as many other morphological parameters in a cell is not random. Centriole positioning as centrosomes or ciliary basal bodies is not an exception to this rule in cell biology. Indeed, centriole positioning is a tightly regulated process that occurs during development, and it is critical for many organs to function properly, not just during development but also in the adulthood. In this book chapter, we overview our knowledge on centriole positioning in different and highly specialized animal cells like photoreceptor or ependymal cells. We will also discuss recent advances in the discovery of molecular pathways involved in this process, mostly related to the cytoskeleton and the cell polarity pathways. And finally, we present quantitative methods that have been used to assess centriole positioning in different cell types although mostly in epithelial cells.

Adipose tissue has emerged as an important regulator of whole-body metabolism, and its capacity to dissipate energy in the form of heat has acquired a special relevance in recent years as potential treatment for obesity. In this context, the p38MAPK pathway has arisen as a key player in the thermogenic program because it is required for the activation of brown adipose tissue (BAT) thermogenesis and participates also in the transformation of white adipose tissue (WAT) into BAT-like depot called beige/brite tissue. Here, using mice that are deficient in p38α specifically in adipose tissue (p38αFab-KO), we unexpectedly found that lack of p38α protected against high-fat diet (HFD)-induced obesity. We also showed that p38αFab-KO mice presented higher energy expenditure due to increased BAT thermogenesis. Mechanistically, we found that lack of p38α resulted in the activation of the related protein kinase family member p38δ. Our results showed that p38δ is activated in BAT by cold exposure, and lack of this kinase specifically in adipose tissue (p38δ Fab-KO) resulted in overweight together with reduced energy expenditure and lower body and skin surface temperature in the BAT region. These observations indicate that p38α probably blocks BAT thermogenesis through p38δ inhibition. Consistent with the results obtained in animals, p38α was reduced in visceral and subcutaneous adipose tissue of subjects with obesity and was inversely correlated with body mass index (BMI). Altogether, we have elucidated a mechanism implicated in physiological BAT activation that has potential clinical implications for the treatment of obesity and related diseases such as diabetes., Author summary Accumulation of fat in adipose tissue is essential to store energy and insulate the body; however, excessive body fat leads to obesity. Of the 2 existing types of adipose tissue, white adipose tissue (WAT) stores energy, whereas brown adipose tissue (BAT) can produce heat. Activation of BAT and transformation of WAT into brown-like ‘brite/beige’ adipocytes have recently emerged as novel strategies against obesity. The uncoupling protein 1 (UCP1) is a hallmark of BAT and is responsible for triggering these 2 processes under the regulation of the p38 MAP kinase (p38MAPK) pathway, but the underlying mechanisms remain unknown. Here, we have analysed this process in detail and demonstrate that a protein kinase called p38α directly correlates with UCP1 levels in human adipose tissue, while it inversely correlates with body mass index (BMI). We find that mice lacking p38α in adipose tissue are protected against diet-induced obesity due to increased body temperature. In addition, another p38 family member, p38δ, is activated in these adipocytes lacking p38α and reduces their thermogenic capacity. Our results suggest that these 2 members of the p38 family have opposite roles in controlling thermogenesis.

The purpose of this paper is to discuss the contextual aspects of the norm, inserted in the article 174 of the New Civil Process Code and in the Mediation Act, which determines the creation of the chambers of mediation and conciliation, aiming to resolve the consensual and extrajudicial conflict in the public administration. In addition, it will also focuses on the perspectives of that legislative innovation, which tends to produce socially relevant results., No contexto da consensualidade administrativa o presente artigo procura abordar aspectos do novo Código de Processo Civil e da Lei de Mediação que preveem a autocomposição de conflitos envolvendo a Administração Pública e determinam a criação de câmaras de mediação e conciliação dirigidas à resolução consensual e extrajudicial de conflitos naquele âmbito, enfocando, ainda, as perspectivas dessas inovações legislativas, que podem produzir resultados sociais relevantes.

Tau is a microtubule-associated protein implicated in the pathogenesis of Alzheimer's disease and other related tauopathies. In this subset of neurodegenerative disorders, Tau auto-assembles into insoluble fibrils that accumulate in neurons as paired helical filaments (PHFs), promoting cellular dysfunction and cytotoxic effects. Growing evidence suggests that abnormal post-translational regulation, mainly hyperphosphorylation and aberrant cleavage, drives Tau to this pathological state. In this work we show that sorbitol-induced hyperosmotic stress promotes Tau proteolysis in SH-SY5Y neuroblastoma cells. The appearance of cleaved Tau was preceded by the activation of μ-calpain, the proteasome system and caspase-3. Tau proteolysis was completely prevented by caspase-3 inhibition but unaffected by neither the proteasome system nor μ-calpain activity blockade. Concomitantly, hyperosmotic stress induced apoptosis in SH-SY5Y cells, which was efficiently avoided by the inhibition of caspase-3 activity. Altogether, our results provide the first evidence that Tau protein is susceptible to caspase-3 proteolysis under hyperosmotic stress and suggest a positive relationship between Tau proteolysis and apoptosis in SH-SY5Y cells. J. Cell. Biochem. 117: 2781-2790, 2016. © 2016 Wiley Periodicals, Inc.

Activation of p38γ modulates the integrity of the complex formed by the human discs large protein (hDlg) with cytoskeletal proteins, which is important for cell adaptation to changes in environmental osmolarity. Here we report that, in response to hyperosmotic stress, p38γ also regulates formation of complexes between hDlg and the nuclear protein polypyrimidine tract-binding protein-associated-splicing factor (PSF). Following osmotic shock, p38γ in the cell nucleus increases its association with nuclear hDlg, thereby causing dissociation of hDlg-PSF complexes. Moreover, hDlg and PSF bind different RNAs; in response to osmotic shock, p38γ causes hDlg-PSF and hDlg-RNA dissociation independently of its kinase activity. These findings identify a novel nuclear complex and suggest a previously unreported function of p38γ, which is independent of its catalytic activity and could affect mRNA processing and/or gene transcription to aid cell adaptation to osmolarity changes in the environment.

Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is a component of the p38γ MAP kinase pathway, which is important for the adaptation of mammalian cells to changes in environmental osmolarity. Here we report a strong decrease in the levels of hDlg protein in the human epithelial cell line HeLa when exposed to osmotic shock. This is independent of the phosphorylation state of hDlg, is prevented by preincubating the cell with the caspase inhibitor z-VAD and is part of the apoptotic process triggered by cellular stress. Although, both caspase 3 and caspase 6 are strongly activated by osmotic shock, the time course of caspase 6 activation parallels hDlg degradation, suggesting that this caspase may be responsible for the proteolysis. Mutating hDlg Asp747 to Ala abolishes caspase-induced cleavage, but does not affect the early stage of apoptosis or cell attachment. Our findings show that osmotic stress triggers hDlg degradation through a mechanism different from the one mediated by proteasomes, and we identify hDlg as a caspase substrate during the apoptotic process, although its proteolysis may not be implicated in the progression of early apoptosis.

Activation of the p38 MAP kinase pathways is crucial for the adaptation of mammalian cells to changes in the osmolarity of the environment. Here we identify SAP97/hDlg, the mammalian homologue of the Drosophila tumour suppressor Dlg, as a physiological substrate for the p38gamma MAP kinase (SAPK3/p38gamma) isoform. SAP97/hDlg is a scaffold protein that forms multiprotein complexes with a variety of proteins and is targeted to the cytoskeleton by its association with the protein guanylate kinase-associated protein (GKAP). The SAPK3/p38gamma-catalysed phosphorylation of SAP97/hDlg triggers its dissociation from GKAP and therefore releases it from the cytoskeleton. This is likely to regulate the integrity of intercellular-junctional complexes, and cell shape and volume in response to osmotic stress.

Canine pyometra often causes glomerulonephritis by immune complex deposition in the glomeruli. Proteinuria, ranging from moderate to severe, may be present secondary to renal damage. To determine urinary protein excretion due to pyometra, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted on urine from 15 bitches with pyometra and 10 healthy bitches. To characterize urinary immunoglobin excretion, Western blot analysis of the urine samples using antibodies to canine IgG and IgA was also performed. Nine bands were detected by electrophoresis in bitches with pyometra, while only four were detected in the healthy animals. The urinary proteins from bitches with pyometra were primarily of glomerular origin; 58% were of medium-high molecular weight (MW), and the remainder were low MW. None of the healthy dogs had IgG or IgA in their urine, whereas three bitches with pyometra had IgG in their urine and another bitch with pyometra had both IgG and IgA. The low proportion of bitches with urinary immunoglobins was probably be due to early diagnosis of the disease. Although only a limited number of dogs was used, this study is apparently the first to characterize the electrophoretic pattern of urinary proteins and to quantify urinary excretion of IgG and IgA in bitches with pyometra.

We investigated apoptosis induced by the green tea component the epigallocatechin-3-gallate (EGCG) and the pathways underlying its activity in a colon cancer cell line. A complete understanding of the mechanism(s) and molecules targeted by green tea polyphenols could be useful in developing novel therapeutic approaches for cancer treatment. EGCG, which is the major polyphenol in green tea, has cytotoxic effects and induced cell death in HT-29 cell death. In this study, we evaluated the effect EGCG on mitogen-activated protein kinase (MAPK) and Akt pathways. EGCG treatment increased phospho-ERK1/2, -JNK1/2 and -p38α, -p38γ and -p38δ, as well as phospho-Akt levels. Using a combination of kinase inhibitors, we found that EGCG-induced cell death is partially blocked by inhibiting Akt, ERK1/2 or alternative p38MAPK activity. Our data suggest that these kinase pathways are involved in the anti-cancer effects of EGCG and indicate potential use of this compound as chemotherapeutic agent for colon cancer treatment.

OBJECTIVES Acute treatment with valproate (VPA) or lithium (Li+) protects cerebellar granule cells (CGC) against apoptosis induced by low potassium (K+) (5 mM). As the protection induced by VPA is absolutely dependent on insulin, in contrast to the observed effects of Li+, we decided to study the different role of the PI3K/PKB pathway in the neuroprotective effects of both drugs. METHODS We have studied the neuroprotection elicited by Li+ or VPA in cultures of rat CGC. We induced the apoptosis by switching to a medium with a low concentration of K+ or by adding C2-ceramide to the cultures. We studied the effect of Li+ and VPA on viability and on the regulation of the PI3K/PKB pathway. RESULTS AND CONCLUSIONS Insulin also protects against low K(+)-induced apoptosis in CGC, probably through its interaction with an insulin-like growth factor receptor. Moreover, whereas Li+ protects against the apoptosis induced by C2-ceramide, VPA cannot, probably due to the inhibition of protein kinase B (PKB) caused in this apoptotic stimulus. These results suggest that VPA protects against low K(+)-induced apoptosis by acting on the PI3K/PKB pathway; however, VPA does not affect the increase of PKB activity caused by insulin in these cells. The protection by Li+ is independent of this transduction pathway. Moreover, Li+ blocks the caspase 3 activation induced by low K+, whereas neither VPA nor insulin affects this activation.

Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mm) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activity in vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine-threonine phosphatase induced by potassium deprivation.

Arginase I is a homotrimeric protein with a binuclear manganese cluster. At the C-terminus of each monomer, the polypeptide chain forms an unusual S-shaped oligomerization motif where the majority of intermonomer contacts are located [Z.F. Kanyo, L.R. Scolnick, D.E. Ash, D.W. Christianson, Nature 383 (1996) 554^557]. In order to study the implication of this motif in the quaternary structure of human arginase I, we have constructed a truncated arginase lacking the 14 C-terminal amino acids, leaving Arg-308 as the last residue in the sequence. The resulting protein retains its trimeric structure, as determined by gel filtration (molecular mass 94 kDa). The same result was obtained in the presence of high ionic strength (KCl 0.5 M). Both data indicate that neither the S-shaped motif nor Arg-308 are fundamental in keeping the trimeric quaternary structure. Data obtained from intrinsic anisotropy and fluorescence intensity studies allow us to predict that the distance between the two unique tryptophans in the sequence is 2.9 nm in the native arginase and 4.1 nm for the truncated mutant. These distances allow us to assume a different conformational state in the truncated arginase without any change in its quaternary structure, suggesting that the carboxy-terminal motif is not the most prominent domain implicated in the quaternary structure of human arginase. Collisional quenching studies reinforce this possibility, since using I 3 as quenching molecule we were able to distinguish the two tryptophans in the truncated arginase. Moreover, kinetic studies show that the truncated mutant was fully active. In summary, the main conclusion about the structure of the human arginase I, derived from our study, is that the C-terminal S-shaped motif is not basic to the maintenance of the quaternary structure nor to the activity of the protein. fl 2000 Elsevier Science B.V. All rights reserved.

Acute treatment with valproate and Li+ was found to protect cultured cerebellar granule cells against apoptosis induced by low K+ (5 mM). Because the protection was unaffected by MK801 (N-methyl-D-aspartate receptor inhibitor), an increase in glutamate release cannot be responsible for the observed neuroprotection. Insulin also protects against low-K+-induced apoptosis of cerebellar granule cells. This protection is totally dependent on LY294002 (a phosphatidylinositol 3-kinase inhibitor). These results suggest a role for phosphatidylinositol 3-kinase in the neuroprotection induced by insulin. Likewise, and in contrast with the results observed with Li+, the protection induced by valproate is also dependent on insulin and LY294002. Moreover, valproate (a branched-chain fatty acid) does not change the plasma membrane microviscosity under physiological conditions. These results suggest that valproate protects against low-K+-induced apoptosis by acting in the phosphatidylinositol 3-kinase/protein kinase B pathway. The protection by Li+ is independent of this transduction pathway.

Activated murine macrophages metabolize arginine by two alternative pathways involving the enzymes inducible NO synthase (iNOS) or arginase. The balance between the two enzymes is competitively regulated by Th1 and Th2 T helper cells via their secreted cytokines: Th1 cells induce iNOS, whereas Th2 cells induce arginase. Whereas the role of macrophages expressing iNOS as inflammatory cells is well established, the functional competence of macrophages expressing arginase remains a matter of speculation. Two isoforms of mammalian arginases exist, hepatic arginase I and extrahepatic arginase II. We investigated the regulation of arginase isoforms in murine bone marrow-derived macrophages (BMMΦ) in the context of Th1 and Th2 stimulation. Surprisingly, in the presence of either Th2 cytokines or Th2 cells, we observe a specific induction of the hepatic isoform arginase I in BMMΦ. Induction of arginase I was shown on the mRNA and protein levels and obeyed the recently demonstrated synergism among the Th2 cytokines IL-4 and IL-10. Arginase II was detectable in unstimulated BMMΦ and was not significantly modulated by Th1 or Th2 stimulation. Similar to murine BMMΦ, murine bone marrow-derived dendritic cells, as well as a dendritic cell line, up-regulated arginase I expression and arginase activity upon Th2 stimulation, whereas arginase II was never detected. In addition to revealing the unexpected expression of arginase I in the macrophage/monocyte lineage, these results uncover a further intriguing parallelism between iNOS and arginase: both have a constitutive and an inducible isoform, the latter regulated by the Th1/Th2 balance.

Working with detergent-solubilized bacteriorhodopsin we have used a table top preparative centrifuge for determination of M(r) of membrane proteins by sedimentation equilibrium. We demonstrate the use of two new methods to measure protein concentration as a function of distance from rotor axis: (i) peak integration after HPLC on silica gel, and (ii) microdensitometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. These methods, although somewhat lengthier than conventional spectrophotometric methods, are more reliable, especially in the presence of a large amount of detergent and small amount of protein. In addition they provide independent information on the status of the protein after sedimentation equilibrium, the association of the solubilized units being readily detected by gel chromatography and proteolytic cleavage by SDS-PAGE.

Fluorescence quenching and energy-transfer studies have been carried out to determine the position of FAD and FMN groups of NADPH-cytochrome P450 reductase and of the heme and substrate groups of cytochrome P450 with respect to the lipid/water interphase. Quenching by iodine of the fluorescence of the flavins of the reductase shows a biphasic pattern, due to the different accessibility of FAD and FMN to the solvent with Stern-Volmer constants of 7.9 x 10(-4) and 2.7 x 10(-3) mM-1, respectively. Both prosthetic groups appear to be buried within the three-dimensional structure of the native reductase, FAD more deeply embedded than FMN and with a relative contribution to the total fluorescence of flavins of 84% (FAD) and 16% (FMN). The lack of significant energy transfer (less than 5%) from FAD+FMN to the rhodamine group of the N-labeled phosphatidylethanolamine incorporated in membranes reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine points out that both groups are located at a distance greater than 5 nm from the lipid/water interphase. Steady-state fluorescence intensity and anisotropy data obtained with native and FMN-depleted NADPH-cytochrome P450 reductase show that energy transfer between both prosthetic groups occurs in the native reductase with an efficiency of ca. 31%, consistent with a separation between these groups of 2 nm as suggested earlier by Bastiaens, P. I. H., Bonants, P. J. M., Muller, F., & Visser, A. J. W. G. [(1989) Biochemistry 28, 8416-8425] from time-resolved fluorescence anisotropy measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is a component of the p38gamma MAP kinase pathway, which is important for the adaptation of mammalian cells to changes in environmental osmolarity. Here we report a strong decrease in the levels of hDlg protein in the human epithelial cell line HeLa when exposed to osmotic shock. This is independent of the phosphorylation state of hDlg, is prevented by preincubating the cell with the caspase inhibitor z-VAD and is part of the apoptotic process triggered by cellular stress. Although, both caspase 3 and caspase 6 are strongly activated by osmotic shock, the time course of caspase 6 activation parallels hDlg degradation, suggesting that this caspase may be responsible for the proteolysis. Mutating hDlg Asp747 to Ala abolishes caspase-induced cleavage, but does not affect the early stage of apoptosis or cell attachment. Our findings show that osmotic stress triggers hDlg degradation through a mechanism different from the one mediated by proteasomes, and we identify hDlg as a caspase substrate during the apoptotic process, although its proteolysis may not be implicated in the progression of early apoptosis.

Resveratrol (RES), a chemopreventive molecule, inhibits the proliferation of tumor cells of different etiologies. We previously showed that RES alters the cell cycle and induces apoptosis in MCF-7 breast tumor cells by interfering with the estrogen receptor (ERaalpha)-dependent phosphoinositide 3-kinase (PI3K) pathway. Here, we analyzed signaling downstream of PI3K, to understand the mechanisms of RES-induced apoptosis. Apoptotic death by RES in MCF-7 was mediated by Bcl-2 downregulation since overexpression of this protein abolished apoptosis. Decreased Bcl-2 levels were not related to cytochrome c release, activation of caspases 3/8 or poly(ADP-ribose) polymerase proteolysis. However, RES decreased mitochondrial membrane potential and increased reactive oxygen species and nitric oxide production. NF-kappaB, a regulator of Bcl-2 expression, and calpain protease activity, a regulator of NF-kappaB, were both inhibited by RES. The patterns for NF-kappaB and calpain activities followed that of PI3K and were inhibited by LY294002. NF-kappaB inhibition coincided with diminished MMP-9 activity and cell migration. These data suggest that RES-induced apoptosis in MCF-7 could involve an oxidative, caspase-independent mechanism, whereby inhibition of PI3K signaling converges to Bcl-2 through NF-kappaB and calpain protease activity. Therefore, Bcl-2 and NF-kappaB could be considered potential targets for the chemopreventive activity of RES in estrogen-responsive tumor cells.

Human arginase I is a potential target for therapeutic intervention in diseases linked to compromisedl-arginine homeostasis. Here, we report high-affinity binding of the reaction coordinate analogue inhibitors 2(S)-amino-6-boronohexanoic acid (ABH,Kd= 5 nM) andS-(2-boronoethyl)-l-cysteine (BEC,Kd= 270 nM) to human arginase I, and we report x-ray crystal structures of the respective enzyme–inhibitor complexes at 1.29- and 1.94-Å resolution determined from crystals twinned by hemihedry. The ultrahigh-resolution structure of the human arginase I–ABH complex yields an unprecedented view of the binuclear manganese cluster and illuminates the structural basis for nanomolar affinity: bidentate inner-sphere boronate–manganese coordination interactions and fully saturated hydrogen bond networks with inhibitor α-amino and α-carboxylate groups. These interactions are therefore implicated in the stabilization of the transition state forl-arginine hydrolysis. Electron density maps also reveal that active-site residue H141 is protonated as the imidazolium cation. The location of H141 is such that it could function as a general acid to protonate the leaving amino group ofl-ornithine during catalysis, and this is a revised mechanistic proposal for arginase. This work serves as a foundation for studying the structural and chemical biology of arginase I in the immune response, and we demonstrate the inhibition of arginase activity by ABH in human and murine myeloid cells.

SAPK3 (stress-activated protein kinase-3, also known as p38gamma) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of alpha1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38gamma and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38gamma binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290 in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38gamma to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38alpha and SAPK2b/p38beta but not SAPK3/p38gamma, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38gamma with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290 in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38gamma co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38gamma, a first physiological substrate described for SAPK3/p38gamma and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein-protein interactions at the synapse in response to adverse stress- or mitogen-related stimuli.

Canine leptospirosis is a zoonotic disease that can cause interstitial nephritis. As a consequence of the renal damage, proteinuria may occur. To determine the urine protein pattern in the disease we performed sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on the urine from 10 dogs with leptospirosis and 20 healthy dogs. Western blotting analysis of the urine samples with antibodies against canine IgG and IgA was also performed to identify these immunoglobulins in the urine. Urine electrophoresis showed three new bands in the dogs suffering from leptospirosis. Only two of the dogs with leptospirosis showed detectable concentrations of IgG and IgA in urine, while a third animal showed IgG alone. The study showed a 36.7% increase in the excretion of low molecular weight proteins in dogs with leptospirosis but almost no change in the high molecular weight protein pattern. These results, together with the low number of animal with detectable concentrations of IgG and IgA in the urine, support the view that canine leptospirosis is characterized by interstitial nephritis.

International audience; Canine leishmaniasis is an endemic disease in the Mediterranean area caused by the protozoan Leishmania infantum, which usually produces renal failure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot using antibodies to IgG and IgA from dogs were carried out in the urine of 22 dogs with leishmaniasis diagnosed by ELISA and confirmed by PCR, and 20 healthy dogs. The results were compared to renal function laboratory tests and to those from a histopathological study of the kidneys from sick animals that died naturally or were euthanized. Five different bands with molecular weights ranging from 10 to 110 kDa were obtained from the electrophoresis of the urine of healthy dogs. 33.5% of total proteins corresponded to low molecular weight proteins and the other proteins had middle and high molecular weights. However, in the group with leishmaniasis, a maximum of 11 different bands with molecular weights ranging from 10 kDa to 150 kDa were displayed in the electrophoresis of the urine. The urine electrophoretic pattern in the sick dogs was classified as mixed (proteins with high and low molecular weights) because low molecular weight proteins made up 57.9% and the rest of the proteins had middle and high molecular weights. In Western blot, none of the healthy dogs showed excretion of IgG and/or IgA, whereas IgG and IgA were detected in the Western blot of urine of 68% and 55% respectively of dogs with leishmaniasis. The results obtained in the leishmaniasis group agreed with glomerular and tubular damage, which were confirmed by the histopathological findings.

The biochemical mechanism of apoptosis induced by ceramide remains still unclear, although it has been reported that dephosphorylation of PKB at Ser-473 may be a key event. In this article, we show that C(2)-ceramide (N-acetyl-sphingosine) induces the dephosphorylation of both protein kinase B (PKB) and glycogen synthase kinase-3 (GSK3) in cerebellar granule cells (CGC). We also show that lithium protects against the apoptosis induced by C(2)-ceramide by blocking the dephosphorylation of both kinases. Since lithium inhibits in vivo the observed protein phosphatase-2A (PP2A) activation induced by ceramide, we hypothesise that the neuroprotective action of lithium may be due to the inhibition of the PP2A activation by apoptotic stimuli.

Exposure of cerebellar granule cells to 1-methyl-4-phenylpiridinium (MPP + ) results in cell death. We have studied the implication of various membrane transporter systems on MPP + neurotoxicity, including the dopamine transporter system (DAT) and cationic amino acid transporters (CAT). We have showed a partial protection against MPP + toxicity when the dopamine transporter is inhibited by 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]4-(3-phenylpropyl)piperazinedihydrochloride (GBR-12909). However, almost full protection is only achieved by the simultaneous addition of GBR-12909 and cationic amino acids. These results suggest two ways system of MPP + entrance into cerebellar granule cells: the DAT with high activity and the CAT with low activity. We also demonstrated that 5,7-dichlorokynurenic acid (MK-801) failed to protect against MPP + exposure, evidencing that N-methyl-D-aspartate (NMDA) receptor is not involved in the MPP + -induced cell death.

One determinant that could play a role in the quaternary structure of human arginase is the pair of salt links between the strictly conserved residues R255 from one monomer and E256 from every adjacent subunit. In this work, the ionic interaction between monomers was disrupted by expressing a human arginase where Glu-256 had been substituted by Gln. Biochemical analyses of the mutant protein showed that: (i) it shares the wild-type kinetic parameters of the arginine substrate; (ii) E256Q arginase behaves as a monomer by gel filtration; (iii) it is drastically inactivated by dialysis in the presence of EDTA, an inhibitory effect which is reversed by addition of Mn 2+ ; and (iv) the mutant enzyme loses thermal stability. The lack of oligomerisation for E256Q arginase and the conservation of E256 throughout evolution of the protein family suggest that this residue is involved in the quaternary structure of arginases.

PRIMARY cultures of cerebellar granule neurons, maintained in a serum-containing medium, underwent apoptosis when exposed to C 2 -ceramide, as assessed by mitochondrial reduction of MTT and intranucleosomal DNA fragmentation. After an 18 h exposure to 50 μM C 2 -ceramide, cell viability decreased by 25-40%. Addition of lithium together with C 2 -ceramide resulted in a partial protection of apoptosis, which was maximal at 5 mM lithium (37% protection). When lithium was added 5 h before the apoptotic stimulus the neuroprotective effect of the ion was clearly increased (66% protection). This effect was not due to intracellular inositol depletion or inhibition of NMDA receptors. Our data broaden the nature of apoptotic insults being reversed by lithium, stressing the neuroprotective effects of the ion.

International audience; Limited proteolysis by proteinase K of rabbit SERCA1 Ca2+-ATPase generates a number of fragments which have been identified recently. Here, we have focused on two proteolytic C-terminal fragments, p20C and p19C, starting at Gly-808 and Asp-818, respectively. The longer peptide p20C binds Ca2+, as deduced from changes in migration rate by SDS-polyacrylamide gel electrophoresis performed in the presence of Ca2+ as well as from labeling with 45Ca2+ in overlay experiments. In contrast, the shorter peptide p19C, a proteolysis fragment identical to p20C but for 10 amino acids missing at the N-terminal side, did not bind Ca2+ when submitted to the same experiments. Two cluster mutants of Ca2+-ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca2+-ATPase activity. Region 808-818 is thus essential for both Ca2+ binding and enzyme activity, in agreement with similar results recently reported for the homologous gastric H+, K+-ATPase (Swarts, H. G. P., Klaassen, C. H. W., de Boer, M., Fransen, J. A. M. , and De Pont, J. J. H. H. M. (1996) J. Biol. Chem. 271, 29764-29772). However, the accessibility of proteinase K to the peptidyl link between Leu-807 and Gly-808 clearly shows that the transmembrane segment M6 ends before region 808-818. It is remarkable that critical residues for enzyme activity are located in a cytoplasmic loop starting at Gly-808.Limited proteolysis by proteinase K of rabbit SERCA1 Ca2+-ATPase generates a number of fragments which have been identified recently. Here, we have focused on two proteolytic C-terminal fragments, p20C and p19C, starting at Gly-808 and Asp-818, respectively. The longer peptide p20C binds Ca2+, as deduced from changes in migration rate by SDS-polyacrylamide gel electrophoresis performed in the presence of Ca2+ as well as from labeling with 45Ca2+ in overlay experiments. In contrast, the shorter peptide p19C, a proteolysis fragment identical to p20C but for 10 amino acids missing at the N-terminal side, did not bind Ca2+ when submitted to the same experiments. Two cluster mutants of Ca2+-ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca2+-ATPase activity. Region 808-818 is thus essential for both Ca2+ binding and enzyme activity, in agreement with similar results recently reported for the homologous gastric H+, K+-ATPase (Swarts, H. G. P., Klaassen, C. H. W., de Boer, M., Fransen, J. A. M. , and De Pont, J. J. H. H. M. (1996) J. Biol. Chem. 271, 29764-29772). However, the accessibility of proteinase K to the peptidyl link between Leu-807 and Gly-808 clearly shows that the transmembrane segment M6 ends before region 808-818. It is remarkable that critical residues for enzyme activity are located in a cytoplasmic loop starting at Gly-808.

International audience; We describe here an easy system for the production of mg amounts of the rabbit Ca(2+)-ATPase SERCA 1a in the yeast S. cerevisiae. The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation. It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca(2+)-dependent manner. Using a temperature-sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.We describe here an easy system for the production of mg amounts of the rabbit Ca(2+)-ATPase SERCA 1a in the yeast S. cerevisiae. The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation. It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca(2+)-dependent manner. Using a temperature-sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.

The overall thermal denaturation of glycogen phosphorylase b is irreversible and our results conform to the theoretical prediction of a reversible process followed by a slower irreversible process. The basic thermodynamic parameters of glycogen phosphorylase b denaturation have been worked out and found to be: critical temperature 57.0 +/- 0.5 degrees C, transition half-width 8 +/- 1 degrees C, and calorimetric enthalpy change and Van't Hoff enthalpy change of the denaturation process 450 +/- 50 and 105 +/- 15 kcal/mol of enzyme monomer, respectively, at pH 7.4. These parameters have been found to be largely altered by the detergents octylglucoside, cholate, and deoxycholate at or below their critical micelle concentration, but not by Triton X-100 nor by lecithin liposomes. Organic solvents, such as dimethyl sulfoxide and methanol, and the presence of sarcoplasmic reticulum membranes produces an alteration of the denaturation thermogram of glycogen phosphorylase b similar to that produced by the above-mentioned detergents. These results allow us to hypothesize that hydrophobic domains of glycogen phosphorylase b are involved in its association to sarcoplasmic reticulum membranes in the sarcoplasmic reticulum/glycogenolytic complex of mammalian skeletal muscle.

Glycosen phosphorylase b at concentrations close to those found in skeletal muscle interacts with sarcoplasmic reticulum membranes, but not with liposomes made of lipids extracted from these membranes, and is inhibited upon binding to the membrane. The interaction of glycogen phosphorylase with the sarcoplasmic reticulum membrane is modulated by phosphorylation, for the a form of this enzyme shows a K0.5 of interaction about 10-fold lower than the b form. Upon association to the membrane the fluorescence properties of the coenzymes of glycogen phosphorylase, pyridoxal-5′-phosphate, are strongly altered, for the fluorescence at 535 nm is partially quenched and the fluorescence at 415–420 nm increases. Using fluorescein labeled sarcoplasmic reticulum membranes we have found that average conformation of the Ca2+ + Mg2+-ATPaze is also altered on binding or phosphorylase b. In conclusion, the results reported in this paper suggest that glycogen phosphorylase and Ca2+ + Mg2+-ATPase directly interact under experimental conditions similar to those found in the sarcoplasm, and that this interaction is modulated by phosphorylation of the phosphorylase.