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Biomedical subjects

Michael A Silverman

Publications and source records attributed to Michael A Silverman.

13 recordsLinked to original sources
PubMed2006

Impaired degranulation but enhanced cytokine production after Fc epsilonRI stimulation of diacylglycerol kinase zeta-deficient mast cells.

Calcium and diacylglycerol are critical second messengers that together effect mast cell degranulation after allergen cross-linking of immunoglobulin (Ig)E-bound FcepsilonRI. Diacylglycerol kinase (DGK)zeta is a negative regulator of diacylglycerol-dependent signaling that acts by converting diacylglycerol to phosphatidic acid. We reported previously that DGKzeta-/- mice have enhanced in vivo T cell function. Here, we demonstrate that these mice have diminished in vivo mast cell function, as revealed by impaired local anaphylactic responses. Concordantly, DGKzeta-/- bone marrow-derived mast cells (BMMCs) demonstrate impaired degranulation after Fc epsilonRI cross-linking, associated with diminished phospholipase Cgamma activity, calcium flux, and protein kinase C-betaII membrane recruitment. In contrast, Ras-Erk signals and interleukin-6 production are enhanced, both during IgE sensitization and after antigen cross-linking of Fc epsilonRI. Our data demonstrate dissociation between cytokine production and degranulation in mast cells and reveal the importance of DGK activity during IgE sensitization for proper attenuation of Fc epsilonRI signals.

Animals↗
PubMed2006

Activity-dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live-cell imaging.

Tissue plasminogen activator (tPA) has been implicated in a variety of important cellular functions, including learning-related synaptic plasticity and potentiating N-methyl-D-aspartate (NMDA) receptor-dependent signaling. These findings suggest that tPA may localize to, and undergo activity-dependent secretion from, synapses; however, conclusive data supporting these hypotheses have remained elusive. To elucidate these issues, we studied the distribution, dynamics, and depolarization-induced secretion of tPA in hippocampal neurons, using fluorescent chimeras of tPA. We found that tPA resides in dense-core granules (DCGs) that traffic to postsynaptic dendritic spines and that can remain in spines for extended periods. We also found that depolarization induced by high potassium levels elicits a slow, partial exocytotic release of tPA from DCGs in spines that is dependent on extracellular Ca(+2) concentrations. This slow, partial release demonstrates that exocytosis occurs via a mechanism, such as fuse-pinch-linger, that allows partial release and reuse of DCG cargo and suggests a mechanism that hippocampal neurons may rely upon to avoid depleting tPA at active synapses. Our results also demonstrate release of tPA at a site that facilitates interaction with NMDA-type glutamate receptors, and they provide direct confirmation of fundamental hypotheses about tPA localization and release that bear on its neuromodulatory functions, for example, in learning and memory.

Animals↗
PubMed2006

SLP76 and SLP65: complex regulation of signalling in lymphocytes and beyond.

SLP76 and SLP65 are adaptor proteins that lack intrinsic enzymatic activity but contain multiple protein-binding domains. These proteins are essential for signalling downstream of integrins and receptors that contain immunoreceptor tyrosine-based activation motifs. The absence of these adaptor proteins profoundly affects various lineages in the haematopoietic compartment and severely compromises vascular development, highlighting their importance as regulators of signalling cascades. In this Review, we discuss the role of SLP76 and SLP65 in several signalling pathways in haematopoietic cells, with an emphasis on recent studies that provide insight into their mechanisms of action.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Disruption of SLP-76 interaction with Gads inhibits dynamic clustering of SLP-76 and FcepsilonRI signaling in mast cells.

We developed a confocal real-time imaging approach that allows direct observation of the subcellular localization pattern of proteins involved in proximal FcepsilonRI signaling in RBL cells and primary bone marrow-derived mast cells. The adaptor protein Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is critical for FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. In this study, we imaged SLP-76 and found it in the cytosol of unstimulated cells. Upon FcepsilonRI cross-linking, SLP-76 translocates to the cell membrane, forming clusters that colocalize with the FcepsilonRI, the tyrosine kinase Syk, the adaptor LAT, and phosphotyrosine. The disruption of the SLP-76 interaction with its constitutive binding partner, Gads, through the mutation of SLP-76 or the expression of the Gads-binding region of SLP-76, inhibits the translocation and clustering of SLP-76, suggesting that the interaction of SLP-76 with Gads is critical for appropriate subcellular localization of SLP-76. We further demonstrated that the expression of the Gads-binding region of SLP-76 in bone marrow-derived mast cells inhibits FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. These studies revealed, for the first time, that SLP-76 forms signaling clusters following FcepsilonRI stimulation and demonstrated that the Gads-binding region of SLP-76 regulates clustering of SLP-76 and FcepsilonRI-induced mast cell responses.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Cutaneous malignant melanoma.

Skin cancer has become the most common neoplasm in the United States. With early diagnosis and appropriate management, most skin cancers have an overall 5-year survival rate of 95%. Cutaneous malignant melanoma (CMM), however, has a significantly higher morbidity and mortality, resulting in 65% of all skin cancer deaths. Although the long-term survival rate for patients with metastatic melanoma is only 5%, early detection of CMM carries an excellent prognosis, with surgical excision often being curative. Primary care physicians can play a critical role in reducing morbidity and mortality from CMM by recognizing patients at risk, encouraging the adoption of risk-reducing behaviors, and becoming adept at identifying suspicious lesions.

Humans↗
PubMed2005

Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide.

A non-natural 16-residue "degron" peptide has been reported to convey proteasome-dependent degradation when fused to proteins expressed in yeast (Gilon, T., Chomsky, O., and Kulka, R. (2000) Mol. Cell. Biol. 20, 7214-7219) or when fused to green fluorescent protein (GFP) and expressed in mammalian cells (Bence, N. F., Sampat, R. M., and Kopito, R. R. (2001) Science 292, 1552-1555). We find that expression of the GFP::degron in Caenorhabditis elegans muscle or neurons results in the formation of stable perinuclear deposits. Similar perinuclear deposition of GFP::degron was also observed upon transfection of primary rat hippocampal neurons or mouse Neuro2A cells. The generality of this observation was supported by transfection of HEK 293 cells with both GFP::degron and DsRed(monomer)::degron constructs. GFP::degron expressed in C. elegans is less soluble than unmodified GFP and induces the small chaperone protein HSP-16, which co-localizes and co-immunoprecipitates with GFP::degron deposits. Induction of GFP::degron in C. elegans muscle leads to rapid paralysis, demonstrating the in vivo toxicity of this aggregating variant. This paralysis is suppressed by co-expression of HSP-16, which dramatically alters the subcellular distribution of GFP::degron. Our results suggest that in C. elegans, and perhaps in mammalian cells, the degron peptide is not a specific proteasome-targeting signal but acts instead by altering GFP secondary or tertiary structure, resulting in an aggregation-prone form recognized by the chaperone system. This altered form of GFP can form toxic aggregates if its expression level exceeds the capacity of chaperone-based degradation pathways. GFP::degron may serve as an instructive "generic" aggregating control protein for studies of disease-associated aggregating proteins, such as huntingtin, alpha-synuclein, and the beta-amyloid peptide.

Amino Acid Sequence↗
PubMed2005

Membrane cholesterol content accounts for developmental differences in surface B cell receptor compartmentalization and signaling.

Recent studies argue for an important role for cholesterol in maintaining plasma membrane heterogeneity and influencing a variety of cellular processes, including signaling, adhesion, and permeability. Here, we document that tolerance-sensitive transitional immature B cells maintain significantly lower membrane unesterified cholesterol levels than mature-stage splenic B cells. In addition, the relatively low level of cholesterol in transitional immature B cells impairs compartmentalization of their B cell receptor (BCR) into cholesterol-enriched domains following BCR aggregation and reduces their ability to sustain certain aspects of BCR signaling as compared with mature B cells. These studies establish an unexpected difference in the lipid composition of peripheral transitional immature and mature B cells and point to a determining role for development-associated differences in cholesterol content for the differential responses of these B cells to BCR engagement.

Animals↗
PubMed2005

Mechanisms of transport and exocytosis of dense-core granules containing tissue plasminogen activator in developing hippocampal neurons.

Dense-core granules (DCGs) are organelles found in specialized secretory cells, including neuroendocrine cells and neurons. Neuronal DCGs facilitate many critical processes, including the transport and secretion of proteins involved in learning, and yet their transport and exocytosis are poorly understood. We have used wide-field and total internal reflection fluorescence microscopy, in conjunction with transport theory, to visualize the transport and exocytosis of DCGs containing a tissue plasminogen activator-green fluorescent protein hybrid in cell bodies, neurites, and growth cones of developing hippocampal neurons and to quantify the roles that diffusion, directed motion, and immobility play in these processes. Our results demonstrate that shorter-ranged transport of DCGs near sites of exocytosis in hippocampal neurons and neuroendocrine cells differs markedly. Specifically, the immobile fraction of DCGs within growth cones and near the plasma membrane of hippocampal neurons is small and relatively unaltered by actin disruption, unlike in neuroendocrine cells. Moreover, transport of DCGs in these domains of hippocampal neurons is unusually heterogeneous, being significantly rapid and directed as well as slow and diffusive. Our results also demonstrate that exocytosis is preceded by substantial movement and heterogeneous transport; this movement may facilitate delivery of DCG cargo in hippocampal neurons, given the relatively low abundance of neuronal DCGs. In addition, the extensive mobility of DCGs in hippocampal neurons argues strongly against the hypothesis that cortical actin is a major barrier to membrane-proximal DCGs in these cells. Instead, our results suggest that extended release of DCG cargo from hippocampal neurons arises from heterogeneity in DCG mobility.

Actin Cytoskeleton↗
PubMed2005

A cytoplasmic motif targets neuroligin-1 exclusively to dendrites of cultured hippocampal neurons.

The formation of neuronal synapses is thought to depend on trans-synaptic interactions between cell adhesion molecules (CAMs) on the surface of axons and dendrites. Synapses are highly asymmetric structures. Pre- and post-synaptic domains might therefore be assembled around heterophilic CAMs which are polarized to axons vs. dendrites. We here investigated the targeting of neuroligin (NLG)-1, a heterophilic CAM, which promotes synapse formation through interaction with its receptor beta-neurexin in axons. We demonstrate that NLG-1 is highly polarized to the dendritic plasma membrane. Dendritic targeting relies on a cytoplasmic amino acid motif. By expressing chimeras of NLG-1 and CD8, an unpolarized protein, we show that the cytoplasmic domain of NLG-1 is necessary and sufficient for dendritic targeting. Furthermore, by truncation analysis we isolated a 32-amino-acid targeting motif. When appended to CD8 this cytoplasmic sequence is sufficient to direct exclusively dendritic localization of the protein. Analysis of yellow fluorescent protein-tagged NLG-1 revealed that vesicular structures containing NLG-1 are excluded from the axon indicating that polarized distribution may be achieved by direct dendritic transport. We propose that the strict polarity of NLG-1 contributes to the directional assembly of synapses during development of the central nervous system.

Animals↗
PubMed2004

Differential requirement for adapter proteins Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and adhesion- and degranulation-promoting adapter protein in FcepsilonRI signaling and mast cell function.

The adapter molecule Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is essential for FcepsilonRI-mediated signaling, degranulation and IL-6 production in mast cells. To test the structural requirements of SLP-76 in mast cell signaling and function, we have studied the functional responses of murine bone marrow-derived mast cells (BMMCs) expressing mutant forms of SLP-76. We found that the N-terminal tyrosines as well as the central proline-rich region of SLP-76 are required for participation of SLP-76 in FcepsilonRI-mediated signaling and function. The C-terminal SH2 domain of SLP-76 also contributes to optimal function of SLP-76 in mast cells. Another adapter molecule, adhesion- and degranulation-promoting adapter protein (ADAP), is known to bind the SH2 domain of SLP-76, and cell line studies have implicated ADAP in mast cell adhesion and FcepsilonRI-induced degranulation. Surprisingly, we found that mast cells lacking ADAP expression demonstrate no defects in FcepsilonRI-induced adhesion, granule release, or IL-6 production, and that ADAP-deficient mice produce a normal passive systemic anaphylactic response. Thus, failure to bind ADAP does not underlie the functional defects exhibited by SLP-76 SH2 domain mutant-expressing mast cells.

Adaptor Proteins, Signal Transducing↗
PubMed2003

SLP-76 regulates Fcgamma receptor and integrin signaling in neutrophils.

While the contribution of intracellular adaptor proteins to lymphocyte activation has been well studied, the function of these molecules in innate immune effector cells such as neutrophils has not been extensively addressed. Here we demonstrate a critical role for the adaptor molecule SH2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) in FcgammaR and integrin signaling. Stimulation of these receptors induces tyrosine phosphorylation and cytoplasmic relocalization of SLP-76 in freshly isolated murine neutrophils. Neutrophils lacking SLP-76 demonstrate decreased FcgammaR-induced calcium flux and reactive oxygen intermediate (ROI) production in response to immune complex stimulation. More dramatically, SLP-76-/- neutrophils fail to produce ROI, spread, or activate critical downstream regulators in response to integrin ligation. These results provide genetic evidence for a critical role of SLP-76 in the regulation of neutrophil function.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

University of Miami Division of Clinical Pharmacology Therapeutic Rounds: medications used to treat anorexia in the frail elderly.

Weight loss and anorexia are common problems that are associated with increased morbidity and mortality in the growing frail elderly population. Orexigenic agents are often prescribed in an attempt to improve appetite and lean muscle mass. Much of the data regarding their benefit comes from studies involving younger patients with illnesses such as acquired immunodeficiency syndrome or cancer. This article reviews the use and potential adverse events associated with these medications in frail elderly patients. This article also discusses the effects of the different antidepressants and antipsychotics on weight gain and appetite.

Aged↗