Mast cells (MC)l are widely distributed throughout vascularized tissues and certain epithelia. They represent a source ofpotent mediators ofinflammation (reviewed in references 1-4). These mediators are released after sensitization with IgE immunoglobulins, which are bound to IgE receptors (FceRI) on the MC, and crosslinking with specific multivalent antigen (4). Such activation causes MC to degranulate releasing histamine, heparin, and other sulphated proteoglycans and certain neutral proteases. Activated MC also elaborate newly synthesized mediators such as products of the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism (reviewed in references 2-4). MC are widely regarded as critical effector cells in the inflammatory reactions underlying disorders of IgE-dependent immediate hypersensitivity and in the expression ofprotective immunity involving IgE (reviewed in references 1-4).Studies in mice indicate that MC are derived from multipotential bone marrowderived hematopoietic precursors which complete their program of differentiation and maturation in vascularized tissues, epithelia, and serosal cavities (reviewed in references 1, 5). This process results in the generation ofmast cell populations which vary in multiple aspects of their phenotype, including morphology, mediator content, and sensitivity to regulation by cytokines affecting proliferation and maturation (reviewed in reference 1). One such population, referred to as "mucosal" mast cells (MMC) because they occur in the mucosal layer of gastrointestinal tissues, appears to be exquisitely sensitive to regulation by the T cell-associated cytokines IL-3 and IL-4 (1). IL-3 probably represents the major cytokine regulating proliferation ofthis subset (6, 7), whereas in vitro studies indicate that IL-4 can act as a costimulant of proliferation (8). Thus, the mouse MMC population is regulated by products I Abbreviations used in this paper: AbMuLV, Abelson murine leukemia virus; Ag, antigen; BMCMC, bone marrow-derived cultured mast cell ; DNP3o-40-HSA 2,4-dinitrophenyl-human serum albumin; FceRI, cell surface receptor for the Fc portion of IgE; GM-CSF, granulocyte/macrophage colony-stimulating factor ; MC, mast cell; MIP, macrophage inflammatory protein; MMC, mucosal mast cell ; PKC, protein kinase C. J. Exp. MED.

M-Ras is a Ras-related protein that shares ϳ55% identity with K-Ras and TC21. The M-Ras message was widely expressed but was most predominant in ovary and brain. Similarly to Ha-Ras, expression of mutationally activated M-Ras in NIH 3T3 mouse fibroblasts or C2 myoblasts resulted in cellular transformation or inhibition of differentiation, respectively. M-Ras only weakly activated extracellular signal-regulated kinase 2 (ERK2), but it cooperated with Raf, Rac, and Rho to induce transforming foci in NIH 3T3 cells, suggesting that M-Ras signaled via alternate pathways to these effectors. Although the mitogen-activated protein kinase/ ERK kinase inhibitor, PD98059, blocked M-Ras-induced transformation, M-Ras was more effective than an activated mitogen-activated protein kinase/ERK kinase mutant at inducing focus formation. These data indicate that multiple pathways must contribute to M-Ras-induced transformation. M-Ras interacted poorly in a yeast two-hybrid assay with multiple Ras effectors, including c-Raf-1, A-Raf, B-Raf, phosphoinositol-3 kinase ␦, RalGDS, and Rin1. Although M-Ras coimmunoprecipitated with AF6, a putative regulator of cell junction formation, overexpression of AF6 did not contribute to fibroblast transformation, suggesting the possibility of novel effector proteins. The M-Ras GTP/GDP cycle was sensitive to the Ras GEFs, Sos1, and GRF1 and to p120 Ras GAP. Together, these findings suggest that while M-Ras is regulated by similar upstream stimuli to Ha-Ras, novel targets may be responsible for its effects on cellular transformation and differentiation.The mammalian Ras superfamily is made up of over 60 GTPases that serve as molecular switches to regulate a diverse array of cellular functions. These include intracellular signal transduction for cell growth and differentiation (Ras subfamily), regulation of the actin cytoskeleton (Rho subfamily), membrane trafficking (Rab subfamily), and nuclear transport (Ran) (1-4). The Ras subfamily consists of Ha-, Ki-, and N-Ras; Krev-1/Rap1A and -1B; Rap2A and -2B; R-Ras; TC21(R-Ras2); Ral A and B; Rheb; Dex-Ras; Rin; and Rit that share several common features outside of the core GTP-binding domain (2).The classic/prototypic Ras proteins, Ha-, Ki-, and N-Ras, transduce signals for growth and differentiation from ligand-bound receptors to the nuclear transcriptional machinery and to the cytoskeleton (2, 3, 5, 6). These proteins can be constitutively activated by point mutation, contributing to the development of a broad spectrum of human malignancies (7). The introduction of equivalent activating mutations into the closely related TC21 and R-Ras proteins also results in transformation in tissue culture models (8, 9), and TC21 mutants have been identified in human tumor cell lines (10, 11). R-Ras has also been associated with apoptosis and integrin activation (12, 13). Overexpression of Rap1A/Krev-1 can induce transformation in some cells (14) but typically has been found to counter Rasinduced activities, due to competitive binding to Ras effectors (15,16). Rheb...

Tat is an 86-to 104-amino-acid viral protein that activates human immunodeficiency virus type 1 expression, modifies several cellular functions, and causes neurotoxicity. Here, we determined the extent to which peptide fragments of human immunodeficiency virus type 1 BRU Tat 1-86 produced neurotoxicity, increased levels of intracellular calcium ([Ca 2؉ ] i), and affected neuronal excitability. Tat 31-61 but not Tat 48-85 dose dependently increased cytotoxicity and levels of [Ca 2؉ ] i in cultured human fetal brain cells. Similarly, Tat 31-61 but not Tat 48-85 depolarized rat hippocampal CA1 neurons in slices of rat brain. The neurotoxicity and increases in [Ca 2؉ ] i could be significantly inhibited by non-N-methyl-D-aspartate excitatory amino acid receptor antagonists. Shorter 15-mer peptides which overlapped by 10 amino acids each and which represented the entire sequence of Tat 1-86 failed to produce any measurable neurotoxicity. Although it remains to be determined if Tat acts directly on neurons and/or indirectly via glial cells, these findings do suggest that Tat neurotoxicity is conformationally dependent, that the active site resides within the first exon of Tat between residues 31 to 61, and that these effects are mediated at least in part by excitatory amino acid receptors.

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We describe, in rheumatoid arthritis (RA), abnormalities in the expression and distribution of heat shock protein (hsp) and dendritic cells (DCs) that are conducive to cross-priming and DC cross-talk. As detected by ELISA, inducible (i)hsp70 was dramatically increased in RA synovial fluid (RASF) vs normal human and RA sera and osteoarthritis and gout synovial fluid. Immunoblot analysis of fresh RASF cells revealed marked increases in ihsp70 and activation of its transcription factor heat shock factor-1, compared with fresh normal peripheral blood cells. Flow cytometry and microscopy demonstrated high levels of ihsp70 on the surface of RASF myeloid DCs (but not normal myeloid DCs) that occurred concurrently with hspRs (CD91/CD14). ihsp70 present in RASF exhibited chaperoning potential, as indicated by the capture of ihsp70 present in RASF on the surface of normal DCs. Binding was partially competitively inhibited by excess α2-macroglobulin, indicating that hspRs in addition to CD91 participate in the capture process. These data indicate that ihsp70 may chaperone autologous Ags into immature RASF DCs via hspRs, and that cross-talk between DCs coexpressing hsp/hspRs reflects a disease process in RA. The induction of surface ihsp70 on normal cells after sublethal heat stress and the release of ihsp70 from normal DCs after inflammatory stress also suggest that the pattern of ihsp70 expression in RASF occurs in response to sustained stress.

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Constitutively activated mutants of the Ras-related protein TC21/R-Ras2 cause tumorigenic transformation of NIH3T3 cells. However, unlike Ras, TC21 fails to bind to and activate the Raf-1 serine-threonine kinase. Thus, whereas Ras transformation is critically dependent on Raf-1 TC21 activity is promoted by activation of Rafindependent signaling pathways. In the present study, we have further compared the functions of Ras and TC21. First we determined the basis for the inability of TC21 to activate Raf-1. Whereas Ras can interact with the two distinct Ras-binding sequences in NH 2 -terminus of Raf-1, designated RBS1 and Raf-Cys, TC21 could only bind Raf-Cys. Thus, the inability of TC21 to bind to RBS1 may prevent it from promoting the translocation of Raf-1 to the plasma membrane. Second, we found that TC21 is an activator of the JNK and p38, but not ERK, mitogen-activated protein kinase cascades and that TC21 transforming activity was dependent on Rac function. Thus, like Ras, TC21 may activate a Rac/JNK pathway. Third, we determined if TC21 could cause the same biological consequences as Ras in three distinct cell types. Like Ras, activated TC21 caused transformation of RIE-1 rat intestinal epithelial cells and terminal dierentiation of PC12 pheochromocytoma cells. Finally, activated TC21 blocked serum starvation-induced dierentiation of C2 myoblasts, whereas dominant negative TC21 greatly accelerated this dierentiation process. Therefore, TC21 and Ras share indistinguishable biological activities in all cell types that we have evaluated. These results support the importance of Rafindependent pathways in mediating the actions of Ras and TC21.

The C5a anaphylatoxin protein plays a central role in inflammation associated with complement activation. This protein is commonly regarded as one of the most potent inducers of the inflammatory response and a C5a peptide agonist was used as a molecular adjuvant. However, the full length C5a protein has not been tested as a potential tumor therapy. In this report, we describe the creation of a mini-gene construct that directs C5a expression to any cell of interest. Functional expression could be demonstrated in the murine mammary sarcoma, EMT6. When C5a expressing cells were injected into syngeneic mice, most C5a-expressing clones had significantly reduced tumor growth. Further characterization of a clone expressing low levels of C5a demonstrated that one-third of mice injected with this line had complete tumor regression. The mice whose tumors regressed were immune to subsequent challenge with unmodified EMT6 cells, suggesting that a component of the innate immune response can be used to augment adaptive immunity. Cellular analyses demonstrated that a significant difference in actual tumor cell number could be detected as early as day 10. A block in cell cycle progression was evident at all time points and high levels of apoptosis were observed early in the regression event. These data demonstrate that the complement protein C5a can play a significant protective role in tumor immunity.