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To accomplish their critical task of removing infected cells and fighting pathogens, leukocytes activate by forming specialized interfaces with other cells. The physics of this key immunological process are poorly understood, but it is important to understand them because leukocytes have been shown to react to their mechanical environment. Using an innovative micropipette rheometer, we show in three different types of leukocytes that, when stimulated by microbeads mimicking target cells, leukocytes become up to 10 times stiffer and more viscous. These mechanical changes start within seconds after contact and evolve rapidly over minutes. Remarkably, leukocyte elastic and viscous properties evolve in parallel, preserving a well-defined ratio that constitutes a mechanical signature specific to each cell type. Our results indicate that simultaneously tracking both elastic and viscous properties during an active cell process provides a new, to our knowledge, way to investigate cell mechanical processes. Our findings also suggest that dynamic immunomechanical measurements can help discriminate between leukocyte subtypes during activation.

To accomplish their critical task of removing infected cells and fighting pathogens, leukocytes activate by forming specialized interfaces with other cells. Using an innovative micropipette rheometer, we show in three different cell types that when stimulated by microbeads mimicking target cells, leukocytes become up to ten times stiffer and more viscous. These mechanical changes initiate within seconds after contact and evolve rapidly over minutes. Remarkably, leukocyte elastic and viscous properties evolve in parallel, preserving a well-defined ratio that constitutes a mechanical signature specific to each cell type. The current results indicate that simultaneously tracking both elastic and viscous properties during an active cell process provides a new way to investigate cell mechanical processes. Our findings also suggest that dynamic immuno-mechanical measurements provide an identifier of leukocyte type and an indicator of the cell’s state of activation.

APE1 is a multifunctional protein that has recently been implicated in protecting cells from oxidative stress. In the current study, we confirmed that APE1׳s effect on cellular antioxidant capacity is related to its redox activity through the use of an APE1 functional mutant, and we investigated the mechanism through which this multifunctional protein affects the function of the transcription factor Nrf-2 in regulating oxidative stress-induced genes. Using a pair of mutants for both the redox activity and the acetylation-regulated activity of APE1, in vitro assays showed that the redox activity of APE1 is crucial for its nuclear association with Nrf-2 and subsequent activation of Nrf-2׳s transcription of several downstream genes during oxidative challenge. Important oxidative stress genes are affected by APE1 redox activity, including Hmox1, Gstm1, and Txnrd1. In addition, utilizing human non-small-cell lung cancer sample tissue as well as a nude mouse xenograft model, we determined that APE1 expression levels are inversely correlated to oxidative stress in vivo. These findings indicated that interference with these crucial functions of APE1 shows promise in preventing resistance to certain radiotherapies and that further research is necessary to understand APE1׳s complex roles in regulating both the basal redox status and the oxidative stress state of the cellular environment.

International audience; Due to the intrinsic molecular Brownian agitation within plasma membrane and the vast diversity of membrane components, it is expected that the plasma membrane organization is highly heterogeneous with the formation of local complex multicomponent assemblies of lipids and proteins on different time scales. Still, deciphering this lateral organization on living cells and on the appropriate length and temporal scales has been challenging but is crucial to advance our knowledge on the biological function of the plasma membrane. Among the methodological developments based on biophotonics, the spot variation FCS (svFCS), a fluorescent correlation spectroscopy (FCS)-based method, has allowed the significant progress in the characterization of cell membrane lateral organization at the suboptical level, including to providing compelling evidence for the in vivo existence of lipid-dependent nanodomains. The aim of this chapter is to serve as a guide for setting and applying the svFCS methodology to study the plasma membrane of both adherent and nonadherent cell types.

Objective To explore the effects of heroin on purine nucleotides metabolism in rat brain. Methods Biochemical changes in association with heroin administration were compared between heroin-administered rats and non-heroin rats. HPLC method was used to detect the absolute content of purine nucleotides in brain tissues. Concentrations of uric acid (UA), blood urea nitrogen (BUN) and creatinine (Cre) in plasma were measured. Enzymatic activities of adenosine deaminase (ADA) and xanthine oxidase (XO) in brain tissue were analyzed. Real-time PCR was used to determine the relative level of transcripts of ADA, XO, adenine phosphoribosyl transferase (APRT), hypoxanthine-guaninephosphoribosyl transferase (HGPRT) and adenosine kinase (AK) in brain tissue. Results Compared with those in the saline group, the content of AMP and GTP of heroin group decreased significantly; the UA concentration in plasma, ADA and XO activities and the mRNA level of ADA and XO in brain tissues in heroin group increased significantly; the mRNA level of AK, APRT and HGPRT in brain tissues in heroin group decreased significantly (P Conclusion Heroin administration may enhance the catabolism and inhibit the anabolism of purine nucleotides in brain. There may be a deficiency of purine nucleotides, especially GTP and AMP in rat brain exposed to heroin. Our findings may provide a new potential approach to study the mechanism of heroin addiction.

Steady-state polarization-resolved fluorescence imaging is used to analyze the molecular orientational order behavior of rigidly labeled major histocompatibility complex class I (MHC I) proteins and lipid probes in cell membranes of living cells. These fluorescent probes report the orientational properties of proteins and their surrounding lipid environment. We present a statistical study of the molecular orientational order, modeled as the width of the angular distribution of the molecules, for the proteins in the cell endomembrane and plasma membrane, as well as for the lipid probes in the plasma membrane. We apply this methodology on cells after treatments affecting the actin and microtubule networks. We find in particular opposite orientational order changes of proteins and lipid probes in the plasma membrane as a response to the cytoskeleton disruption. This suggests that MHC I orientational order is governed by its interaction with the cytoskeleton, whereas the plasma membrane lipid order is governed by the local cell membrane morphology.

International audience; Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-gamma receptor (IFN-gamma R) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-gamma R2 subunit, this mutation blocks IFN-gamma activity by unknown mechanisms. We show that the lateral diffusion of IFN-gamma R2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-gamma R2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-gamma could not occur. Removing IFN-gamma R2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo.

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (∅

The role of lipid rafts in T cell signalling has recently been extensively studied and hotly debated. In this review we wish to present recent advances in the study of lipid rafts with regards to the initiation of TCR signal transduction. We also wish to clarify some common misconceptions regarding certain models and experimental methods reported in the literature. Based on the relevant data supporting the functional importance of lipid rafts in early TCR mediated signalling events, we propose a model for TCR signal initiation involving a "TCR signalosome" organized in a specific raft subset, which is highly dynamic and evolving upon TCR activation.

The transmembrane protein LAT (linker for activation of T cells) couples the T cell receptor (TCR) to downstream signaling effectors. Mice homozygous for a mutation of a single LAT tyrosine residue showed impeded T cell development. However, later they accumulated polyclonal helper T (T H ) cells that chronically produced type 2 cytokines in large amounts. This exaggerated T H 2 differentiation caused tissue eosinophilia and massive maturation of plasma cells secreting to immunoglobulins of the E and G1 isotypes. This paradoxical phenotype establishes an unanticipated inhibitory function for LAT that is critical for the differentiation and homeostasis of T H cells.

Recent studies suggest that rafts are involved in numerous cell functions, including membrane traffic and signaling. Here we demonstrate, using a polyoxyethylene ether Brij 98, that detergent-insoluble microdomains possessing the expected biochemical characteristics of rafts are present in the cell membrane at 37 degrees C. After extraction, these microdomains are visualized as membrane vesicles with a mean diameter of approximately 70 nm. These findings provide further evidence for the existence of rafts under physiological conditions and are the basis of a new isolation method allowing more accurate analyses of raft structure. We found that main components of T cell receptor (TCR) signal initiation machinery, i.e. TCR-CD3 complex, Lck and ZAP-70 kinases, and CD4 co-receptor are constitutively partitioned into a subset of rafts. Functional studies in both intact cells and isolated rafts showed that upon ligation, TCR initiates the signaling in this specialized raft subset. Our data thus strongly indicate an important role of rafts in organizing TCR early signaling pathways within small membrane microdomains, both prior to and following receptor engagement, for efficient TCR signal initiation upon stimulation.

Plasma membrane microdomains that are enriched in sphingolipids, cholesterol and glycosylphosphatidylinositol-anchored proteins appear to be important in T-cell signalling. Here, Subburaj Ilangumaran, Hai-Tao He and Daniel Hoessli discuss the possible mechanisms involved in T-cell activation via such microdomains.

While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells. Here, we describe a method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization. The method is based on questioning the relationship between the diffusion time τd and the squared waist of observation w2. Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations. These investigations have allowed significant progress in the characterization of cell membrane lateral organization at the suboptical level, and have provided, for instance, compelling evidence for the in vivo existence of raft nanodomains.

Despite intensive investigation, the mechanisms of T cell receptor (TCR)-mediated signal generation remain poorly understood. Here we review various dynamic processes at the cell membrane that might critically control this signaling. Firstly, we summarize recent reports providing new information on the sensitivity of TCR/ligand interaction to the membrane environment and particularly to applied forces. Secondly, we review recent evidence that forces and displacements are continuously generated at cell surfaces. Thirdly, we summarize recent experimental evidence demonstrating the capacity of forces to generate signals. Lastly, we provide a quantitative model to exemplify the capacity of dynamic processes to modulate TCR properties such as specificity that were previously difficult to explain with conventional models. It is concluded that the described dynamic processes must be integrated into current models of TCR signaling.

The plasma membrane delineating the cell is a complex multicomponent assembly of lipids and proteins. Deciphering the lateral organization of this supramolecular complex on appropriate length and temporal scales is necessary to unravel its implication in biological function. Here, we show how measurements of diffusion may be taken to shed light on membrane function. We first describe the current methods used to report lateral diffusion of membrane components. We then focus on one fluorescent correlation spectroscopy (FCS)-based method, namely, the spot variation FCS (svFCS), which allows the characterization of the modes of molecular confinement within the plasma membrane of living cells. We next illustrate with different biological systems the progress made toward improving our understanding of cell membrane function. We also discuss the findings with regard to the current view of nanoscale domains/assemblies as a general membrane-organizing principle.

International audience; Cell membranes actively participate in numerous cellular functions. Inasmuch as bioactivities of cell membranes are known to depend crucially on their lateral organization, much effort has been focused on deciphering this organization on different length scales. Within this context, the concept of lipid rafts has been intensively discussed over recent years. In line with its ability to measure diffusion parameters with great precision, fluorescence correlation spectroscopy (FCS) measurements have been made in association with innovative experimental strategies to monitor modes of molecular lateral diffusion within the plasma membrane of living cells. These investigations have allowed significant progress in the characterization of the cell membrane lateral organization at the suboptical level and have provided compelling evidence for the in vivo existence of raft nanodomains. We review these FCS-based studies and the characteristic structural features of raft nanodomains. We also discuss the findings in regards to the current view of lipid rafts as a general membrane-organizing principle.

The aim of this study was to explore whether Thy-1, like other members of the Ig-like superfamily (e.g., CD2 and neural cell adhesion molecule), participates in cell-cell adhesion. This was investigated by measuring the binding of Thy-1+ probe cells (thymocytes or AKR1 T lymphoma cells) to Thy-1- cloned mouse thymic epithelial (MTE) cells using a quantitative cell adhesion assay. The results were as follows: (a) the thymo-epithelial cell interaction was found to be inhibitable (by 25-40%) by soluble Thy-1 molecules purified from phosphatidylinositol-specific phospholipase C-treated mouse thymocytes as well as by Fab' fragments of a Thy-1-specific mAb; (b) the binding of the Thy-1- AKR1 (Thy-1-d) mutant to MTE cells was found to be reduced (by 50%) as compared with that of the wild type T lymphoma; (c) the Thy-1-mediated adhesion pathway did not require Ca2+ and promoted the initial thymo-epithelial binding measured at 4 degrees C. These data provide the first direct evidence of an adhesive function of Thy-1 and suggest that this molecule, in addition to its T cell triggering properties, might play a role during the early T cell maturation by promoting thymocyte adhesion to thymic stroma.

T-cell antigen receptor triggering mechanisms and lipid rafts are of broad interest, but are also controversial topics. Here, we review some recent progress in these two research fields, which has been accomplished mostly in live cells and with the use of advanced technologies. We then discuss the potential relationship between membrane-domain organization and T-cell antigen receptor-triggering mechanisms. On the basis of the relevant experimental observations, we argue that the key to achieving a better understanding of both processes is the ability to monitor the molecular dynamics and interactions taking place in the membrane of T cells at a spatial scale of tens to hundreds of nanometres, with a subsecond-to-second temporal resolution.

Cell membranes are fascinating supramolecular aggregates that not only form a barrier between compartments but also harbor many chemical reactions essential to the existence and functioning of a cell. Here, it is proposed to review the molecular dynamics and mosaic organization of the plasma membrane, which are thought to have important functional implications. We will first summarize the basic concepts of Brownian diffusion and lipid domain formation in model membranes and then track the development of ideas and tools in this field, outlining key results obtained on the dynamic processes at work in membrane structure and assembly. We will focus in particular on findings made using fluorescent labeling and imaging procedures to record these dynamic processes. We will also discuss a few examples showing the impact of lateral diffusion on cell signal transduction, and outline some future methodological challenges which must be met before we can answer some of the questions arising in this field of research.

The lipopolysaccharides (LPS) of intracellular Proteobacteria such as Brucella, Chlamydia, Legionella and Rickettsia, have properties distinct from enterobacterial LPSs. These properties include deficient LPS induction of host cell activation, low endotoxicity and resistance to macrophage degradation. Together these constitute key virulence mechanisms for intracellular survival and replication. We previously demonstrated that B. abortus LPS captured by macrophages was recycled back to the plasma membrane where it was found associated with macrodomains. Furthermore, this LPS interferes with the MHC class II (MHC-II) presentation of peptides to specific T cell hybridomas. Here, we characterized the Brucella LPS macrodomains by microscopy and biochemistry approaches. We show for the first time that LPS macrodomains act as detergent resistant membranes (DRMs), segregating several lipid-raft components, LPS-binding proteins and MHC-II molecules. Brucella LPS macrodomains remain intact for several months in macrophages and are resistant to the disruptive effects of methyl β-cyclodextrin. Fluorescent anisotropy measurements show that B. abortus LPS is responsible for the formation of rigid surface membrane complexes. In addition, relocalization of MHC-II molecules is observed in these structures. The effects of B. abortus LPS on membrane properties could be responsible for pathogenic effects such as the inhibition of MHC-II-dependent antigen presentation. © 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd. Los lipopolisacáridos (LPS) de Proteobacterias intracelulares como Brucella, Chlamydia, Legionella y Rickettsia, tienen propiedades distintas a los LPS de las enterobacterias. Estas propiedades incluyen una inducción deficiente de los LPS a la activación de las células del huésped, una baja endotoxicidad y una resistencia a la degradación de los macrófagos. En conjunto, constituyen mecanismos de virulencia clave para la supervivencia y replicación intracelular. Anteriormente demostramos que el LPS de B. abortus capturado por los macrófagos se reciclaba de nuevo a la membrana plasmática, donde se encontraba asociado a macrodominios. Además, este LPS interfiere con la presentación de péptidos del MHC clase II (MHC-II) a hibridomas de células T específicos. Aquí, caracterizamos los macrodominios de LPS de Brucella mediante enfoques de microscopía y bioquímica. Demostramos por primera vez que los macrodominios de LPS actúan como membranas resistentes a los detergentes (DRM), segregando varios componentes de los lípidos, proteínas de unión a LPS y moléculas MHC-II. Los macrodominios de LPS de Brucella permanecen intactos durante varios meses en los macrófagos y son resistentes a los efectos disruptivos de la metil β-ciclodextrina. Las mediciones de anisotropía fluorescente muestran que el LPS de B. abortus es responsable de la formación de complejos rígidos de membrana superficial. Además, se observa la relocalización de las moléculas MHC-II en estas estructuras. Los efectos del LPS de B. abortus sobre las propiedades de la membrana podrían ser responsables de efectos patógenos como la inhibición de la presentación del antígeno dependiente del MHC-II Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

Coronin has been described as an actin-binding protein of Dictyostelium discoideum, and it has been demonstrated to play a role in cell migration, cytokinesis and phagocytosis. Coronin-related proteins are found in many eukaryotic species, including Coronin-1 in mammals whose expression is enriched in the hematopoietic tissues. Here, we characterize Coronin-1 gene and protein expression in mouse embryonic and adult T lymphocytes. Coronin-1 is expressed throughout T cell ontogeny and in peripheral alphabeta T cells. Expression varies along thymic cell development, with maximum levels observed in embryonic early thymocytes and, in the adults, the selected TCRalphabeta(+) single-positive thymocytes. Subcellular localization analysis indicates that Coronin-1 is in equilibrium between the cytosol and the cell cortex, where it accumulates in F-actin-rich membrane protrusions induced by polarized activation of TCR-CD3-stimulated T cells. These data are consistent with a role of Coronin-1 in T cell differentiation/activation events involving membrane dynamisms and the cortical actin cytoskeleton.

Several patients with Mendelian susceptibility to mycobacterial diseases (MSMD) were shown to present a common mutation T168N creating an additional N-glycosylation site in the extracellular domain of the interferon γ receptor subunit IFNGR2 [1] . This inherited modification of the IFNGR2 subunit caused a complete lack of gene response to IFN-γ, a key cytokine for host defense, and resulted in children death. In patient cells, IFN-γ-induced JAK/STAT signaling was fully inhibited. Spot variation fluorescence correlation spectroscopy (SvFCS) in live cells revealed that the IFNGR complex associates with membrane lipid nanodomains of the raft type upon IFN-γ stimulation. In contrast, the IFNGR2 T168N mutant did not associate with lipid nanodomains. Bioluminescence resonance energy transfer (BRET) experiments found a lack of IFNGR conformational change induced by IFN-γ binding and defects in JAK kinase association with IFNGR complex in patient cells. Removal of the extra glycan restores normal JAK/STAT signaling and IFN-γ biological activity. Proteomics analysis indicates that abnormal galectin binding to the mutated IFNGR2 subunit is responsible for these defects. Depletion of galectins restores normal IFNGR conformational change and JAK/STAT signaling in patient cells. Our results provide the first positive evidence for the role of raft lipid nanodomains in IFNGR assembly and JAK/STAT signaling by IFN-γ in human cells. They also revealed the key role of receptor glycosylation and galectins in this process.

Somatostatin (SRIF) induces its biological actions by binding to and stimulating membrane-associated receptors. To investigate the molecular mechanisms by which SRIF induces its biological effects, we have characterized the biochemical properties of SRIF receptors. SRIF receptors can be solubilized in an active form with the detergent CHAPS and can be detected with the high-affinity SRIF analog [125I]MK 678. The pharmacological characteristics of solubilized SRIF receptors from brain are similar to the receptors in membranes, suggesting that the solubilized receptors retain their biological activity. Solubilized SRIF receptors appear to be tightly associated with GTP-binding proteins, since analogs of GTP can greatly reduce agonist labeling of the solubilized SRIF receptor. The solubilized SRIF receptor migrates as a mass of approximately 400 kd and is a glycoprotein since it can specifically interact with lectin columns. The solubilization of the SRIF receptor has allowed for its purification by affinity chromatography. The purified SRIF receptor migrates as a mass of 60 kd in denaturing gels. Using affinity chromatography, the receptor can be purified to near homogeneity. Present studies are directed toward sequencing and cloning cDNA encoding the SRIF receptor in order to further characterize its physical properties and expression.

The brain somatostatin (somatotropin release-inhibiting factor; SRIF) receptor was purified by affinity chromatographic techniques. A protein of 60 kDa could be purified from rat brain. The protein was eluted from a [D-Trp8]SRIF affinity column with either sodium acetate (pH 5.5) or free [D-Trp8]SRIF. The binding of the protein to the affinity column was prevented by free [D-Trp8]SRIF or the stable SRIF analogue SMS 201-996 but not by the inactive somatostatin 28-(1-14). The purified receptor could be covalently labeled by the 125I-labeled SRIF analogue CGP 23996. Excess [D-Trp8]SRIF blocked the binding of 125I-labeled CGP 23996 to the purified receptor, but somatostatin 28-(1-14) did not affect the binding. A 60-kDa protein was also purified from the anterior pituitary cell line AtT-20, which has a high expression of SRIF receptors. In contrast, no 60-kDa protein could be purified from CHO cells, which have no detectable SRIF receptors. These findings present evidence for the purification of the SRIF receptor.

The neural cell adhesion molecule (N-CAM) of rodents comprises three distinct proteins of Mr 180,000, 140,000, and 120,000 (designated N-CAM-180, -140, and -120). They are expressed in different proportions by different tissues and cell types. but the individual contribution of each form to cell adhesion is presently unknown. Previous studies have shown that the two N-CAM species of higher relative molecular mass span the membrane whereas N-CAM-120 lacks a transmembrane domain and can be released from the cell surface by phosphatidylinositol-specific phospholipase C. In this report, we provided evidence that N-CAM-120 contained covalently bound phosphatidylinositol and studied N-CAM-120 from its biosynthesis to its membrane insertion and finally to its release from the cell surface. Evidence was presented showing that the lipid tail of N-CAM-120 contained ethanolamine as is the case for other lipid-linked molecules. The phospholipid anchor was attached to the protein during the first minutes after completion of the polypeptide chain. This process took place in the endoplasmic reticulum as judged from endoglycosidase H digestion experiments. Immediately after a 2-min pulse with [35S]methionine, we detected also a short-lived precursor that had not yet acquired the lipid tail. Pulse-chase studies established that N-CAM-120 was transported to the cell surface from which it was slowly released into the extracellular milieu. The molecules recovered in the incubation medium appeared to have lost all of their bound fatty acid but only around half of the ethanolamine. Upon fractionation of brain tissue, approximately 75% of N-CAM-120 was recovered with a membrane fraction and approximately 25% in a membrane-free supernatant. A small proportion (approximately 6%) was found to be resistant to extraction by non-ionic detergent. A major posttranslational modification of N-CAM is polysialylation. Our results showed that also N-CAM-120 was polysialylated in the young postnatal brain and released in this form from cultured cerebellar cells. The presence of N-CAM in a form that can be released from the cell surface and accumulates in the extracellular fluid suggests a novel mechanism by which N-CAM-mediated adhesion may be modulated.

The rodent neural cell adhesion molecule (NCAM) consists of three glycoproteins with Mr of 180,000, 140,000 and 120,000. The Mr 120,000 protein (NCAM-120) has been shown to exist in membrane-bound and soluble forms but the nature of its membrane association and release has remained obscure. We show here that phosphatidylinositol-specific phospholipase C (PI-PLC), but not a phospholipase C of different specificity, releases a substantial proportion of NCAM-120 from brain membranes and solubilizes almost quantitatively NCAM-120 present at the surface of C6 astroglial cells. The PI-PLC effect was highly selective since only one other protein species was detectably released from C6 cells. These results suggest that NCAM-120 is held in the membrane by covalently bound phosphatidylinositol or a closely related lipid in a way similar to several other surface proteins from eukaryotic cells. The presence of NCAM in a form which can be released from the cell surface by a highly selective mechanism raises additional possibilities for modulation and control of cell--cell adhesion.

We evaluated the importance of CD3-zeta ITAMs in T cell responses by breeding the P14 transgenic TCR into mice in which CD3-zeta chains lacking all or part of their ITAMs were genetically substituted for wild-type CD3-zeta chains. In contrast to the H-Y TCR, the P14 TCR permitted the development of peripheral CD8+ T cells harboring signaling-defective CD3-zeta subunits. The absence of functional CD3-zeta ITAMs did not reduce the spectrum of activation events and effector functions that constitute the normal attributes of mature CD8+ T cells. The only detectable differences were quantitative and noted only when T cells were challenged with suboptimal peptide concentrations. Therefore, the ITAMs present in the CD3-gammadeltaepsilon module are sufficient for qualitatively normal TCR signaling and those present in CD3-zeta have no exclusive role during T cell activation.

The physical properties of brain and pituitary somatostatin receptors were characterized using photocrosslinking techniques. Somatostatin receptors in rat corpus striatum and anterior pituitary membranes were covalently bound to the non-reducible somatostatin analog, [125I]CGP 23996, using the crosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate and ultraviolet light. In striatal membranes, a protein of 60,000 mol. wt was labeled by [125I]CGP 23996. The binding was potently inhibited by somatostatin analogs but not by other biologically active peptides. The labeling of the 60,000 mol. wt protein by [125I]CGP 23996 was diminished by guanine triphosphate gamma thiol, which is consistent with the labeling of a somatostatin receptor coupled to guanine triphosphate binding proteins. The migration of the [125I]CGP 23996 labeled 60,000 mol. wt protein in native sodium dodecyl sulfate-gels was not affected by the reducing agent dithiothreitol, indicating that there is a general lack of disulfide bridges in the striatal somatostatin receptor. The striatal somatostatin receptor was solubilized with the detergent 3-[(3-cholamidopropyl)-dimethylaminoio]-1-propanesulfonate and specifically bound to the lectin wheat germ agglutinin, suggesting that the striatal somatostatin receptor is a glycoprotein. [125I]CGP 23996 also labeled a 60,000 mol. wt protein in anterior pituitary membranes. The characteristics of [125I]CGP 23996 binding to anterior pituitary membranes were consistent with the labeling of a somatostatin receptor. Interestingly, a comparison of the [125I]CGP 23996 labeled material from striatal and anterior pituitary membranes by two-dimensional polyacrylamide gel electrophoresis revealed the presence of several striatal somatostatin receptors of varying charge (pI values between 6 and 6.5) but only a single pituitary receptor. These findings indicate that physical differences may exist between subtypes of somatostatin receptors.