Messenger Levels Research Articles

Phosphatidylinositol 5-Phosphate Links Dehydration Stress to the Activity of ARABIDOPSIS TRITHORAX-LIKE Factor ATX1

BackgroundChanges in gene expression enable organisms to respond to environmental stress. Levels of cellular lipid second messengers, such as the phosphoinositide PtdIns5P, change in response to a variety of stresses and can modulate the localization, conformation and activity of a number of intracellular proteins. The plant trithorax factor (ATX1) tri-methylates the lysine 4 residue of histone H3 (H3K4me3) at gene coding sequences, which positively correlates with gene transcription. Microarray analysis has identified a target gene (WRKY70) that is regulated by both ATX1 and by the exogenous addition of PtdIns5P in Arabidopsis. Interestingly, ATX1 contains a PtdIns5P interaction domain (PHD finger) and thus, phosphoinositide signaling, may link environmental stress to changes in gene transcription.Principal FindingsUsing the plant Arabidopsis as a model system, we demonstrate a link between PtdIns5P and the activity of the chromatin modifier ATX1 in response to dehydration stress. We show for the first time that dehydration leads to an increase in cellular PtdIns5P in Arabidopsis. The Arabidopsis homolog of myotubularin (AtMTM1) is capable of generating PtdIns5P and here, we show that AtMTM1 is essential for the induced increase in PtdIns5P upon dehydration. Furthermore, we demonstrate that the ATX1-dependent gene, WRKY70, is downregulated during dehydration and that lowered transcript levels are accompanied by a drastic reduction in H3K4me3 of its nucleosomes. We follow changes in WRKY70 nucleosomal K4 methylation as a model to study ATX1 activity at chromatin during dehydration stress. We found that during dehydration stress, the physical presence of ATX1 at the WRKY70 locus was diminished and that ATX1 depletion resulted from it being retained in the cytoplasm when PtdIns5P was elevated. The PHD of ATX1 and catalytically active AtMTM1 are required for the cytoplasmic localization of ATX1.Conclusions/SignificanceThe novelty of the manuscript is in the discovery of a mechanistic link between a chromatin modifying activity (ATX1) and a lipid (PtdIns5P) synthesis in a signaling pathway that ultimately results in altered expression of ATX1 dependent genes downregulated in response to dehydration stress.

Redox Control of Protein Kinase C: Cell- and Disease-Specific Aspects

Hormones, growth factors, electrical stimulation, and cell-cell interactions regulate numerous cellular processes by altering the levels of second messengers, thus influencing biochemical reactions inside the cells. The Protein Kinase C family (PKCs) is a group of serine/threonine kinases that are dependent on calcium (Ca(2+)), diacylglycerol, and phospholipids. Signaling pathways that induce variations on the levels of PKC activators have been implicated in the regulation of diverse cellular functions and, in turn, PKCs are key regulators of a plethora of cellular processes, including proliferation, differentiation, and tumorigenesis. Importantly, PKCs contain regions, both in the N-terminal regulatory domain and in the C-terminal catalytic domain, that are susceptible to redox modifications. In several pathophysiological conditions when the balance between oxidants, antioxidants, and alkylants is compromised, cells undergo redox stress. PKCs are cell-signaling proteins that are particularly sensitive to redox stress because modification of their redox-sensitive regions interferes with their activity and, thus, with their biological effects. In this review, we summarize the involvement of PKCs in health and disease and the importance of redox signaling in the regulation of this family of kinases.

Thrombin-Induced Endothelin-1 Synthesis and Secretion in Retinal Pigment Epithelial Cells Is Rho Kinase Dependent

The retinal pigment epithelium (RPE) is a major source for endothelin-1 (ET-1), a potent vasoactive peptide, at the outer blood–retinal barrier. Factors that regulate ET-1 synthesis at this site may help identify its normal function and its role in pathologic states accompanying retinal injury. Thrombin is one such factor that might act on the RPE after injury and breakdown of the blood–retinal barrier. The present study was conducted to identify signaling intermediates in thrombin-induced ET-1 synthesis and secretion in primary human RPE (hRPE) and transformed RPE cells (ARPE-19) and a possible pharmacological strategy to block excess release of ET-1. Cultured hRPE cells were treated with different concentrations of thrombin and thrombin receptor agonists, and a time course to measure levels of preproET-1 (ppET-1) mRNA and secreted mature ET-1 was performed. Levels of secondary messengers [Ca²+]i and RhoA were measured and pharmacologically inhibited to determine how receptor-mediated thrombin activity lead to changes in ET-1 levels. Thrombin primarily acts via the protease-activated receptor-1 (PAR-1) subtype in RPE to induce ET-1 synthesis. Thrombin and other receptor agonists increased both [Ca²+]<]i and active RhoA. PAR-1-dependent rho/Rho kinase activation led to increase in ppET-1 mRNA and mature ET-1 secretion. Transient intracellular calcium mobilization and protein kinase C activation by thrombin play a minor role, if any, in ET-1 synthesis in RPE. Instead, rho/Rho kinase activation after PAR-1 stimulation strongly increased ppET-1 mRNA and ET-1 secretion in hRPE cells.

Plasticity of vagal brainstem circuits in the control of gastric function

Sensory information from the viscera, including the gastrointestinal (GI) tract, is transmitted through the afferent vagus via a glutamatergic synapse to neurons of the nucleus tractus solitarius (NTS), which integrate this sensory information to regulate autonomic functions and homeostasis. The integrated response is conveyed to, amongst other nuclei, the preganglionic neurons of the dorsal motor nucleus of the vagus (DMV) using mainly GABA, glutamate and catecholamines as neurotransmitters. Despite being modulated by almost all the neurotransmitters tested so far, the glutamatergic synapse between NTS and DMV does not appear to be tonically active in the control of gastric motility and tone. Conversely, tonic inhibitory GABAergic neurotransmission from the NTS to the DMV appears critical in setting gastric tone and motility, yet, under basal conditions, this synapse appears resistant to modulation. Here, we review the available evidence suggesting that vagal efferent output to the GI tract is regulated, perhaps even controlled, in an 'on-demand' and efficient manner in response to ever-changing homeostatic conditions. The focus of this review is on the plasticity induced by variations in the levels of second messengers in the brainstem neurons that form vago-vagal reflex circuits. Emphasis is placed upon the modulation of GABAergic transmission to DMV neurons and the modulation of afferent input from the GI tract by neurohormones/neurotransmitters and macronutrients. Derangement of this 'on-demand' organization of brainstem vagal circuits may be one of the factors underlying the pathophysiological changes observed in functional dyspepsia or hyperglycemic gastroparesis.

A Blue Light‐inducible Phosphodiesterase Activity in the Cyanobacterium Synechococcus elongatus

A blue light-inducible phosphodiesterase (PDE) activity, specific for the hydrolysis of cyclic di-GMP (c-di-GMP), has been identified in a recombinant protein from Synechococcus elongatus. Blue light (BL) activation is accomplished by a light, oxygen, voltage (LOV) domain, found in plant phototropins and bacterial BL photoreceptors. The genome of S. elongatus contains two genes coding for proteins with LOV domains fused to EAL domains (SL1 and SL2). In both cases, a GGDEF motif is placed in between the LOV and the EAL motifs. Such arrangement is frequently found with diguanylate-cyclase (DGC) functions that form c-di-GMP. Cyclic di-GMP acts as a second messenger molecule regulating biofilm formation in many microbial species. Both enzyme activities modulate the intracellular level of this second messenger, although in most proteins only one of the two enzyme functions is active. Both S. elongatus LOV-GGDEF-EAL proteins were expressed in full length or as truncated proteins. Only the SL2 protein, expressed as a LOV-GGDEF-EAL construct, showed an increase of PDE activity upon BL irradiation, demonstrating this activity for the first time in a LOV-domain protein. Addition of GTP or c-di-GMP did not affect the observed enzymatic activity. In none of the full-length or truncated proteins was a DGC activity detected.

Differential regulation of TNF receptors by vagal nerve stimulation protects heart against acute ischemic injury

Vagal nerve stimulation (VS) has been reported to improve the survival after both acute and chronic myocardial infarction through the release of neurotransmitter ACh. However, the precise mechanism behind its beneficial effect is still unknown. In this study, we demonstrate the upregulation of tumor necrosis factor-alpha (TNF-α) and its cell survival TNF receptor-2 (TNFR2) as the mechanism behind VS induced myocardial protection.We investigated the effects of efferent VS on myocardial ischemic injury with in vivo and in vitro mouse models. In in vivo hearts VS significantly increased the expression of TNF-α both at the messenger and protein level after 3-hours of myocardial ischemia. In the in vitro studies ACh treatment before hypoxia, induced a significant upregulation of TNF-α compared to the untreated cardiomyocytes. Immunofluorescence analysis confirmed the synthesis of TNF-α by cardiomyocytes both in vivo and in vitro. VS also significantly reduced the myocardial infarct size (23.9±5.7% vs. 56±1.9%) and activated the cell survival Akt cascade system. Further, ACh upregulated the cell survival TNFR2 expression, while downregulating the cell destructive TNF receptor 1 (TNFR1) expression. These results were confirmed using the TNF receptors deficient mice, where the VS mediated protection was lost both in vivo and in vitro in TNFR2 (TNFR2-/-) and TNF receptors double knock out (TNFR1-/-2-/-) mice.VS and ACh protects the heart against acute ischemia or hypoxic injury by differentially regulating the TNF receptor subtypes.

SHIP Is Required for Dendritic Cell Maturation

Although several groups have investigated the role of SHIP in macrophage (M) development and function, SHIP's contribution to the generation, maturation, and innate immune activation of dendritic cells (DCs) is poorly understood. We show herein that SHIP negatively regulates the generation of DCs from bone marrow precursors in vitro and in vivo, as illustrated by the enhanced expansion of DCs from SHIP(-/-) GM-CSF cultures, as well as increased numbers of DCs in the spleens of SHIP-deficient mice. Interestingly, however, these SHIP(-/-) DCs display a relatively immature phenotype and secrete substantially lower levels of IL-12 after TLR ligand stimulation than wild type DCs. This, in turn, leads to a dramatically reduced stimulation of Ag-specific T cell proliferation and Th1 cell responses in vitro and in vivo. This immature phenotype of SHIP(-/-) DCs could be reversed with the PI3K inhibitors LY294002 and wortmannin, suggesting that SHIP promotes DC maturation by reducing the levels of the PI3K second messenger phosphatidylinositol-3,4,5-trisphosphate. These results are consistent with SHIP being a negative regulator of GM-CSF-derived DC generation but a positive regulator of GM-CSF-derived DC maturation and function.

Inositol trisphosphate 3-kinases: focus on immune and neuronal signaling.

The localized control of second messenger levels sculpts dynamic and persistent changes in cell physiology and structure. Inositol trisphosphate [Ins(1,4,5)P(3)] 3-kinases (ITPKs) phosphorylate the intracellular second messenger Ins(1,4,5)P(3). These enzymes terminate the signal to release Ca(2+) from the endoplasmic reticulum and produce the messenger inositol tetrakisphosphate [Ins(1,3,4,5)P(4)]. Independent of their enzymatic activity, ITPKs regulate the microstructure of the actin cytoskeleton. The immune phenotypes of ITPK knockout mice raise new questions about how ITPKs control inositol phosphate lifetimes within spatial and temporal domains during lymphocyte maturation. The intense concentration of ITPK on actin inside the dendritic spines of pyramidal neurons suggests a role in signal integration and structural plasticity in the dendrite, and mice lacking neuronal ITPK exhibit memory deficits. Thus, the molecular and anatomical features of ITPKs allow them to regulate the spatiotemporal properties of intracellular signals, leading to the formation of persistent molecular memories.

Enhanced NMDA R1, NMDA 2B and mGlu 5 receptors gene expression in the cerebellum of insulin induced hypoglycaemic and streptozotocin induced diabetic rats

Glucose homeostasis in humans is an important factor for the functioning of the nervous system. A decrease in glucose content below a minimal level or hypoglycemia is dangerous for cells of the central and peripheral nerve system. In the present study we showed the effects of insulin induced hypoglycaemia and streptozotocin induced diabetes on the cerebellar glutamate receptor subunits and glutamate transporter. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. We found an up regulation in NMDA receptor number and gene expression of N-methyl- d-aspartic acid (NMDA R1), NMDA 2B, metabotrophic glutamate 5 (mGlu 5) glutamate receptor subunits in experimental rats. The glutamate content was shown to be increased with decreased glutamate aspartate transporter (GLAST) gene expressions indicating lower reuptake of glutamate. The enhanced gene expression of NMDA R1, NMDA 2B, mGlu 5 glutamate receptors were confirmed by immunohistochemistry studies. At the second messenger level, the IP3 content and IP3 receptors were enhanced in the cerebellum of both hypoglycaemic and diabetic rats increased. The present study showed that the enhanced glutamate content activates NMDA receptors, increasing the inositol triphosphate (IP3) content which mediates Ca 2+ overload in cells causing cell damage and neurodegeneration. Our results also showed that the enhanced glutamate receptor activity were more prominent in hypoglycaemic group compared to diabetic group. Further the neurodegeneration by the up regulation of glutamate receptor activity causing motor dysfunction was demonstrated by the Rotarod test. Thus our results suggest that enhanced NMDA receptor mediated neurodegeneration affect the motor learning and memory ability of an individual.

Pheromone Transduction in Moths

Calling female moths attract their mates late at night with intermittent release of a species-specific sex-pheromone blend. Mean frequency of pheromone filaments encodes distance to the calling female. In their zig-zagging upwind search male moths encounter turbulent pheromone blend filaments at highly variable concentrations and frequencies. The male moth antennae are delicately designed to detect and distinguish even traces of these sex pheromones amongst the abundance of other odors. Its olfactory receptor neurons sense even single pheromone molecules and track intermittent pheromone filaments of highly variable frequencies up to about 30 Hz over a wide concentration range. In the hawkmoth Manduca sexta brief, weak pheromone stimuli as encountered during flight are detected via a metabotropic PLCβ-dependent signal transduction cascade which leads to transient changes in intracellular Ca2+ concentrations. Strong or long pheromone stimuli, which are possibly perceived in direct contact with the female, activate receptor-guanylyl cyclases causing long-term adaptation. In addition, depending on endogenous rhythms of the moth's physiological state, hormones such as the stress hormone octopamine modulate second messenger levels in sensory neurons. High octopamine levels during the activity phase maximize temporal resolution cAMP-dependently as a prerequisite to mate location. Thus, I suggest that sliding adjustment of odor response threshold and kinetics is based upon relative concentration ratios of intracellular Ca2+ and cyclic nucleotide levels which gate different ion channels synergistically. In addition, I propose a new hypothesis for the cyclic nucleotide-dependent ion channel formed by insect olfactory receptor/coreceptor complexes. Instead of being employed for an ionotropic mechanism of odor detection it is proposed to control subthreshold membrane potential oscillation of sensory neurons, as a basis for temporal encoding of odors.

Aven and Survivin Expression in Egyptian Acute Leukemia and Their Relation to Apoptosis.

Abstract 4717 BackgroundSeveral anti apoptotic signals have been recently identified. Aven and Survivin are broadly expressed and are conserved in mammalian species. Patients and MethodsSixty-four patients with acute leukemia [39 with acute myeloblastic leukemia (AML) and 25 with acute lymphoblastic leukemia (ALL)] were used as the study group. Reverse transcriptase (RT –PCR) was used, Aven and Survivin expression were detected at the messenger (mRNA) level .DNA fragmentation was carried out on daily basis before &24 hours after the therapeutic dose every day for 4 times during the initial induction of chemotherapy (everyday for ALL& every otherday for AML). ResultsSurvivin expression was found in (66%) of acute leukemia, near significantly (p=0.06) more in AML (74%) than in ALL (52%). While, Aven expression was (40.6%) in acute leukemia equally expressed in AML (41%) and ALL (40%).Patients were categorized into 3 groups based on DNA fragmentation, whereby group I: DNA fragmentation found on day 1 or day 1+day 2, group II: DNA fragmentation found on days 1, 2 and 3, group III where fragmentation was found on days 1+2+3+4 or/and day5. Absence of Aven expression significantly (p<0.001) contributed to DNA fragmentation as 24 /35 (68.6%) of acute leukemia with Aven negative were in group III (the best group). Absence of Survivin did not contribute as much, Where 18 out of 31 (58%) of group III were Survivin positive (P= 0.1).None of the concordant both positive Survivin and Aven were in group III (the good 5 day fragmentation), this was statistically highly significant (P< 0.001) relation. Survivin was statistically related to CD7 expression (P<0.001) in AML only, while Aven was statistically related to CD34 expression (P<0.014) in the whole acute leukemia cases. Aven was statistically correlated to alkaline phosphatase (P< 0.036).There was a significant dissociation between Aven and Survivin in AML (p=0.03) and near significant dissociation in ALL (p=0.07). ConclusionAven seems to be more important as an inhibitor of apoptosis than survivin in acute leukemia. The presence of both Aven & survivin confers a survival disadvantage & a significantly worse DNA fragmentation pattern, thus suggesting a synergistic inhibition of apoptosis when present together, although, they tend not to be expressed together. The highly significant relation between CD7 & surviving expression, might suggest their involvement in a common signal transduction pathway.Key words: Survivin, Aven, AML, ALL and DNA fragmentation. Disclosures:No relevant conflicts of interest to declare.

Corneal aldehyde dehydrogenases: Multiple functions and novel nuclear localization

Aldehyde dehydrogenases (ALDHs) represent a superfamily of NAD(P) +-dependent enzymes which catalyze the oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding acids. Some ALDHs have been identified as corneal crystallins and thereby contribute to the protective and refractive properties of the cornea. ALDH3A1 is highly expressed in the cornea of most mammals with the exception of rabbit that abundantly expresses ALDH1A1 in the cornea instead of ALDH3A1. In this study, we examined the gene expression of other ALDHs and found high messenger levels of ALDH1B1, ALDH2 and ALDH7A1 in mouse cornea and lens. Substantial evidence supports a protective role for ALDH3A1 and ALDH1A1 against ultraviolet radiation (UVR)-induced oxidative damage to ocular tissues. The mechanism by which this protection occurs includes UVR filtering, detoxification of reactive aldehydes generated by UVR exposure and antioxidant activity. We recently have identified ALDH3A1 as a nuclear protein in corneal epithelium. Herein, we show that ALDH3A1 is also found in the nucleus of rabbit keratocytes. The nuclear presence of ALDH3A1 may be involved in cell cycle regulation.

Doxorubicin induces ceramide and diacylglycerol accumulation in rat hepatocytes through independent routes

Doxorubicin (DOX) is a potent anticancer drug, whose clinical use is limited due to its toxicity. This toxicity has been associated with free radicals generated during the drug metabolism. We previously found that DOX increased the intracellular diacylglycerol (DAG) levels at 1 h in isolated rat hepatocytes, probably by mobilizing choline-enriched phospholipids. In this work, we studied the effects of DOX on oxidative stress markers, and the possible contribution of ceramide metabolism to DAG accumulation. Other possible routes of DAG production, such as impairment of triacylglycerol (TAG) synthesis, and their connection with oxidative stress were also investigated. Time-course experiments revealed that DOX decreased intracellular GSH at 2 h, but did not affect cell viability, ATP or malondialdehyde (MDA) levels at any time. DOX did not modify the intracellular levels of [ 3H]-ceramide during the first 90 min of exposure, but increased it significantly at 2 h. [ 3H]-Sphingomyelin remained unchanged during the whole period. These results indicate that ceramide metabolism is not involved in the early DAG response to DOX. The drug markedly increased the incorporation of [ 3H]-oleate into intracellular DAG from 60 min. In contrast, DOX reduced the incorporation of [ 3H]-oleate into intracellular phospholipids and TAG. DOX inhibited TAG synthesis at the DAG acyltransferase step. These results suggest that DOX increases the intracellular levels of the lipid messengers, ceramide and DAG, by independent mechanisms. Activation of the de novo synthesis of ceramide is probably involved in the sphingolipid accumulation, while inhibition of TAG synthesis contributes to DAG accumulation, this response being independent of oxidative damage.

P4.10 Serotonin availability increases in rat small intestine during a high-fat diet

A high fat diet is associated with subtle changes in the gastrointestinal function and in the levels of intestinal messengers secreted from enteroendocrine cells. Enterochromaffin (EC) cells contain serotonin (5-HT) which is a key regulator of gut function. Our recent electrochemical studies have shown that 5-HT levels are increased in a rat model of obesity but the molecular basis for this increased is unknown. We hypothesised that changes in 5-HT synthesis or transport proteins could account for these observations.

Abl knockout differentially affects p130 Crk-associated substrate, vinculin, and paxillin in blood vessels of mice

Actin polymerization has recently emerged as an important cellular process that regulates smooth muscle contraction. Abelson tyrosine kinase (Abl) has been implicated in the regulation of actin dynamics and force development in vascular smooth muscle. In the present study, the systolic blood pressure was lower in Abl(-/-) knockout mice compared with wild-type mice. The knockout of Abl diminished the tyrosine phosphorylation of p130 Crk-associated substrate (CAS, an adapter protein associated with smooth muscle contraction) in resistance arteries upon stimulation with phenylephrine or angiotensin II. The agonist-elicited enhancement of F-actin-to-G-actin ratios in arteries assessed by fluorescent microscopy was also reduced in Abl(-/-) mice. It has been known that vinculin is a structural protein that links actin filaments to extracellular matrix via transmembrane integrins, whereas paxillin is a signaling protein associated with focal contacts mediating actin cytoskeleton remodeling. The expression of vinculin and paxillin at protein and messenger levels was lower in arterial vessels from Abl knockout mice. However, the agonist-induced increase in myosin phosphorylation was not attenuated in arteries from Abl knockout mice. These results indicate that Abl differentially regulates Crk-associated substrate, vinculin, and paxillin in arterial vessels. The Abl-regulated cellular process and blood pressure are independent of myosin activation in vascular smooth muscle.

Calcium Homeostasis Is Dysregulated in Parkinsonian Patients With l-DOPA-Induced Dyskinesias

Long-term treatment of Parkinson disease (PD) is frequently associated with l-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias (LIDs). L-DOPA-induced dyskinesias are likely due to changes in the signal transduction pathways, at the striatal level, related to pulsatile stimulation of dopamine receptors. We investigated whether markers of this phenomenon can also be detected peripherally. We analyzed mRNA expression for D5 (D1-like) and D3 (D2-like) receptors and levels of second messengers, such as cAMP and free intracellular Ca2+ ([Ca2+]i), in peripheral blood lymphocytes of PD patients with (LID+) or without LIDs (LID-). Patients with PD showed depressed [Ca2+]i rise in response to mitogen-induced activation. The defect was more pronounced in LID+ (-33% with respect to healthy controls) than in LID- patients (-20%). Peripheral blood lymphocyte levels of cAMP were decreased in both LID+ (3.8 +/- 2.9 pmol/10 cells) and LID- patients (4.2 +/- 2.4 pmol/10(6) cells), with respect to controls (6 +/- 2.6 pmol/10(6) cells). No differences were found in dopamine receptor mRNA expression. Our results demonstrate that second messenger levels are altered in the peripheral blood lymphocytes of PD patients treated with dopaminergic agents and that patients with LIDs show further alterations in the regulation of [Ca2+]i homeostasis. This may represent a distinctive trait of patients prone to develop dyskinetic movements.

Oxidative-Dependent Integration of Signal Transduction with Intercellular Gap Junctional Communication in the Control of Gene Expression

Research on oxidative stress focused primarily on determining how reactive oxygen species (ROS) damage cells by indiscriminate reactions with their macromolecular machinery, particularly lipids, proteins, and DNA. However, many chronic diseases are not always a consequence of tissue necrosis, DNA, or protein damage, but rather to altered gene expression. Gene expression is highly regulated by the coordination of cell signaling systems that maintain tissue homeostasis. Therefore, much research has shifted to the understanding of how ROS reversibly control gene expression through cell signaling mechanisms. However, most research has focused on redox regulation of signal transduction within a cell, but we introduce a more comprehensive-systems biology approach to understanding oxidative signaling that includes gap junctional intercellular communication, which plays a role in coordinating gene expression between cells of a tissue needed to maintain tissue homeostasis. We propose a hypothesis that gap junctions are critical in modulating the levels of second messengers, such as low molecular weight reactive oxygen, needed in the transduction of an external signal to the nucleus in the expression of genes. Thus, any comprehensive-systems biology approach to understanding oxidative signaling must also include gap junctions, in which aberrant gap junctions have been clearly implicated in many human diseases.

Expression of HLA-G in patients with B-cell chronic lymphocytic leukemia (B-CLL).

The expression of HLA-G was reported in certain malignancies and its role in escaping from immunosurveillance in cancers was proposed since HLA-G is a nonconventional HLA class I molecule that protects fetus from immunorecognition during pregnancy. Recent studies proposed HLA-G as novel prognostic marker for patients with B-CLL. HLA-G was showed to bear even better prognostic information compared to Zeta-chain associated protein of 70kDa (ZAP-70) and CD38 although some other authors did not find HLA-G expression in CLL. Therefore in this study we characterized the expression of HLA-G on both RNA and protein level. In most of 20 B-CLL patients we were able to detect signal from HLA-G using flow cytometry analysis. The expression of HLA-G was confirmed on messenger level by real-time RT-PCR experiments. No correlation between HLA-G expression and expression of well established prognostic factors such as ZAP-70 and CD38 was detected. These results confirm that HLA-G is expressed on CLL leukemic cells. Furthermore the expression of HLA-G on CLL cells suggests that this molecule might be involved in escaping of CLL cells from immunosurveillance.

Platelets from Sickle Cell Disease Individuals Demonstrate Increased Adhesive Properties That Are Reversed by Hydroxyurea Therapy in Association with Alterations in Intraplatelet cAMP and αIIbα3 Integrin Activation

Sickle cell disease (SCD) is a chronic inflammatory disease that results in hemolytic anemia and vaso-occlusive events, the principal cause of morbidity in SCD patients. Whilst roles for red cells and leukocytes in the vaso-occlusive process in SCD are well established, the contribution of platelets to this process remains unclear. Increased platelet activation has been described in SCD patients, contributing to the hemostatic activation observed in the disease. Since the adhesion of platelets to the vascular endothelium and extracellular matrix (ECM) could potentially contribute to vaso-occulusion, this study compared the adhesive properties of platelets (PLTs) from healthy control subjects (CON), steady-state SCD patients (SCD) and SCD patients on hydroxyurea therapy (SCDHU; 20–30 mg/kg/day). Washed PLTs were ressuspended in Krebs solution (1.2×108 PLT/ml) and their adhesion to fibrinogen (FB, 50 μg/ml) coated 96-well plates evaluated utilizing static adhesion assays (15 min, 37°C). SCD PLTs demonstrated a significantly greater adhesion to FB than CON PLTs (26.0±4.0%; 15.8±2.0%, respectively, n≥13; p<0.05, Mann-Whitney test). In contrast, SCDHU PLT adhesion was significantly lower than that of SCD PLTs, being similar to that of CON PLTs (11.7±1.5%, n=13; p<0.01 comp. SCD). A thrombin stimulus (50 mU/ml) significantly increased CON, SCD and SCDHU adhesion to FB (26.2±2.1%; 33.8±3.7%; 23.5±3.7%, respectively, n≥13; p<0.001 comp. to basal, paired t test). Levels of the second messenger, cyclic adenosine monophosphate (cAMP), the reduction of which potentiates platelet activation, were found to be significantly decreased in the PLTs of SCD individuals compared to those of CON and SCDHU (5.4±0.6; 8.0±0.7; 7.1±0.4 pmol/108 PLT, respectively, n=7; P<0.05 for SCD comp. to CON and SCDHU). Interestingly, thrombin significantly reduced cAMP levels in CON and SCDHU (6.0±0.9; 5.4±0.5 pmol/108 PLTs, respectively, n=7, P<0.05 comp. to basal), but not SCD PLTs (4.7±0.6 pmol/108 PLT, n=7; P>0.05), providing further evidence that PLTs circulate in an activated state in SCD individuals. Flow cytometry studies demonstrated no alterations in the expressions of CD42b (GPIb molecule), CD62P (P-selectin), CD49b (α2 integrin subunit) and CD41a (αIIb integrin subunit) on the surface of SCD PLTs compared to CON and SCDHU (P<0.05, data not shown). However, binding of the PAC-1 antibody, which specifically recognizes activated αIIbβ3 integrin, was significantly higher on SCD PLTs than on CON and SCDHU PLTs (40.7±8.9%; 17.1±6.5%; 20.3±5.4%, respect., n≥8, p<0.05). The αIIbβ3 integrin, in its activated affinity, can bind to numerous soluble ligands that in turn may bridge the interaction between PLTs, endothelial cells and other cell types. As such, PLTs from SCD individuals in steady state circulate in an activated state and present an increased ability to adhere to the blood component, FB, compared to control PLTs. Increased adhesive properties were found associated with decreased intraplatelet cAMP and high affinity αIIbβ3 integrin expression. Interestingly, HU therapy was associated with a reversal in increased PLT adhesion coupled with augmented intraplatelet cAMP and reduced αIIbβ3 activation. Thus, SCD platelets may have an increased capacity for adhesion to the vascular wall and ECM components, where the presence of adhered and activated platelets may contribute to the vaso-occlusive process by facilitating leukocyte accumulation at the endothelium and the release of platelet factors that may stimulate further inflammation at the site.

Oral phosphodiesterase-5 inhibitors and sperm functions

This review aims to elucidate the possible effects of phosphodiesterase-5 (PDE5) inhibitors on sperm functions. PDEs hydrolyze cyclic nucleotides, and together with adenylyl and guanylyl cyclase, which catalyze the formation of cAMP and cGMP, regulate the levels of these second messengers in cells. cGMP-specific PDE5 is one of the PDEs that have been intensively studied because of its fundamental pharmacological relevance, as oral PDE5 inhibitors are used successfully in treating erectile dysfunction. In addition, they have shown diverse beneficial actions in different disease categories. Specific relevance of the cGMP system in reproductive functions has been recently proposed. Its use was shown to be devoid of effects on semen volume, concentration, sperm membrane integrity or sperm penetration assay. Most available studies demonstrated a significant increase in sperm motility and viability both in vivo and in vitro, which seems to be enhanced at low doses and reduced at high concentrations. Also, these molecules showed a role in capacitation and a debated one concerning acrosome reaction. However, due to the relative short period since the launching of oral PDE5 inhibitors, more investigations should be carried out in wider scales to assess their effect(s) on variant sperm function that could be beneficial as potential therapeutic approaches.