PLD Protein-Protein Interactions With Signaling Molecules and Modulation by PA.

We describe methods for studying phospholipase D (PLD) interactions with signaling proteins and modulation of these interactions by the PLD reaction product, phosphatidic acid (PA). PLD is fundamental to the physiological maintenance of cellular/intracellular membranes, protein trafficking, cytoskeletal dynamics, membrane remodeling, cell proliferation, meiotic division and sporulation. PA is an acidic phospholipid involved in the biosynthesis of many other lipids that affects the enzymatic activities of many different signaling proteins via protein-lipid interactions or as a substrate. The involvement of PLD as an effector of protein-protein interactions and downstream signaling via PA-mediated processes has led to the investigation of PA-binding domains in target protein partners. We present here data and protocols detailing the interaction between PLD2-Rac2 interaction and modulation of this interaction by PA. We describe biochemical techniques to measure interactions between PLD, PA, and the small GTPase Rac2, which are associated in the cell. We found two maxima concentrations of PA that contributed to association or dissociation of Rac2 with PLD2, as well as the PLD2 lipase and guanine nucleotide exchange factor (GEF) activities. Fluctuations in the Rac2-PLD2 protein-protein binding interaction facilitate shuttling of Rac2 and/or PLD2 within the cell dependent on local cellular PA concentration. Fluorescence resonance emission transfer stoichiometry for PLD2 and Rac2 binding yielded a 3:1 ratio of Rac2:PLD2. Detection of PA in mammalian cells with a new biosensor showed colocalization in and around the nucleus. We also described methods for quantitation of PA in biological materials by HPLC electrospray ionization tandem mass spectrometry.

How should we address the diagnosis of overactive bladder in women?

OBJECTIVE: Determine the degree of completion, agreement and diagnostic performance of various instruments for assessing the presence and intensity of urgency and other symptoms of idiopathic overactive bladder (OAB) and determine which is the best diagnostic combination. MATERIAL AND METHODS: Observational, noninterventional, cross-sectional multicentre study on 247 women aged 18 years or older, with a clinical diagnosis of OAB, evaluated in 55 functional urology and urodynamic units. The women completed the Patient Perception of Intensity of Urgency Scale questionnaire, an independent bladder control self-assessment questionnaire (B-SAQ), the Overactive Bladder Questionnaire Short-Form and a 3-day voiding diary (VD3d), and they underwent a urodynamic study (UDS). The degree of completion and agreement among the instruments was assessed using the Kappa index (95% CI) and Cramér's V. The diagnostic performance of each tool and their combination was studied using absolute frequencies of positive cases for each OAB symptom. RESULTS: The patients mean age was 57.66 years (SD, 13.43). There was a high degree of completion (>85%). The agreement among the instruments was poor or moderate, and there was no agreement with the UDS. The best combination of tools for the diagnosis of OAB in women was the B-SAQ and VD3d. CONCLUSIONS: The degree of completion of all instruments was high, the agreement between them was poor-moderate and not significant for the UDS. The instruments that had the best diagnostic performance for assessing urgency and other OAB symptoms, providing data on their severity and discomfort, were the B-SAQ and the VD3d.

Síndrome de Kounis tras la administración de Cefditorén / Kounis syndrome after Cefditorén administration

El síndrome de Kounis fue definido, por primera vez, en 1991, como la aparición simultánea de episodios coronarios agudos y reacciones alérgicas anafilácticas o anafilactoides. Los agentes etiológicos asociados a su aparición son múltiples y los más frecuentes son los fármacos, en especial, los antibióticos. Su diagnóstico es eminentemente clínico, no existe ninguna prueba patognomónica. No hay consenso ni guías de práctica clínica específicas; se recomienda el tratamiento específico para el síndrome coronario agudo enfocado en tratar el vasoespasmo y la anafilaxia, con el agravante de que puede haber contraindicaciones cuando se usan conjuntamente y que dichos fármacos antianginosos pueden desencadenar el cuadro. Se presenta un caso clínico de síndrome de Kounis asociado a cefditorén, el primero descrito en la literatura(AU)

Kounis syndrome was first described in 1991 as the simultaneous occurrence of acute coronary events and allergic anaphylactic or anaphylactoid reactions. Multiple etiologic agents are associated with this syndrome, the most common are drugs, especially antibiotics. Diagnosis is eminently clinical, there are not pathognomonic tests. Consensus and specific clinical practice guidelines are lacking; specific acute coronary syndrome treatment is recommended focusing on vasospasm and anaphylaxis, with the aggravating circumstance that contraindications can be present when used together and such antianginal drugs may trigger the condition. We present a case of Kounis syndrome associated with cefditoren, the first reported in the literature.(AU)

Draft Genome Sequence of Xanthomonas arboricola pv. pruni Strain Xap33, Causal Agent of Bacterial Spot Disease on Almond.

We report the annotated genome sequence of Xanthomonas arboricola pv. pruni strain Xap33, isolated from almond leaves showing bacterial spot disease symptoms in Spain. The availability of this genome sequence will aid our understanding of the infection mechanism of this bacterium as well as its relationship to other species of the same genus.

Validación de la versión española del cuestionario «Beneficio, satisfacción y disposición para continuar el tratamiento» en pacientes con vejiga hiperactiva / Validation of the Spanish Version of the Questionnaire «Benefit, Satisfaction and Willingness to Continue the Treatment» in Patients with Overactive Bladder

Objetivos: Realizar la validación lingüística y psicométrica en español del cuestionario Beneficio, satisfacción y disposición para continuar el tratamiento (BSD). Material y métodos: Estudio epidemiológico, observacional, multicéntrico, prospectivo (octubre 2008-febrero 2009) en pacientes ≥ 40 años, con síndrome de vejiga hiperactiva idiopática de novo y que iniciaran tratamiento antimuscarínico bajo criterio clínico. Se recopilaron datos en la visita basal (presencial) y se realizó el seguimiento del estudio a través de encuestas telefónicas cerradas al primer y tercer mes desde el inicio. Se empleó el cuestionario de Morisky-Green para determinar el cumplimiento terapéutico y se completaron los Cuestionarios de Autoevaluación del Control de la Vejiga (CACV) y el BSD, realizándose la validación de este último. Resultados: Se reclutaron 312 pacientes evaluables; en la visita de 3 meses se dispone de información de 93. El 65% y el 71% de los pacientes eran incumplidores con el tratamiento al mes y a los 3 meses, respectivamente. La correlación entre la puntuación del cuestionario BSD y las puntuaciones del cuestionario CACV al mes y a los 3 meses de inicio del estudio fue moderada y estadísticamente significativa. La consistencia interna entre los ítems del cuestionario BSD fue alta (alfa de Cronbach: 0,89 al mes y 0,84 a los 3 meses). El 92% había entendido bien las preguntas y el 84% no tuvo ningún problema para cumplimentar el cuestionario BSD sin instrucciones previas (N = 25). Conclusiones: El cuestionario BSD demostró ser una herramienta factible, válida y fiable para conocer la propia valoración del paciente sobre el tratamiento recibido, de acuerdo a sus propiedades psicométricas (AU)

Objectives: To perform the linguistic and psychometric validation of the Spanish version of the BSW (Benefit, Satisfaction and Willingness to continue) questionnaire. Material and methods: Epidemiologic, observational, multicenter, prospective (October 2008-February 2009) study in patients ≥ 40 years old with de novo overactive bladder syndrome who start treatment with antimuscarinics by physicians assessment. Data was recorded at baseline (face-to-face) and the follow-up of the study after 1 and 3 months (closed surveys by phone). Morisky-Green questionnaire was used to assess compliance. Bladder Control Self-assessment Questionnaire (B-SAQ) and BSW questionnaire were completed, performing the validation of BSW. Results: 312 evaluable patients were recruited, 93 remained until the 3 months visit. 65% and 71% of patients were not compliant with treatment at 1 and 3 months, respectively. The correlation between the BSW and the B-SAQ questionnaires after 1 and 3 months was moderate and statistically significant. The internal consistency between the BSW questionnaire items was high (Cronbach alpha: 0,89 at 1 month and 0,84 at 3 months). 92% of patients understood the questions and 84% were able to fill the BSW questionnaire without need of previous instructions (N = 25). Conclusions: The BSW questionnaire has been shown to be a feasible, valid and reliable tool to know the patient self-assessment of the treatment, according to its psychometric properties (AU)

Perfil de síntomas del tracto urinario inferior en el varón y su impacto en la calidad de vida / Profile of Lower Urinary Tract Symptoms in the Male and Their Impact on Quality of Life

Objetivos: La etiología multifactorial de los síntomas del tracto urinario inferior (STUI) justifica su evaluación detallada para un adecuado abordaje terapéutico según las guías europeas actuales. Para conocer el perfil sintomático e impacto en la calidad de vida relacionada con la salud (CVRS) de pacientes varones que acuden a consulta de urología se desarrolló el siguiente estudio. Material y métodos: Estudio epidemiológico, transversal en 826 varones adultos con ≥ 1 STUI de novo no tratados previamente. Se recogieron variables sociodemográficas y clínicas. Los pacientes cumplimentaron la Puntuación internacional de síntomas prostáticos (IPSS), Cuestionario de autoevaluación del control vesical (CACV) y cuestionario SF-12. Resultados: La edad media (DE) fue de 65 (10) años. El 69% presentaba una combinación de síntomas de llenado, vaciado y posmiccionales. El 30% tenía ≥ 2 episodios de urgencia y ≥ 8 micciones al día (subpoblación con síntomas de llenado [SLL]). Los SLL fueron el motivo de consulta en el 86% de los casos. El flujo urinario máximo medio fue 11,4 ml/s y el 44% tenía volumen prostático entre 20-40 cc y el 91% síntomas moderados o graves (IPSS) con puntuación media (DE) de 17 (7). La subpoblación con SLL tenía puntuaciones mayores del CACV (síntomas 6,9 vs 4,8; molestias 7,8 vs 5,1). La subpoblación con SLL presentaba peor CVRS (IPSS ítem 8). Conclusiones: Estos hallazgos evidencian que es importante conocer el perfil de síntomas de cada paciente y el grado de molestia e impacto en la calidad de vida para orientar adecuadamente el tratamiento (AU)

Objectives: Following current European Guidelines, Lower Urinary Tract Symptoms (LUTS) are produced by several causes and, thus, thorough clinical assessment should be undertaken for a correct therapeutic management. This study was conducted in order to assess the symptoms profile and their impact on Health-Related Quality of Life (HRQL) of male patients attending urology outpatient clinics.Material and methodsEpidemiological, cross-sectional study including adult male patients (n = 826) presenting with at least one de novo previously untreated LUTS. Socio-demographic and clinical variables were collected. Patients completed IPSS, Bladder Control Self-Assessment Questionnaire (B-SAQ) and SF-12 questionnaires.Results: Mean age (SD) was 65 (10) years. A combination of storage, voiding and post-micturition symptoms were present in 69% of subjects and 30% showed ≥ 2 urgency episodes and ≥6 micturitions daily (storage symptoms –SS— sub-population). Storage symptoms were the reason for consultation in 86%. Mean peak urinary flow (Qmax) was 11.4 mL/s, in 44% of patients,prostate volume was 20-40 mL and 91% showed moderate or severe symptoms on IPSS with an overall mean (SD) score of 17 (7). SS sub-population had higher B-SAQ scores (6,9 vs 4,8 for symptoms; 7,8 vs 5,1 for bother), and worse HRQL (IPSS item 8).Conclusions: These findings support the importance of addressing treatment adequately according to patient profile, bothersomeness and impact on HRQL (AU)

Validation of the Spanish version of the questionnaire «Benefit, satisfaction and willingness to continue the treatment¼ in patients with overactive bladder.

OBJECTIVES: To perform the linguistic and psychometric validation of the Spanish version of the BSW (Benefit, Satisfaction and Willingness to continue) questionnaire. MATERIAL AND METHODS: Epidemiologic, observational, multicenter, prospective (October 2008-February 2009) study in patients ≥40 years old with de novo overactive bladder syndrome who start treatment with antimuscarinics by physicians assessment. Data was recorded at baseline (face-to-face) and the follow-up of the study after 1 and 3 months (closed surveys by phone). Morisky-Green questionnaire was used to assess compliance. Bladder Control Self-assessment Questionnaire (B-SAQ) and BSW questionnaire were completed, performing the validation of BSW. RESULTS: 312 evaluable patients were recruited, 93 remained until the 3 months visit. 65% and 71% of patients were not compliant with treatment at 1 and 3 months, respectively. The correlation between the BSW and the B-SAQ questionnaires after 1 and 3 months was moderate and statistically significant. The internal consistency between the BSW questionnaire items was high (Cronbach alpha: 0,89 at 1 month and 0,84 at 3 months). 92% of patients understood the questions and 84% were able to fill the BSW questionnaire without need of previous instructions (N=25). CONCLUSIONS: The BSW questionnaire has been shown to be a feasible, valid and reliable tool to know the patient self-assessment of the treatment, according to its psychometric properties.

Phospholipase D (PLD) drives cell invasion, tumor growth and metastasis in a human breast cancer xenograph model.

Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based short hairpin RNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, P<0.05) and their onset delayed when compared with control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (5-fluoro-2-indolyl des-chlorohalopemide and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide). These inhibitors led to significant (>70%, P<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid, Wiscott-Aldrich Syndrome protein, growth receptor-bound protein 2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows for the first time that PLD2 has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target.

Mirabegron for the treatment of overactive bladder: a prespecified pooled efficacy analysis and pooled safety analysis of three randomised, double-blind, placebo-controlled, phase III studies.

INTRODUCTION: To examine pooled efficacy data from three, large phase III studies comparing mirabegron (50 and 100 mg) with placebo, and pooled safety data including additional mirabegron 25 mg and tolterodine extended release (ER) 4 mg results. METHODS: This prespecified pooled analysis of three randomised, double-blind, placebo-controlled, 12-week studies, evaluated efficacy and safety of once-daily mirabegron 25 mg (safety analysis), 50 or 100 mg (efficacy and safety analyses) and tolterodine ER 4 mg (safety analysis) for the treatment of symptoms of overactive bladder (OAB). Co-primary efficacy measures were change from baseline to Final Visit in the mean number of incontinence episodes/24 h and mean number of micturitions/24 h. Key secondary efficacy end-points included mean number of urgency episodes/24 h and mean volume voided/micturitions, while other end-points included patient-reported outcomes according to the Treatment Satisfaction-Visual Analogue Scale (TS-VAS) and responder analyses [dry rate (posttreatment), ≥ 50% reduction in incontinence episodes/24 h, ≤ 8 micturitions/24 h (post hoc analysis)]. The safety analysis included adverse event (AE) reporting, laboratory assessments, ECG, postvoid residual volume and vital signs (blood pressure, pulse rate). RESULTS: Mirabegron (50 and 100 mg once daily) demonstrated statistically significant improvements compared with placebo for the co-primary end-points, key secondary efficacy variables, TS-VAS and responder analyses (all comparisons p < 0.05). Mirabegron is well tolerated and demonstrates a good safety profile. The most common AEs (≥ 3%) included hypertension, nasopharyngitis and urinary tract infection (UTI); the incidence of hypertensive events and UTIs decreased with increasing dose. For mirabegron, the incidence of the bothersome antimuscarinic AE, dry mouth, was at placebo level and of a lesser magnitude than tolterodine. CONCLUSION: The efficacy and safety of mirabegron are demonstrated in this large pooled clinical trial dataset in patients with OAB.

Profile of lower urinary tract symptoms in the male and their impact on quality of life.

OBJECTIVES: Following current European Guidelines, Lower Urinary Tract Symptoms (LUTS) are produced by several causes and, thus, thorough clinical assessment should be undertaken for a correct therapeutic management. This study was conducted in order to assess the symptoms profile and their impact on Health-Related Quality of Life (HRQL) of male patients attending urology outpatient clinics. MATERIAL AND METHODS: Epidemiological, cross-sectional study including adult male patients (n=826) presenting with at least one de novo previously untreated LUTS. Socio-demographic and clinical variables were collected. Patients completed IPSS, Bladder Control Self-Assessment Questionnaire (B-SAQ) and SF-12 questionnaires. RESULTS: Mean age (SD) was 65 (10) years. A combination of storage, voiding and post-micturition symptoms were present in 69% of subjects and 30% showed >2 urgency episodes and >6 micturitions daily (storage symptoms -SS- sub-population). Storage symptoms were the reason for consultation in 86%. Mean peak urinary flow (Q(max)) was 11.4 mL/s, in 44% of patients,prostate volume was 20-40 mL and 91% showed moderate or severe symptoms on IPSS with an overall mean (SD) score of 17 (7). SS sub-population had higher B-SAQ scores (6,9 vs 4,8 for symptoms; 7,8 vs 5,1 for bother), and worse HRQL (IPSS item 8). CONCLUSIONS: These findings support the importance of addressing treatment adequately according to patient profile, bothersomeness and impact on HRQL.

The elucidation of novel SH2 binding sites on PLD2.

Our laboratory has recently reported that the enzyme phospholipase D2 (PLD2) exists as a ternary complex with PTP1b and the growth factor receptor bound protein 2 (Grb2). Here, we establish the mechanistic underpinnings of the PLD2/Grb2 association. We have identified residues Y(169) and Y(179) in the PLD2 protein as being essential for the Grb2 interaction. We present evidence indicating that Y(169) and Y(179) are located within two consensus sites in PLD2 that mediate an SH2 interaction with Grb2. This was demonstrated with an SH2-deficient GSTGrb2 R86K mutant that failed to pull-down PLD2 in vitro. In order to elucidate the functions of the two neighboring tyrosines, we created a new class of deletion and point mutants in PLD2. Phenylalanine replacement of Y(169) (PLD2 Y169F) or Y(179) (PLD2 Y179F) reduced Grb2 binding while simultaneous mutation completely abolished it. The role of the two binding sites on PLD2 was found to be functionally nonequivalent: Y(169) serves to modulate the activity of the enzyme, whereas Y(179) regulates total tyrosine phosphorylation of the protein. Interestingly, binding of Grb2 to PLD2 occurs irrespectively of lipase activity, since Grb2 binds to catalytically inactive PLD2 mutants. Finally, PLD2 residues Y(169) and Y(179) are necessary for the recruitment of Sos, but only overexpression of the PLD2 Y179F mutant resulted in increased Ras activity, p44/42(Erk) phosphorylation and enhanced DNA synthesis. Since Y(169) remains able to modulate enzyme activity and is capable of binding to Grb2 in the PLD2 Y179F mutant, we propose that Y(169) is kept under negative regulation by Y(179). When this is released, Y(169) mediates cellular proliferation through the Ras/MAPK pathway.

Modulation of MEK activity during G-CSF signaling alters proliferative versus differentiative balancing.

Previous studies of the granulocyte colony stimulating factor (G-CSF) receptor have demonstrated that discrete signals direct proliferative and maturation signaling. Receptor deletion/mutant studies have shown that although activation of the ras-mitogen activated protein (MAP) kinase pathway is necessary for G-CSF directed proliferation, it is not necessary for maturation induced by this cytokine. We have assessed the effects of selective inhibition or overexpression of MAP kinase kinase (MEK) in a cell line model of G-CSF-induced neutrophil progenitor growth. Using the human G-CSF responsive MPD cell line, we specifically inhibited MEK using PD 98059 and also transfected MPD cells with a constitutively active MEK construct. We then exposed the cells to G-CSF and assessed the effects of MEK inhibition and forced expression on proliferation and differentiation. Inhibition of MEK followed by G-CSF stimulation consistently resulted in an early 2.5-fold increase in morphologically differentiated neutrophils expressing CD11b and CD16 and containing lactoferrin over that produced by G-CSF alone. MEK inhibition alone had little effect on the differentiation stage of these cells, although proliferation was impaired. Forced expression of activated MEK resulted in a three- to five-fold decrease in differentiated, lactoferrin containing neutrophilic cells resultant from G-CSF induction, and a commensurate increase in cell proliferation. These observations suggest that modulation of MAPK activation may be a control point for altering the balance between proliferation and differentiation in response to G-CSF. Physiologically, this control is likely exerted by costimulatory cytokines.

A systematic approach to the complete study of a signaling molecule: ribosomal p90rsk as an example.

Ribosomal p90rsk is a kinase of central importance in transducing mitogenic signals from an activated receptor to the cell nucleus and for protein synthesis. Here, we analyze the optimal steps to fully describe this kinase in both normal neutrophils and leukemic cell lines. These are: (i) immunological analyses (immunoblotting and immunoprecipitation); (ii) enzyme activity assays (in vitro and "in-gel"); and (iii) immunobiochemical combination methods (immunoprecipitation/kinase assay, immunoprecipitation/"in-gel" assay and ion exchange chromatography/immunoblotting). For the enzyme assays, we describe a novel method to measure ribosomal p90rsk kinase activity "in-gel", based on a renatured-protein method that allows for the direct quantitation of enzyme activity. Finally, we present an algorithm that can be readily implemented to the quantification of the extent of stimulation of a kinase in response to a particular extracellular stimuli. In our case, it was found that activation of p90rsk was higher in proliferating leukemic cells than in mature neutrophils, indicating that a suppression of key signal transduction links could contribute to the maturational arrest typical of acute leukemia. All the techniques and strategies described here for p90rsk could be easily extrapolated to the study of any signal transduction molecule, provided it has a phosphotransferase activity.

Presence of a phospholipase D (PLD) distinct from PLD1 or PLD2 in human neutrophils: immunobiochemical characterization and initial purification.

Utilizing the transphosphatidylation reaction catalyzed by phospholipase D (PLD) in the presence of a primary alcohol and the short-chain phospholipid PC8, we have characterized the enzyme from human neutrophils. A pH optimum of 7.8-8.0 was determined. PIP(2), EDTA/EGTA, and ATP were found to enhance basal PLD activity in vitro. Inhibitory elements were: oleate, Triton X-100, n-octyl-beta-glucopyranoside, divalent cations, GTPgammaS and H(2)O(2). The apparent K(m) for the butanol substrate was 0.1 mM and the V(max) was 6.0 nmol mg(-1) h(-1). Immunochemical analysis by anti-pan PLD antibodies revealed a neutrophil PLD of approximately 90 kDa and other bands recognized minimally by anti-PLD1 or anti-PLD2 antibodies. The 90-kDa protein is tyrosine-phosphorylated upon cell stimulation with GM-CSF and formyl-Met-Leu-Phe. Protein partial purification using column liquid chromatography was performed after cell subfractionation. Based on the enzyme's regulatory and inhibitory factors, and its molecular weight, these data indicate an enzyme isoform that might be different from the mammalian PLD1/2 forms described earlier. The present results lay the foundation for further purification of this granulocyte PLD isoform.

Phospholipase D (PLD) is present in Leishmania donovani and its activity increases in response to acute osmotic stress.

We report here that the signaling molecule phospholipase D (PLD) is present in the parasitic protozoan Leishmania donovani. In vitro enzymatic activity is dependent on Ca2+ and Mg2+ ions, its basal activity is stimulated by phosphatidyl-inositol-4,5-bisphosphate (PIP2) and its pH optima are pH 8.0 and pH 6.0. PLD activity increases 3-fold about 5 min after an abrupt decrease in osmolality from 317 mOsm (isosmotic) to 155 mOsm and increases 1.5-fold in response to an abrupt increase in osmolality to 617 mOsM. Cells grown for > 24 h under the anisosmotic conditions showed only marginal changes in activity compared to the controls grown under isosmotic conditions, indicating an adaptation to long-term exposure to hypo- or hyper-osmolarity. Immunologically, two isoforms, PLD1 and PLD2, are present. An analysis of in vitro PLD activity in anti-PLD immunocomplexes revealed that either hypotonic (cell swelling) or hypertonic stress (cell shrinking) causes an increase in PLD1 activation but a reduction in PLD2 activity. The interplay between these two isoforms results in a predominance for PLD1 in the observed increase when measuring total PLD activity. Finally, the increase in enzymatic activity in acute hyposmotic shock is accompanied by tyrosyl phosphorylation of the PLD1 isoform, suggesting a role for protein tyrosine kinase in the control of PLD activity in response to osmotic stress.

MAP kinase upregulation after hematopoietic differentiation: role of chemotaxis.

Mitogen-activated protein kinase (MAPK) isoform p42 is known to be active in exponentially growing cells at several points of the cell cycle. A high basal activity was present in three cell lines representative of immature myeloid cells tested: uHL-60, AML-14, and MPD. However, DMSO-induced differentiation of HL-60 cells (dHL-60) and subsequent expression of the neutrophilic phenotype occurred with a concomitant reduction on the basal level of MAPK activity. Simultaneously, extracellular stimuli like the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) induced a fast (<10 min) and robust response. In terms of MAPK activity, the more mature the cell was, the higher the corresponding activity, in the three differentiation series considered: AML-14 < 3D10; MPD < G-MPD; uHL-60 < dHL-60 < neutrophils. Interestingly, peripheral blood neutrophils expressed the highest (16-fold) MAPK activation level in response to GM-CSF. Finally, using the specific MAPK inhibitor PD-98059, we demonstrated that MAPK activation is needed for neutrophil chemotaxis toward interleukin-8 and its priming by GM-CSF. Since neutrophils are terminally differentiated cells, GM-CSF does not serve a purpose in proliferation, and it must trigger the recruitment of selective signal transduction pathways particular to that final stage that includes enhanced physiological functions such as chemotaxis.

Complicaciones médicas por el consumo de drogas de uso lúdico / Medical complications derived from drug consume

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Direct inhibition of in vitro PLD activity by 4-(2-aminoethyl)-benzenesulfonyl fluoride.

While conducting a purification protocol of phospholipase D (PLD) from human granulocytes, we observed that PLD activity was inhibited by a commonly-used protease inhibitor cocktail. Of the six inhibitors present in the cocktail, the serine protease inhibitor, 4-(2-aminoethyl)-benezensulfonyl fluoride (AEBSF), was found to be the sole inhibitor of PLD. AEBSF caused a loss of neutrophil and purified plant PLD activities in vitro, but not in intact cells at the concentrations used, nor did it affect the related phospholipases A(2) and C, that were utilized as specificity controls. The compound AEBSNH(2), which has the fluoride replaced by an -NH(2) group, failed to affect PLD activity as did other compounds structurally related to AEBSF with known protease inhibitory capabilities. Finally, basal- and agonist-stimulated PLD activity was inhibited in phosphatidylcholine-specific anti-PLD immunoprecipitates (IC(50) = 75 microM). These results suggest that AEBSF, in an effect probably unrelated to its anti-proteolytic ability, directly interferes with PLD enzymatic activity, making it a significant compound to begin analyzing the role of PLD in mammalian cell signaling.