Increased expression of microRNA-155 in peripheral blood mononuclear cells from psoriasis patients is related to disease activity.

BACKGROUND: MicroRNAs (miRNAs) gene expression regulators are altered in psoriasis suggesting their role in the pathogenesis. OBJECTIVE: To study expression changes of inflammation and toll-like receptor (TLR)-related miRNAs, miRNA-155, let-7i, miRNA-21, miRNA-146a and miRNA-223 in peripheral mononuclear cells (PBMCs) and miRNA-21, miRNA-146a and miRNA-223 in plasma, from chronic plaque-type psoriasis patients who were treatment-naive or had undergone a washout period (n = 11). MiRNAs were evaluated at baseline and after 11 (9-12) months [median (25th-75th percentile range)] of methotrexate (MTX) or topical (betamethasone plus calcipotriene) treatment. METHODS: MiRNA expression was analysed with quantitative real-time reverse transcription-polymerase chain reaction. Matched controls were studied. RESULTS: Psoriasis patients presented, at baseline, increased expression of miRNA-155, let-7i, miRNA-146a, miRNA-21 and miRNA-223 in PBMCs, plus miRNA-21, miRNA-146a and miRNA-223 in plasma. Receiver-operator characteristic (ROC) curve analysis and area under the curve (AUC) showed that expression of these miRNAs have the potential to distinguish between psoriasis and controls. At baseline, miRNA-155 expression in PBMCs correlated with Psoriasis Area Severity Index (PASI) [12 (8-14)] (Spearman r: 0.7140, P < 0.05) suggesting a role in psoriasis. After MTX or topical treatment, reduction in PASI was observed [87.5% (75-100)]; miRNA-155 expression in PBMCs decreased; plasma miRNA-21, miRNA-146a and miRNA-223 were down-regulated. ROC analysis showed that miRNA-155 expression in PBMCs from psoriasis patients have the potential to distinguish between patients' samples at baseline and after treatment (AUC: 0.942, sensitivity: 0.91; specificity: 0.91 values; maximum likelihood ratio =10). After treatment, miRNA-146a expression in PBMCs increased; miRNA-155/miRNA-146a ratio decreased, suggestive of a regulatory feedback; let-7i expression decreased; miRNA-21 and miRNA-223 remained elevated. CONCLUSION: In this exploratory study, psoriasis patients presented increased expression of miRNA-155 in PBMCs that correlated with PASI and decreased with disease remission. MiRNA-21, miRNA-146a and miRNA-223 in PBMCs and plasma were increased at baseline and differentially modulated, underscoring different roles of TLR-related miRNAs in psoriasis.

Supporting data for the MS identification of distinct transferrin glycopeptide glycoforms and citrullinated peptides associated with inflammation or autoimmunity.

This data article presents the results of all the statistical analyses applied to the relative intensities of the detected 2D-DiGE protein spots for each of the 3 performed DiGE experiments. The data reveals specific subsets of protein spots with significant differences between WT and CD38-deficient mice with either Collagen-induced arthritis (CIA), or with chronic inflammation induced by CFA, or under steady-state conditions. This article also shows the MS data analyses that allowed the identification of the protein species which serve to discriminate the different experimental groups used in this study. Moreover, the article presents MS data on the citrullinated peptides linked to specific protein species that were generated in CIA(+) or CFA-treated mice. Lastly, this data article provides MS data on the efficiency of the analyses of the transferrin (Tf) glycopeptide glycosylation pattern in spleen and serum from CIA(+) mice and normal controls. The data supplied in this work is related to the research article entitled "identification of multiple transferrin species in spleen and serum from mice with collagen-induced arthritis which may reflect changes in transferrin glycosylation associated with disease activity: the role of CD38" [1]. All mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifiers PRIDE: PXD002644, PRIDE: PXD002643, PRIDE: PXD003183 and PRIDE: PXD003163.

Identification of multiple transferrin species in the spleen and serum from mice with collagen-induced arthritis which may reflect changes in transferrin glycosylation associated with disease activity: The role of CD38.

UNLABELLED: Collagen type II-induced arthritis (CIA) is an inflammatory and autoimmune disease. Spleen protein extracts were subjected to 2D-DiGE and MS-MALDI-TOF/TOF analysis to identify protein species that differ in abundance in CD38-KO versus B6 WT mice either with arthritis or with inflammation. Using multivariate analyses, in Col-II-immunized mice, 23 distinct spleen protein species were able to discriminate between WT and CD38-KO mice. Among them, several citrullinated proteins and multiple serotransferrin (Tf) species were identified. In contrast, in CFA/IFA-treated mice, the distinct protein profile, which discriminates between CD38-KO and WT mice, was unrelated with Tf, but not with citrullination. Unexpectedly, non-immunized CD38-KO mice showed a distinct proteome profile as compared with that in non-immunized WT mice, and again multiple protein species were identified as Tf. By using a µLC-TOF-MS method to separate and detect Tf glycopeptide glycoforms, increases in fucosylation and glycan branching was observed in sera from mice CIA(+) versus non-immunized, and between WT and CD38-KO with arthritis. Data on 2-DE Tf spots indicated differences in glycosylation related with NeuGc content. Thus, Tf changed significantly in its glycosylation pattern in arthritic mice. The MS data have been deposited to the ProteomeXchange Consortium with the dataset identifiers: PXD002644, PXD002643, PXD003183, and PXD003163. SIGNIFICANCE: 2-DE followed by µLC-TOF-MS could be implemented to identify Tf glycoforms linked to specific protein species, and correlate a particular Tf species to a function. To gain insight into the relationship between transferrin glycoforms and its biological function it is particularly interesting to study putative differences in the glycosylation pattern of Tf in specific tissues associated with the disease (i.e.: joints), or in specific compartments such as exosomes/microvesicles, which are highly enriched in Tf receptors.

Análisis del desarrollo motor en escolares brasileños con medidas corporales de obesidad y sobrepeso / Analysis of the motor development in brazilian schoolchildren with corporal measures of obesity and overweight

El objetivo de este estudio fue analizar el perfil de las características motoras de niños con sobrepeso/obesidad. Se midieron las características antropométricas y las características motoras de 284 niños sanos de 6 a 10 años, dividiéndose en tres grupos: normales, con sobrepeso y con obesidad. El instrumento utilizado es la Escala de Desarrollo Motor EDM, desarrollado por Rosa Neto en 1996. Los niños obesos mostraron una deficiencia significativa (p<0,05) en todas las estructuras motoras y en relación con su edad cronológica. Los niños con sobrepeso mostraron un retraso en el equilibrio (p<0,01), esquema corporal (p<0,02), organización espacial (p≤0,01) y organización temporal (p≤0,01). Se puede concluir que los niños con sobrepeso y obesidad tienen un retraso con respecto a sus compañeros de peso normal (AU)

The aim of this study was to analyse the profile of the motor features of children with overweight/obesity. The anthropometric and motor characteristics of 284 healthy children 6 to 10 years were measured, divided in three groups: with normal weight, with overweight and with obesity. The used instrument is the Scale of Motor Development EDM, developed by Rosa Neto in 1996. Obese children showed a significant deficiency (p<0.5) in all structures in relation to motor and chronological age. Overweight children showed a delay in equilibrium (p<0.01), body image (p<0.02), spatial organization (p<0.01) and temporal organization (p<0.01). We conclude that children with overweight and obesity are lagging behind their peers of normal weight (AU)

Alteraciones en los niveles de expresión del microARN-33 en plasma de pacientes con psoriasis / Abnormal Levels of Expression of Plasma MicroRNA-33 in Patients With Psoriasis

INTRODUCCIÓN Y OBJETIVOS: Se han identificado microARN (miARN) circulantes implicados en la regulación postranscripcional de genes del metabolismo de lípidos (miARN-33) y de la función vascular y angiogénesis (miARN-126). El objetivo de este estudio exploratorio ha sido evaluar los niveles plasmáticos de miARN-33 y miARN-126 en pacientes con psoriasis en placas y su relación con parámetros clínicos. MATERIAL Y MÉTODOS: Se estudiaron once pacientes con psoriasis en placas (PASI [mediana] [25 - 75% percentil] 13 [9-14] y BSA 12 [11-15]) y un grupo pareado en edad y sexo de 11 controles sanos. Se analizaron factores de riesgo cardiovascular y la ateromatosis carotídea subclínica. Los miARN plasmáticos se evaluaron mediante la reacción en cadena de la polimerasa cuantitativa a tiempo real (qRT-PCR). RESULTADOS: La media del grosor de la íntima carotídea (GIM) estaba aumentada en pacientes (0,57 mm [0,54-0,61], n = 11) respecto a controles (0,50 mm [0,48-0,54], datos disponibles n = 9) (test Mann-Whitney, p = 0,0055). La expresión de miARN-33 en pacientes (5,34 [3,12-7,96], n = 11) estaba significativamente aumentada respecto a controles (2,33 [1,71-2,84], n = 7; solo se pudo detectar en 7 de 11) (test de Wilcoxon signed Rank, p = 0,0049). No se observaron diferencias en los niveles de miARN-126 entre pacientes y controles. En pacientes se observó una correlación positiva entre miARN-33 e insulina (r = 0,7289, p = 0,0109, n = 11); y una correlación negativa entre miARN-126 y GIM (r = -0,6181, p = 0,0426, n = 11). CONCLUSIÓN: Los pacientes con psoriasis presentaban niveles plasmáticos aumentados de miARN-33 (metabolismo de lípidos y glucosa), que se correlacionaban con los niveles de insulina. La valoración de miARN-33 circulante puede contribuir al conocimiento de las alteraciones inflamatorias sistémicas en psoriasis

INTRODUCTION AND OBJECTIVES: Circulating microRNAs (miRNA) are involved in the posttranscriptional regulation of genes associated with lipid metabolism (miRNA-33) and vascular function and angiogenesis (miRNA-126). The objective of this exploratory study was to measure plasma levels of miRNA-33 and miRNA-126 in patients with plaque psoriasis and evaluate their association with clinical parameters. MATERIAL AND METHODS: We studied 11 patients with plaque psoriasis. The median Psoriasis Area Severity Index (PASI) was 13 (interquartile range [IQR], 9-14) and body surface area involvement was 12 (IQR, 11-15). Eleven healthy controls matched for age and sex were also included. We analyzed cardiovascular risk factors and subclinical carotid atheromatosis. Plasma miRNAs were evaluated using quantitative real-time polymerase chain reaction. RESULTS: Carotid intima-media thickness was greater in patients (0.57 mm; IQR, 0.54-0.61; n = 11) than in controls (0.50 mm; IQR, 0.48-0.54; data available for 9 controls) (P = 0.0055, Mann-Whitney). Expression of miRNA-33 in patients (5.34; IQR, 3.12-7.96; n = 11) was significantly higher than in controls (2.33; IRQ, 1.71-2.84; only detected in 7 of 11 controls) (P = 0.0049, Wilcoxon signed rank). No differences in miRNA-126 levels were observed between patients and controls. In patients (n = 11), we observed a positive correlation between miRNA-33 and insulin levels (r = 0.7289, P = 0.0109) and a negative correlation between miRNA-126 and carotid intima-media thickness (r = -0.6181, P = 0.0426). CONCLUSION: In psoriasis patients plasma levels of lipid and glucose metabolism-related miRNA-33 are increased and correlated with insulin. The study of circulating miRNA-33 in psoriasis may provide new insights about the associated systemic inflammatory abnormalities

Abnormal levels of expression of plasma microRNA-33 in patients with psoriasis.

INTRODUCTION AND OBJECTIVES: Circulating microRNAs (miRNA) are involved in the posttranscriptional regulation of genes associated with lipid metabolism (miRNA-33) and vascular function and angiogenesis (miRNA-126). The objective of this exploratory study was to measure plasma levels of miRNA-33 and miRNA-126 in patients with plaque psoriasis and evaluate their association with clinical parameters. MATERIAL AND METHODS: We studied 11 patients with plaque psoriasis. The median Psoriasis Area Severity Index (PASI) was 13 (interquartile range [IQR], 9-14) and body surface area involvement was 12 (IQR, 11-15). Eleven healthy controls matched for age and sex were also included. We analyzed cardiovascular risk factors and subclinical carotid atheromatosis. Plasma miRNAs were evaluated using quantitative real-time polymerase chain reaction. RESULTS: Carotid intima-media thickness was greater in patients (0.57mm; IQR, 0.54-0.61; n=11) than in controls (0.50mm; IQR, 0.48-0.54; data available for 9 controls) (P=.0055, Mann-Whitney). Expression of miRNA-33 in patients (5.34; IQR, 3.12-7.96; n=11) was significantly higher than in controls (2.33; IQR, 1.71-2.84; only detected in 7 of 11 controls) (P=.0049, Wilcoxon signed rank). No differences in miRNA-126 levels were observed between patients and controls. In patients (n=11), we observed a positive correlation between miRNA-33 and insulin levels (r=0.7289, P=.0109) and a negative correlation between miRNA-126 and carotid intima-media thickness (r=-0.6181, P=.0426). CONCLUSION: In psoriasis patients plasma levels of lipid and glucose metabolism-related miRNA-33 are increased and correlated with insulin. The study of circulating miRNA-33 in psoriasis may provide new insights about the associated systemic inflammatory abnormalities.

Disminución de los niveles plasmáticos de clusterina en pacientes con psoriasis / Decreased Plasma Levels of Clusterin in Patients With Psoriasis

Introducción y objetivos: La psoriasis es una enfermedad inflamatoria crónica que se ha asociado a un incremento del riesgo cardiovascular. La clusterina (apolipoproteína J) es un componente de las lipoproteínas de alta densidad (HDL) y tiene un papel protector de la ateroesclerosis. El objetivo del estudio ha sido evaluar la clusterina y el factor inhibitorio de la migración del macrófago (MIF) plasmáticos en pacientes con psoriasis grave comparando grupos de pacientes con distintos riesgos cardiovasculares asociados. Material y métodos: Se estudiaron 21 pacientes con psoriasis grave (Psoriasis Area Severity Index [PASI] y Body Surface Area [BSA] > 10) y 11 controles sin enfermedad dermatológica. Se evaluaron los factores de riesgo cardiovascular según criterios del síndrome metabólico del Adult Treatment Panel III (ATP- III ) y la ateromatosis carotídea subclínica mediante ecografía doppler de carótidas. La clusterina y MIF plasmáticos se midieron mediante Enzyme-Linked Immuno Sorbent Assay (ELISA). Resultados: El 47% de los pacientes con psoriasis presentaba criterios de síndrome metabólico y el 33% presentó placa de ateroma carotídea. Se observó una disminución significativa de la clusterina plasmática (μg/ml) en pacientes con psoriasis respecto a controles (81,39 ± 27,30; n = 21, versus 117 ± 21,6, n = 11; p = 0,0017). El MIF plasmático (ng/ml) estaba aumentado significativamente en los pacientes con psoriasis y placa de ateroma carotídea respecto a los controles (53,22 ± 29,02; n = 6, versus 34,21 ± 9,65; n = 11; p = 0,0394). Conclusiones: La disminución de clusterina en pacientes con psoriasis sugiere una relación con la enfermedad y con la situación inflamatoria sistémica. El aumento de MIF en pacientes parece relacionarse con la presencia de factores de riesgo cardiovascular asociados y placa de ateroma carotídea (AU)

Introduction and objectives: Psoriasis is a chronic inflammatory disease that has been linked to increased cardiovascular risk. The glycoprotein clusterin (apolipoprotein J) is a component of high-density lipoproteins and has a protective role in atherosclerosis. The aim of the present study was to evaluate the plasma levels of clusterin and the proinflammatory cytokine macrophage migration inhibitory factor (MIF) in patients with severe psoriasis, comparing groups of patients with different risks of cardiovascular disease. Material and methods: Twenty-one patients with severe psoriasis (psoriasis area severity index and body surface area > 10) and 11 healthy controls with no dermatologic disease were studied. Cardiovascular risk factors were assessed according to the Adult Treatment Panel (ATP) IIIcriteria. Subclinical carotid atheromatosis was assessed by Doppler ultrasonography of the carotid arteries. Plasma clusterin and MIF levels were measured by enzyme-linked immunosorbent assay. Results: ATP-III criteria for metabolic syndrome were met by 47% of the patients, and 33% had carotid atheromatous plaque. Mean (SD) clusterin plasma levels were significantly lower in patients with psoriasis compared with controls (81.39 [27.30] micreg/mL for the 21 patients vs 117[21.6] g/mL for the 11 controls; P = .0017). MIF plasma levels (ng/ml) were significantly higherin patients with atheromatous plaque compared with controls (53.22 [29.02] for the 6 patientswith plaque vs 34.21 [9.65] for the 11 controls; P = 0.0394).Conclusions: The decreased plasma levels of clusterin in psoriatic patients suggested an association with the disease and might be an indicator of systemic inflammatory activity. Increased levels of MIF appear to be associated with cardiovascular risk factors and carotid atheromatous plaque (AU)

Decreased plasma levels of clusterin in patients with psoriasis.

INTRODUCTION AND OBJECTIVES: Psoriasis is a chronic inflammatory disease that has been linked to increased cardiovascular risk. The glycoprotein clusterin (apolipoprotein J) is a component of high-density lipoproteins and has a protective role in atherosclerosis. The aim of the present study was to evaluate the plasma levels of clusterin and the proinflammatory cytokine macrophage migration inhibitory factor (MIF) in patients with severe psoriasis, comparing groups of patients with different risks of cardiovascular disease. MATERIAL AND METHODS: Twenty-one patients with severe psoriasis (psoriasis area severity index and body surface area>10) and 11 healthy controls with no dermatologic disease were studied. Cardiovascular risk factors were assessed according to the Adult Treatment Panel (ATP) III criteria. Subclinical carotid atheromatosis was assessed by Doppler ultrasonography of the carotid arteries. Plasma clusterin and MIF levels were measured by enzyme-linked immunosorbent assay. RESULTS: ATP-III criteria for metabolic syndrome were met by 47% of the patients, and 33% had carotid atheromatous plaque. Mean (SD) clusterin plasma levels were significantly lower in patients with psoriasis compared with controls (81.39 [27.30] µg/mL for the 21 patients vs 117 [21.6] µg/mL for the 11 controls; P=.0017). MIF plasma levels (ng/ml) were significantly higher in patients with atheromatous plaque compared with controls (53.22 [29.02] for the 6 patients with plaque vs 34.21 [9.65] for the 11 controls; P=.0394). CONCLUSIONS: The decreased plasma levels of clusterin in psoriatic patients suggested an association with the disease and might be an indicator of systemic inflammatory activity. Increased levels of MIF appear to be associated with cardiovascular risk factors and carotid atheromatous plaque.

Señalización mediada por el receptor para el antígeno de los linfocitos T (TCR): CD3-epsilon es algo más que un amplificador de señales / Insights into T cell antigen receptor (TCR) signaling: CD3-epsilon is more than a signal amplifier

Para que se produzca el reclutamiento de tirosincinasas y otras moléculas adaptadoras o efectoras al receptor para el antígeno de los linfocitos T (complejo TCR/CD3) se necesita la fosforilación de las tirosinas de un motivo de activación presente en todas las subunidades CD3 del complejo. Este motivo es conocido por su acrónimo ITA M (del inglés Immuno receptor Ty rosine-based Activation Motif). En los últimos diez años se ha investigado a fondo el papel de los ITAM de CD3 en la activación de los linfocitos T y en la selección tímica. Sin embargo, la función de los ITAM de las otras subunidades CD3 es menos conocida. En esta revisión se pondrá de manifiesto el potencial señalizador del dominio intracitoplásmico de CD3 con especial hincapié en el papel que tienen las interacciones proteína - proteína y proteína-lípidos en la regulación de la activación de linfocito T. También se discutirá la capacidad del ITAM de CD3 para actuar como un regulador negativo de la señalización mediada por el T C R (AU)

Signaling through CD38 induces NK cell activation.

Human CD38 is a signal transduction molecule, and, concurrently, an ectoenzyme catalyzing the synthesis and degradation of cyclic ADP-ribose (cADPR), a potent Ca2+ mobilizer. One facet of CD38 that has not yet been addressed is its role in NK cells. To this end, the events triggered by CD38 ligation with agonistic mAb were analyzed on freshly purified human NK cells. Ligation was followed by (i) a significant rise in the intracellular level of Ca2+, (ii) increased expression of HLA class II and CD25, and (iii) tyrosine phosphorylation of discrete cytoplasmic substrates. The phosphorylation cascade involved CD3-zeta and FcepsilonRIgamma chains, zeta-associated protein (ZAP)-70 and the proto-oncogene product c-Cbl. NK effector functions were then analyzed: CD38 signaling was able (iv) to induce release of IFN-gamma and, more prominently, of granulocyte macrophage colony stimulating factor, as assessed by measuring both mRNA and protein products; and, lastly, (v) to induce cytolytic effector functions on target cells after IL-2 activation, as shown both by cytotoxicity assays and ultrastructural changes. The tyrosine-phosphorylated substrates and all the effects mediated by CD38 were similar to those observed following triggering via CD16 (FcgammaRIIIA); moreover, Ca2+ mobilization via CD38 no longer operated in NK-derived cell lines lacking CD16. These results suggest that the activation signals transduced by CD38 in NK cells elicit relevant cellular events. The effects are similar to those elicited via CD16 and possibly rely on common signaling pathways.

Complexation between tert-butyl ketones and beta-cyclodextrin. Structural study by NMR and MD simulations

A structural study (NMR and MD) of the complexation between tert-butyl ketones and beta-cyclodextrin has been performed. A priority order for the alkyl and phenyl groups composing the ketones has been determined based on association constants: Ph- > C(6)H(11)- = t-Bu- > Bu-, Pr-, Me-. Geometries for the complexes are proposed based on NOE values and on the MD simulations. Bimodal complexation occurs in all the compounds studied.

Learning volleyball serves: a preliminary study of the effects of knowledge of performance and of results.

Recently, doubts have begun to surface about the emphasis that for years has been given to the variable knowledge of results in motor learning, and a view has been expressed that information on how an action has been made (knowledge of performance) may be of more use. This study compared the two types of information in learning a volleyball serve by eight subjects, who were given the two kinds of feedback at various points in the process. Analysis seems to show that knowledge of performance tends to be more effective for learning and that there may be interference across information if knowledge of results is provided after knowledge of performance.

The CD3-gamma delta epsilon transducing module mediates CD38-induced protein-tyrosine kinase and mitogen-activated protein kinase activation in Jurkat T cells.

We have examined the ability of the CD3-gamma delta epsilon and CD3-zeta signaling modules of the T cell receptor (TCR) to couple CD38 to intracellular signaling pathways. The results demonstrated that in TCR+ T cells that express the whole set of CD3 subunits CD38 ligation led to complete tyrosine phosphorylation of both CD3-zeta and CD3-epsilon polypeptide chains. In contrast, in TCR+ cells with a defective CD3-zeta association CD38 engagement caused tyrosine phosphorylation of CD3-epsilon but not of CD3-zeta. Despite these differences, in both cell types CD38 ligation resulted in protein-tyrosine kinase and mitogen-activated protein kinase activation. However, in cells expressing chimerical CD25-zeta or CD25-epsilon receptors or in a TCR-beta- Jurkat T cell line, CD38 ligation did not result in tyrosine phosphorylation of the chimeric receptors, or CD3 subunits, or protein-tyrosine kinase or mitogen-activated protein kinase activation. In summary, these results support a model in which CD38 transduces activating signals inside the cell by means of CD3-epsilon and CD3-zeta tyrosine phosphorylation. Moreover, these data identify the CD3-gamma delta epsilon signaling module as a necessary and sufficient component of the TCR/CD3 complex involved in T cell activation through CD38.

CD38 is functionally dependent on the TCR/CD3 complex in human T cells.

One of the functions of surface CD38 is the induction of phosphorylation of discrete cytoplasmic substrates and mobilization of cytoplasmic calcium (Ca2+). The present work addresses the issue of whether the signaling mediated via CD38 operates through an independent pathway or, alternatively, is linked to the TCR/CD3 signaling machinery. We studied the signals elicited through CD38 by the specific agonistic IB4 monoclonal antibody (mAb) by monitoring the levels of cytoplasmic Ca2+ and the induced phenotypic and functional variations in T cell growth. IB4 mAb presented the unique ability to increase cytoplasmic Ca2+ levels, which correlated with the phosphorylation of the PLC-gamma1. These effects were blocked by phorbol 12-myristate 13-acetate (PMA) and were dependent on the presence of a functional TCR/CD3 surface complex, no effects being recorded on mutant Jurkat cells lacking part of the CD3 structures. CD38 signaling appeared to share with TCR/CD3 the ability to induce apoptotic cell death in Jurkat T cells, an event paralleled by specific up-regulation of the Fas molecule and inhibited by cyclosporin A. CD28, a costimulatory molecule, is synergized by increasing CD38-induced apoptotic cell death. The results indicate the existence of a strong functional interdependence between CD38 and TCR/CD3.

CD38 ligation results in activation of the Raf-1/mitogen-activated protein kinase and the CD3-zeta/zeta-associated protein-70 signaling pathways in Jurkat T lymphocytes.

CD38 ligation with the specific mAb IB4 induced early and late signaling events in Jurkat T cells, as judged by the transient induction of tyrosine phosphorylation of phospholipase C-gamma1, c-Cbl, zeta-associated protein (ZAP)-70, Shc, extracellular signal-regulated protein kinase-2 (Erk-2) as mitogen-activated protein (MAP) kinase, and increased expression of the activation Ag CD69. In addition, CD38 ligation induced Ras-dependent events such as Erk-2 mobility shift and increased Erk-2 kinase activity. Further evidence that Erk-2 activation is regulated by CD38 ligation was obtained indirectly with the observed induction of Raf-1, Lck, and Sos-1 mobility shifts, processes that are believed to be dependent, at least in part, on MAP kinase activation. Using a protein tyrosine kinase inhibitor, herbimycin A, or a protein kinase C inhibitor, Ro-31-8220, we found that the anti-CD38-induced Erk-2 activation is both protein tyrosine kinase and protein kinase C dependent. CD38 ligation also resulted in increased CD3-zeta tyrosine phosphorylation and its association with ZAP-70. CD38 ligation in a Jurkat Lck-deficient mutant, JCam1, failed to induce substrate tyrosine phosphorylation and activation of Erk-2. These data indicated that in Jurkat T cells, CD38 receptor triggering results in Lck-regulated activation of both Raf-1/MAP kinase and CD3-zeta/ZAP-70/phospholipase C-gamma1 signaling pathways.

An octamer element functions as a regulatory element in the differentiation-responsive CD11c integrin gene promoter: OCT-2 inducibility during myelomonocytic differentiation.

The integrin CD11c/CD18 mediates leukocyte adhesion to endothelium and other cell types and is a receptor for LPS, iC3b, and fibrinogen. CD11c expression is restricted to myeloid and activated B cells, is regulated during leukocyte differentiation, and constitutes a diagnostic tool for hairy cell leukemia. Mapping of in vivo DNA-protein interactions in the CD11c proximal promoter revealed three adjacent myeloid-specific interactions, one of which lies on an octamer consensus sequence, ATTT GCAT (Oct185). Oct185 disruption increased the CD11c promoter activity while decreasing its myeloid differentiation responsiveness, indicating that Oct185 contributes to the activity of the CD11c promoter and suggesting that Oct185 is a negative regulatory element whose function changes during myeloid differentiation. Oct185 is recognized by the ubiquitous Oct-1 factor in all cell lineages and by Oct-2 in B lymphoid lineage cells. Unexpectedly, Oct-2 binding to Oct185 was induced de novo upon monocytic differentiation of U937 and HL-60 cells but not during HL-60 granulocytic differentiation, as determined by electrophoretic mobility shift assays and immunochemical studies, and Oct-2 complexes were also observed in cultured adherent monocytes. Western blotting showed that the pattern of Oct-2 isoforms in myeloid cells is similar to that seen in B cells. The Oct-2 up-regulated expression in differentiating myeloid cells and its binding to the Oct185 negative regulatory element suggests its involvement in the differentiation-regulated activity of the CD11c promoter and might represent an important parameter for the myeloid- and B cell-restricted expression of the CD11c/CD18 integrin and other molecules with similar patterns of expression.

A role for activated p21 ras in inhibition/regulation of platelet-derived growth factor (PDGF) type-beta receptor activation.

Ligand-stimulated Platelet-Derived Growth Factor (PDGF) type-beta receptor autophosphorylation, and tyrosine phosphorylation of receptor-associated signalling proteins, is blocked in cells expressing activated Ras genes. A factor present in membrane fractions of v-ras-expressing fibroblasts (Kbalb cells) dominantly inhibits the autophosphorylation of the PDGF type-beta receptor. Purification of this factor, via ion exchange, reveals that the inhibitor can be physically separated from the PDGF type-beta receptor, with reconstitution of PDGF type-beta receptor kinase activity in response to ligand binding. The inhibitor exhibited specificity for the PDGF type-beta receptor, and consistently co-purified with activated p21 ras, with Syp/PTP-2, and with Grb2. Neutralization of the p21 ras protein from the Kbalb cell membranes by p21 ras-specific monoclonal antibodies, however, completely removed the inhibition of PDGF type-beta receptor, rendering the PDGF type-beta receptor molecule capable of autophosphorylation in response to ligand. These results indicate that activated p21 ras either interacts directly with the PDGF type-beta receptor to inhibit autokinase activity, or complexes with different molecules such as Syp and/or Grb2 at the cell membrane to act on another effector which then inhibits PDGF type-beta receptor function.

A small GTP-binding protein, Rho, associates with the platelet-derived growth factor type-beta receptor upon ligand binding.

Ligand binding to the platelet-derived growth factor (PDGF) receptor initiates a complex and diverging cascade of signaling pathways. GTP-binding proteins with intrinsic GTPase activity (G-proteins) frequently link cell surface receptors to intracellular signaling pathways, but no close associations of the PDGF receptor and any small G-proteins, nor any such associations activated by ligand binding to the receptor have been previously reported. We demonstrate that a small GTP-binding protein binds specifically to the murine and human PDGF type-beta receptor. In response to PDGF-BB stimulation, there is an increase in the amount of labeled small G-protein associated with the PDGF type-beta receptor. The GTP-binding protein did not undergo ligand-induced association with a mutant receptor protein that was unable to bind ATP. Proteolytic cleavage analysis, together with two-dimensional separation techniques, identified the small G-protein specifically associating with the PDGF type-beta receptor after ligand binding as a member of the Rho family. This was confirmed by demonstration that the small G-protein coimmunoprecipitated by the anti-PDGF receptor antibody was a substrate for the ADP-ribosyltransferase C3 exoenzyme. Thus, the PDGF type-beta receptor may form a complex with one or more small G-proteins upon binding PDGF-BB, and the Rho small G-protein is likely to be an important component of the proteins making up the multimeric signaling complex of the PDGF type-beta receptor.

G(O), a guanine nucleotide binding protein, is expressed during neurite extension in the embryonic mouse.

The developmental pattern of expression of the G protein alpha o subunit and GAP43 were compared by immunohistochemical staining of mouse embryos. Staining for alpha o and GAP43 was identical and detected throughout the developing nervous system, and the antigens first appeared in neurons at the beginning of neuronal differentiation. GAP43 and alpha o were not detected in regions containing only neuroblasts. These observations suggest that alpha o and GAP43 may not be required for the decision to pass from neuroblast to differentiated neuron, but may play a role in signal transduction during early neuronal development.

Nerve growth factor changes G protein levels and localization in PC12 cells.

Growth cones at the growing tips of developing neurites contain the machinery to transmit information from receptors to a variety of intracellular enzymes and ion channels. In order to understand how signals are transmitted across the membrane, we asked whether the multiplicity of signalling pathways in the growth cone is reflected by the diversity of G proteins found in this organelle. Our immunohistochemical analysis indicated that growth cones of differentiated PC12 cells contain at least 4 alpha G protein subunits, 3 that are pertussis toxin substrates (alpha o, alpha i-1, alpha i-2) and 1 that is not (alpha q). In addition to localization in the neurites and growth cones, alpha o, alpha i-1, alpha i-2, and alpha q were detected in intracellular perinuclear structures. We also analyzed the temporal change in G proteins in PC12 cells differentiated by treatment with nerve growth factor (NGF). Time course experiments have shown that alpha o and beta proteins coordinately increase after 2 days of treatment with NGF, reach a maximum at 4 days, and remain elevated. In contrast to alpha o, alpha i-2 reached a peak at 4 days, then declined to almost the basal level by day 7 of treatment with NGF. These data indicated that the levels of alpha o, alpha i-2, and beta are differentially regulated during NGF-induced neuronal differentiation in PC12 cells. The alpha o protein was highly concentrated at the tips of the growth cones before the cellular level of alpha o had increased appreciably, suggesting that the alpha subunits are translocated during the first stage of neurite development. In addition, not every neural process has the same high level of alpha o, suggesting that G proteins may help define the specialized functions of particular neurites within a single cell.