BRAF inhibitors reverse the unique molecular signature and phenotype of hairy cell leukemia and exert potent antileukemic activity.

Hairy cell leukemia (HCL) shows unique clinicopathological and biological features. HCL responds well to purine analogs but relapses are frequent and novel therapies are required. BRAF-V600E is the key driver mutation in HCL and distinguishes it from other B-cell lymphomas, including HCL-like leukemias/lymphomas (HCL-variant and splenic marginal zone lymphoma). The kinase-activating BRAF-V600E mutation also represents an ideal therapeutic target in HCL. Here, we investigated the biological and therapeutic importance of the activated BRAF-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway in HCL by exposing in vitro primary leukemic cells purified from 26 patients to clinically available BRAF (vemurafenib; dabrafenib) or MEK (trametinib) inhibitors. Results were validated in vivo in samples from vemurafenib-treated HCL patients within a phase 2 clinical trial. BRAF and MEK inhibitors caused, specifically in HCL (but not HCL-like) cells, marked MEK/ERK dephosphorylation, silencing of the BRAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene expression signature, downregulation of the HCL markers CD25, tartrate-resistant acid phosphatase, and cyclin D1, smoothening of leukemic cells' hairy surface, and, eventually, apoptosis. Apoptosis was partially blunted by coculture with bone marrow stromal cells antagonizing MEK-ERK dephosphorylation. This protective effect could be counteracted by combined BRAF and MEK inhibition. Our results strongly support and inform the clinical use of BRAF and MEK inhibitors in HCL.

Macrophage induced gelsolin in response to Group B Streptococcus (GBS) infection.

Group B Streptococcus (GBS) has evolved several strategies to avoid host defences. We have shown that interaction of macrophages with GBS causes macrophage calpain activation, cytoskeletal disruption and apoptosis, consequences of intracellular calcium increase induced by membrane permeability alterations provoked by GBS-ß-haemolysin. Open question remains about what effect calcium influx has on other calcium-sensing proteins such as gelsolin, involved in cytoskeleton modulation and apoptosis. Therefore we analysed the effect of GBS-III-COH31:macrophage interaction on gelsolin expression. Here we demonstrate that an early macrophage response to GBS-III-COH31 is a very strong gelsolin increase, which occurs in a time- and infection-ratio-dependent manner. This is not due to transcriptional events, translation events, protein turnover alterations, or protein-kinase activation, but to calcium influx, calpain activation and caspase-3 degradation. In fact, EGTA and PD150606 (calpain inhibitor) prevented gelsolin increase while BAF (caspase inhibitor) enhanced it. Since gelsolin increase is induced by highly ß-haemolytic GBS-III-NEM316 and GBS-V-10/84, but not by weakly ß-haemolytic GBS, or GBS-III-COH31 in conditions suppressing ß-haemolysin expression/activity and the presence of dipalmitoylphosphatidylcholine (ß-haemolysin inhibitor), GBS-ß-haemolysin is solely responsible for gelsolin increase causing, through membrane permeability defects, calcium influx and calpain activation. Early gelsolin increase could represent a macrophage response to antagonize apoptosis since gelsolin knockdown increases macrophage susceptibility to GBS-induced apoptosis. This response seems to be GBS specific because macrophage apoptosis by Staurosporine or Cycloeximide does not induce gelsolin.

BRAF mutations in hairy-cell leukemia.

BACKGROUND: Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. METHODS: We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. RESULTS: Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

IL-17-producing CD4-CD8- T cells are expanded in the peripheral blood, infiltrate salivary glands and are resistant to corticosteroids in patients with primary Sjogren's syndrome.

OBJECTIVES: It has been recently observed that a T-cell subset, lacking of both CD4 and CD8 molecules and defined as double negative (DN), is expanded in the blood of patients with systemic lupus erythematosus, produces IL-17 and accumulates in the kidney during nephritis. Since IL-17 production is enhanced in salivary gland infiltrates of primary Sjögren's syndrome (SS) patients, we investigated whether DN T cells may be involved in the pathogenesis of salivary gland damage. METHODS: Phenotypic characterisation of peripheral blood mononuclear cells from SS patients and controls was performed by flow cytometry in freshly isolated and anti-CD3-stimulated cells. SS minor salivary glands were processed for immunofluorescence staining. RESULTS: CD3(+)CD4(-)CD8(-) DN T cells were major producers of IL-17 in SS and expressed ROR-γt. They were expanded in the peripheral blood, spontaneously produced IL-17 and infiltrated salivary glands. In addition, the expansion of αß-TCR(+) DN T cells was associated with disease activity. Notably, IL-17-producing DN T cells from SS patients, but not from healthy controls, were strongly resistant to the in vitro effect of dexamethasone. CONCLUSIONS: These findings appear to be of great interest since the identification of a peculiar T-cell subset with pro-inflammatory activity, but resistant to corticosteroids, in an autoimmune disorder such as SS may help to design new specific treatments for the disease.

H(2)O(2) disposal in cardiolipin-enriched brain mitochondria is due to increased cytochrome c peroxidase activity.

The mitochondrial electron transport chain is a source of oxygen superoxide anion (O(2)(-)) that is dismutated to H(2)O(2). Although low levels of ROS are physiologically synthesized during respiration, their increase contributes to cell injury. Therefore, an efficient machinery for H(2)O(2) disposal is essential in mitochondria. In this study, the ability of brain mitochondria to acquire cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylserine (PS) in vitro through a fusion process was exploited to investigate lipid effects on ROS. MTT assay, oxygen consumption, and respiratory ratio indicated that the acquired phospholipids did not alter mitochondrial respiration and O(2)(-) production from succinate. However, in CL-enriched mitochondria, H(2)O(2) levels where 27% and 47% of control in the absence and in the presence of antimycin A, respectively, suggesting an increase in H(2)O(2) elimination. Concomitantly, cytochrome c (cyt c) was released outside mitochondria. Since free oxidized cyt c acquired peroxidase activity towards H(2)O(2) upon interaction with CL in vitro, a contribution of cyt c to H(2)O(2) disposal in mitochondria through CL conferred peroxidase activity is plausible. In this model, the accompanying CL peroxidation should weaken cyt c-CL interactions, favouring the detachment and release of the protein. Neither cyt c peroxidase activity was elicited by PS in vitro, nor cyt c release was observed in PS-enriched mitochondria, although H(2)O(2) levels were significantly decreased, suggesting a cyt c-independent role of PS in ROS metabolism in mitochondria.

Group B Streptococcus (GBS) disrupts by calpain activation the actin and microtubule cytoskeleton of macrophages.

Group B Streptococcus (GBS) has evolved several strategies to avoid host defences where macrophages are one of main targets. Since pathogens frequently target the cytoskeleton to evade immune defences, we investigated if GBS manipulates macrophage cytoskeleton. GBS-III-COH31 in a time- and infection ratio-dependent manner induces great macrophage cytoskeleton alterations, causing degradation of several structural and regulatory cytoskeletal proteins. GBS ß-haemolysin is involved in cytoskeleton alterations causing plasma membrane permeability defects which allow calcium influx and calpain activation. In fact, cytoskeleton alterations are not induced by GBS-III-COH31 in conditions that suppress ß-haemolysin expression/activity and in presence of dipalmitoylphosphatidylcholine (ß-haemolysin inhibitor). Calpains, particularly m-calpain, are responsible for GBS-III-COH31-induced cytoskeleton disruption. In fact, the calpain inhibitor PD150606, m-calpain small-interfering-RNA and EGTA which inhibit calpain activation prevented cytoskeleton degradation whereas µ-calpain and other protease inhibitors did not. Finally, calpain inhibition strongly increased the number of viable intracellular GBS-III-COH31, showing that cytoskeleton alterations reduced macrophage phagocytosis. Marked macrophage cytoskeleton alterations are also induced by GBS-III-NEM316 and GBS-V-10/84 through ß-haemolysin-mediated plasma membrane permeability defects which allow calpain activation. This study suggests a new GBS strategy to evade macrophage antimicrobial responses based on cytoskeleton disruption by an unusual mechanism mediated by calcium influx and calpain activation.

A plant secretory signal peptide targets plastome-encoded recombinant proteins to the thylakoid membrane.

Plastids are considered promising bioreactors for the production of recombinant proteins, but the knowledge of the mechanisms regulating foreign protein folding, targeting, and accumulation in these organelles is still incomplete. Here we demonstrate that a plant secretory signal peptide is able to target a plastome-encoded recombinant protein to the thylakoid membrane. The fusion protein zeolin with its native signal peptide expressed by tobacco (Nicotiana tabacum) transplastomic plants was directed into the chloroplast thylakoid membranes, whereas the zeolin mutant devoid of the signal peptide, Δzeolin, is instead accumulated in the stroma. We also show that zeolin folds in the thylakoid membrane where it accumulates as trimers able to form disulphide bonds. Disulphide bonds contribute to protein accumulation since zeolin shows a higher accumulation level with respect to stromal Δzeolin, whose folding is hampered as the protein accumulates at low amounts in a monomeric form and it is not oxidized. Thus, post-transcriptional processes seem to regulate the stability and accumulation of plastid-synthesized zeolin. The most plausible zeolin targeting mechanism to thylakoid is discussed herein.

Human CD1-restricted T cell recognition of lipids from pollens.

Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex-related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4(+) T cell receptor for antigen (TCR)alphabeta(+), some CD4(-)CD8(-) TCRgammadelta(+), but rarely Valpha24i(+) natural killer-T cells, and required CD1a(+) and CD1d(+) antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-gamma, in some cases IL-10 and transforming growth factor-beta, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis-exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids.

The benefits of exercise in cancer patients and the criteria for exercise prescription in cardio-oncology.

Cancer and cardiovascular diseases are the leading causes of death in high-income countries. Cardiovascular complications can be found in cancer patients, being the result of so-called 'cardio-toxicity'. Therefore, it becomes essential to thoroughly investigate the origin of cardiac damage and the strategy to prevent it or to reverse the negative remodelling associated with cardiotoxicity. In this review the beneficial effects of physical exercise in cancer patients were analysed, particularly to prevent cardio-toxicity before its clinical manifestation. According to the relevance of exercise, we suggest strategies for exercise prescription with a tailored approach in these patients. In conclusion, physical exercise seems to be a promising and effective treatment for cancer patients during and after therapy and seems to counteract the negative effects induced by drugs on the cardiovascular system. Exercise prescription should be tailored according to patient's individual characteristics, to the drugs administered, to the personal history, and to his/her response to exercise, taking into account that different types of training can be prescribed according also to the patient's choice. A cardiological evaluation including exercise testing is essential for an appropriate prescription of exercise in these patients.

Born to be exported: COOH-terminal nuclear export signals of different strength ensure cytoplasmic accumulation of nucleophosmin leukemic mutants.

Creation of a nuclear export signal (NES) motif and loss of tryptophans (W) 288 and 290 (or 290 only) at the COOH terminus of nucleophosmin (NPM) are both crucial for NPM aberrant cytoplasmic accumulation in acute myelogenous leukemia (AML) carrying NPM1 mutations. Hereby, we clarify how these COOH-terminal alterations functionally cooperate to delocalize NPM to the cytoplasm. Using a Rev(1.4)-based shuttling assay, we measured the nuclear export efficiency of six different COOH-terminal NES motifs identified in NPM mutants and found significant strength variability, the strongest NES motifs being associated with NPM mutants retaining W288. When artificially coupled with a weak NES, W288-retaining NPM mutants are not exported efficiently into cytoplasm because the force (W288) driving the mutants toward the nucleolus overwhelms the force (NES) exporting the mutants into cytoplasm. We then used this functional assay to study the physiologic NH(2)-terminal NES motifs of wild-type NPM and found that they are weak, which explains the prominent nucleolar localization of wild-type NPM. Thus, the opposing balance of forces (tryptophans and NES) seems to determine the subcellular localization of NPM. The fact that W288-retaining mutants always combine with the strongest NES reveals mutational selective pressure toward efficient export into cytoplasm, pointing to this event as critical for leukemogenesis.

NCX-4016, a nitric oxide-releasing aspirin, protects endothelial cells against apoptosis by modulating mitochondrial function.

We investigated the effect of a nitric oxide (NO)-releasing derivative of aspirin (NCX-4016) on a mitochondria-dependent model of apoptosis in human umbilical endothelial cells (HUVEC). Exposure of HUVEC to staurosporine caused a progressive fall in mitochondrial membrane potential (DeltaPsi(m)) and apoptosis. Exposure to an NO donor, (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), caused an early (1-3h) hyperpolarization of DeltaPsi(m) and reduction of apoptosis that was followed (at 4-8 h) by an accelerated collapse of DeltaPsi(m) and cell death. In contrast, treatment with NCX-4016, but not with aspirin or a non-NO-releasing analogue of NCX-4016, protected HUVEC against the apoptotic actions of staurosporine for up to 8 h. Confocal microscopy demonstrated that although NCX-4016 released NO in subcellular compartments, DETA-NO caused a generalized increase in cytosolic fluorescence. Exposure to DETA-NO resulted in a rapid and profound inhibition of cell respiration (78.3 +/- 6.4%), whereas NCX-4016 caused a less pronounced reduction in oxygen consumption (43.5 +/- 5.3%). Staurosporine caused a time-dependent activation of proapoptotic caspases. NCX-4016 prevented this activation, whereas DETA-NO failed to inhibit caspase activity. In contrast to DETA-NO, NCX-4016 did not increase mitochondrial oxidative stress. These data demonstrated that NCX-4016 conveys NO directly inside endothelial cells and modulates mitochondrial function.

Ser-256 phosphorylation dynamics of Aquaporin 2 during maturation from the ER to the vesicular compartment in renal cells.

Aquaporin 2 (AQP2) phosphorylation at Ser-256 by protein kinase A (PKA) is a key signal for vasopressin-stimulated AQP2 insertion into the plasma membrane in renal cells. This study underscores the possible role of phosphorylation at Ser-256 in regulating AQP2 maturation. AQP2-transfected renal CD8 cells were incubated with brefeldin A (BFA) to accumulate newly synthesized AQP2 in the endoplasmic reticulum (ER), and AQP2 flow from ER to the vesicular compartment was analyzed after BFA washout. We found that a) in the ER, AQP2 is weakly phosphorylated; b) the amount of phosphorylated AQP2 (p-AQP2) at Ser-256 increased significantly during transit in the Golgi, even in the presence of the PKA inhibitor H89; and c) AQP2 transport from the Golgi to the vasopressin-regulated vesicular compartment occurred with a concomitant decrease in p-AQP2 at Ser-256. These results support the hypothesis that AQP2 transition in the Golgi apparatus is associated with a PKA-independent increase in AQP2 phosphorylation at Ser-256. Conversely, impaired constitutive phosphorylation in a Golgi-associated compartment occurring in cells expressing mutated S256A-AQP2 or E258K-AQP2 causes phosphorylation-defective AQP2 routing to lysosomes. This result might explain the molecular basis of the dominant form of nephrogenic diabetes insipidus caused by the mutation E258K-AQP2, in which the phenotype is caused by an impaired routing of AQP2.

Lifestyle, nutrition and breast cancer: facts and presumptions for consideration.

Breast cancer is the most common cancer in women worldwide, and the high incidence of this cancer coupled with improvements in initial treatments has led to an ever-increasing number of breast cancer survivors. Among the prospective epidemiological studies on diet and breast cancer incidence and recurrence, to date, there is no association that is strong, reproducible and statistically significant, with the exception of alcohol intake, overweight, and weight gain. Nevertheless, many beliefs about food and breast cancer persist in the absence of supporting scientific evidence. After a comprehensive review regarding the role of lifestyle on breast cancer outcomes and a thorough study of the dissemination field including mass media, clinical institutions, and academic figures, we briefly reported the most common presumptions and also facts from the literature regarding lifestyle, nutrition, and breast cancer. The randomised controlled trial is the best study-design that could provide direct evidence of a causal relationship; however, there are methodological difficulties in applying and maintaining a lifestyle intervention for a sufficient period; consequently, there is a lack of this type of study in the literature. Instead, it is possible to obtain indirect evidence from observational prospective studies. In this article, it becomes clear that for now the best advice for women's health is to follow the World Cancer Research Fund/American Institute of Cancer Research (WCRF/AICR) recommendations on diet, nutrition, physical activity, and weight management for cancer prevention, because they are associated with a lower risk of developing most types of cancer, including breast cancer. Despite current awareness of the role of nutrition in cancer outcomes, there is inadequate translation from research findings into clinical practice. We suggest the establishment of a multidisciplinary research consortium to demonstrate the real power of lifestyle interventions.

Involvement of A1 adenosine receptors in the acquisition of fertilizing capacity.

Ejaculated mammalian spermatozoa acquire competence to fertilize oocytes by a two-step process: capacitation followed by acrosome reaction. The biochemical and biophysical modifications occurring in vivo in the female reproductive tract can be reproduced in vitro, and previous studies have suggested a capacitative role for adenosine A(1) receptor (A(1)R). Mice with a targeted disruption of the Adora 1 gene (A(1)R-/- mice) provide a useful model for better understanding the role of the A(1)R in fertility. Murine spermatozoa express A(1)R in the head, neck, midpiece region, and tail. The number of capacitated spermatozoa incubated in human tubal fluid was significantly reduced in A(1)R-/- compared with A(1)R+/+ and A(1)R+/- spermatozoa. The difference between A(1) R+/+ and A(1)R-/- mouse spermatozoa was mainly in the time necessary to reach the maximum percentage of capacitation. A(1)R+/+ murine sperm obtained the full state of capacitation within 90 minutes whereas A(1)R-/- sperm required 240 minutes. Caffeine, a known antagonist of A(1) and A(2A) adenosine receptors, lowered the number of capacitated sperm and affected the time of capacitation in a dose-dependent manner, mimicking the effects of the lack of A(1) receptors. Although number, motility, and viability of A(1)R-/- murine sperm was not significantly different from A(1)R+/+ mouse spermatozoa, a significant reduction of the number of pups produced by A(1)R-/- male mice suggests that A(1) receptors must be fully operative to accomplish the optimal degree of capacitation and thereby fertilization.

Methods to discriminate the distribution of acidic glycohydrolases between the endosomal-lysosomal systems and the plasma membrane.

The endosomal-lysosomal system plays important roles in cellular physiology. Beyond the well-known function as terminal degradative compartment, necessary to maintain the health of the cell, lysosomes are critical for many other cellular processes, such as termination of signaling mediated by cell surface receptors and processing of internalized peptides in antigen-presenting cells. Moreover, the intracellular membrane trafficking related to the endosomal-lysosomal system plays a pivotal role in diverse physiological and pathological processes, such as exocytosis, plasma membrane repair, and endocytosis. Increasing evidences suggest that several lysosomal glycohydrolases, together with nonlysosomal glycohydrolases, are associated with cell membranes in their active form, and they are localized into lipid microdomains. The role of these forms in physiological and pathological conditions, such as differentiation and aging, neurodegenerative diseases, and cancer spreading, is under investigation. Here we provide general methods to purify lipid microdomain proteins and to discriminate cell surface lipid microdomains-associated glycohydrolases from those not exposed on cell surface. The methods reported here have been developed to characterize the membrane-associated forms of the acidic glycohydrolases ß-hexosaminidase and ß-galactosidase, but they may be applied to any other protein of interest.

NGF induces the expression of group IIA secretory phospholipase A2 in PC12 cells: the newly synthesized enzyme is addressed to growing neurites.

We proposed that group IIA secretory phospholipase A(2) (GIIA) participates in neuritogenesis based on our observations that the enzyme migrates to growth cones and neurite tips when PC12 cells are induced to differentiate by nerve growth factor (NGF) (Ferrini et al., Neurochem Res 35:2168-2174, 2010). The involvement of other secretory PLA(2) isoforms in neuronal development has been suggested by others but through different mechanisms. In the present study, we compared the subcellular distribution of GIIA and group X sPLA(2) (GX) after stimulation of PC12 cells with NGF. We found that GIIA, but not GX, localized at the neuritic tips after treatment with NGF, as demonstrated by immunofluorescence analysis. We also found that NGF stimulated the expression and the activity of GIIA. In addition, NGF induced the expressed myc-tagged GIIA protein to migrate to neurite tips in its active form. We propose that GIIA expression, activity, and subcellular localization is regulated by NGF and that the enzyme may participate in neuritogenesis through intracellular mechanisms, most likely by facilitating the remodelling of glycerophospholipid molecular species by deacylation-reacylation reactions necessary for the incorporation of polyunsaturated fatty acids.

Contribution of the central hydrophobic residue in the PXP motif of voltage-dependent K+ channels to S6 flexibility and gating properties.

Shaker-like (KV1.1) channels contain a highly conserved Pro-Val-Pro (PVP) motif at the base of S6 that produces a kink in the S6 helices and provides a flexible element thought to be essential for channel gating. The role of proline-induced kinks in transmembrane helices is well known, but the contribution of the small hydrophobic valine between these two prolines is not known, and interestingly, Shab-like (KV2.1) channels possess an isoleucine at this position (PIP). Here we show that the exact nature of this central hydrophobic residue within the PXP motif confers unique functional properties to KV1 channels, including changes in activation and deactivation kinetics, voltage-dependent properties and open probabilities, but single-channel conductance and cell expression levels are not affected. In support of these functional changes, molecular dynamic simulations demonstrate that valine and isoleucine contribute differently to S6 flexibility within this motif. These results therefore indicate that the nature of the central hydrophobic residue in the PXP motif is an important functional determinant of KV channel gating by contributing, at least in part, to the relative flexibility of this motif.

Trafficking and phosphorylation dynamics of AQP4 in histamine-treated human gastric cells.

BACKGROUND INFORMATION: AQP4 (aquaporin 4) internalization and a concomitant decrease in the osmotic water permeability coefficient (Pf) after histamine exposure has been reported in AQP4-transfected gastric HGT1 cells. RESULTS: In the present study we report that AQP4 internalization is followed by an increase in AQP4 phosphorylation. Histamine treatment for 30 min resulted in an approx. 10-fold increase in AQP4 phosphorylation that was inhibited by 1 microM H89, a specific PKA (protein kinase A) inhibitor, but not by PKC (protein kinase C) and CK2 inhibitors. Moreover, measurement of PKA activity after 30 min of histamine treatment showed that PKA activity was approx. 3-fold higher compared with basal conditions. AQP4 phosphorylation was prevented in cells treated with histamine for 30 min after pre-incubation with PAO (phenylarsine oxide), an inhibitor of protein endocytosis. Using an endo-exocytosis assay we showed that, after histamine washed out, internalized AQP4 recycled back to the cell surface, even in cells in which de novo protein synthesis was inhibited by cycloheximide. CONCLUSIONS: Phosphorylation experiments, combined with immunolocalization studies, indicated that AQP4 phosphorylation is mediated by PKA and occurs subsequently to its internalization in late endosomes. We suggest that phosphorylation might be a mechanism involved in retaining AQP4 in a vesicle-recycling compartment.

Group B Streptococcus induces macrophage apoptosis by calpain activation.

Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mphi) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mphi apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, alpha-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mphi apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mphi apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.

Mutated nucleophosmin detects clonal multilineage involvement in acute myeloid leukemia: Impact on WHO classification.

Because of a lack of specific clonality markers, information on lineage involvement and cell of origin of acute myeloid leukemia with normal karyotype (AML-NK), is missing. Because Nucleophosmin (NPM) gene is frequently mutated in AML-NK and causes aberrant NPM cytoplasmic localization (NPMc+), it was used as an AML lineage clonality marker. Clonal NPM exon 12 mutations were detected in myeloid, monocytic, erythroid, and megakaryocytic cells but not in fibroblasts or endothelia that were laser-microdissected from 3 patients with NPMc+ AML. Aberrant cytoplasmic expression of mutated NPM proteins was identified with anti-NPM antibodies in 2 or more myeloid hemopoietic cell lineages in 99 (61.5%) of 161 of NPMc+ AML paraffin-embedded bone marrow biopsies; lymphoid involvement was excluded in 3 investigated cases. These findings suggest that NPMc+ AML derives from either a common myeloid or earlier progenitor. Immunohistochemical studies show that varying combinations and ratios of NPMc+ leukemic cells from distinct lineages are responsible for heterogeneity within each French-American-British (FAB) classification type and for NPMc+ AML falling into different FAB categories. These findings question the value of FAB criteria in subdividing the WHO category of "AML not otherwise characterized" and suggest that, for clinical use, NPMc+ AML be provisionally regarded as a separate AML with prognostic significance.