In this report, we demonstrate that myogenic cultures inhibited from differentiating by treatment with fibroblast growth factor or transforming growth factor 11 show reduced levels of MyoDl mRNA. Although this repression may contribute to the inhibition of myogenesis by growth factors, additional regulatory pathways must be affected, since inhibition still occurs in cultures engineered to constitutively express MyoDI mRNA.Skeletal muscle differentiation involves the transcriptional activation of the contractile protein gene set and the formation of multinucleated muscle fibers. The contractile protein genes are controlled by cis and tran.s regulatory systems which appear to respond to exogenous growth factors, since addition of fibroblast growth factor (FGF) or transforming growth factor 3 (TGF-3) to differentiating muscle cells in culture inhibits both fusion and the accumulation of these muscle-specific proteins (7,9,13,14,16,21). It has been hypothesized that FGF and TGF-, may influence one or perhaps several master regulatory pathways. Although the regulatory genes that control the induction of skeletal myogenesis have not been defined completely, at least three genes, MyoDl (8,20), inyd (18), and myogenin (22). play an important role in establishing the myogenic lineage and inducing the expression of muscle-specific genes.To determine whether these regulatory genes are modulated by exogenous growth factors, we investigated the transcriptional activity of the MyoDl gene in cells exposed to FGF or TGF-3. 23A2 myoblasts (12) were induced to differentiate in medium containing insulin, transferrin, and selenium (ITS) (23) to establish the normal expression pattern of MyoDl and the contractile protein gene, troponin I (TnI), during skeletal myogenesis. RNA was isolated from proliferating and differentiation-induced 23A2 cultures, electrophoresed through formaldehyde-agarose gels, and hybridized with nick-translated TnI (cM113) (10) and MyoD1 (pEMC11s) (8) probes as described previously (11). TnI mRNA was detected within 24 h after ITS induction and reached maximal levels by 48 h (Fig. 1) (Fig. 2) showed no TnI mRNA (Fig. 2), no myosin heavy-chain protein, and no fusion (data not shown). Interestingly, MyoD1 mRNA levels also were reduced in growth-factortreated cultures so that by 48 h, MyoD1 mRNA was no longer detected. This growth-factor-induced inhibition of differentiation is reversible, however, since removal of FGF from inhibited cultures restored MyoD1 expression, increased TnI mRNA levels (Fig. 2), and led to a resumption of myoblast fusion. Similar results were obtained when TGF-, was removed, although the recovery time was delayed (data not shown). The growth-factor-induced repression of MyoDi mRNA appears to be specific, since the control pAL15 gene (2, 3) was expressed equally in all experimental groups. In addition. the effects of FGF and TGF-3 on myogenesis and MyoD1 expression are not unique to the 23A2 cell line. C2C12 myoblasts (4) behaved in an identical fashion when treated with these growth fac...

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Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS) is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR) has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS) failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-α, IL-1β and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has offered novel opportunities to cell biologists to study two mutually exclusive activation pathways of macrophages being mediated through a single receptor.

. Expression of the oncogenic form of H-ras p21 in the mouse myogenic cell line, 23A2, blocks myogenesis and inhibits expression of the myogenic regulatory factor gene, MyoDl. Previous studies from a number of laboratories have demonstrated that the activation of ras p21 is associated with changes in phospholipid metabolism that directly, or indirectly, lead to elevated levels of intracellular diacylglycerol and the subsequent activation of protein kinase C (PKC) . To assess the importance of PKC activity to the ras-induced inhibition of skeletal myogenesis, we examined the levels of PKC activity associated with the terminal differentiation of wild-type myoblasts and with the differentiation-defective phenotype of 23A2 ras cells. We demonstrate that there is a 50% reduction in PKC activity during normal myogenesis and that PKC activity T HE differentiation of skeletal muscle cells involves the withdrawal of proliferating myoblasts from the cell cycle, the formation of multinucleate myofibers and the transcriptional activation of a complete set of musclespecific genes (13) . Experiments with cultured myogenic cell lines have led to the identification of multiple genetic and environmental factors that influence the establishment and the proper differentiation of the myogenic lineage. The protein products of at least five genes, MyoDl, myd, myogenin, Myf-S, and MRF4, are important to the regulation of myogenic differentiation (5,11,38,40,51) . In addition, depletion ofserum growth factors from the culture medium is essential for myocyte fusion and for the coordinate expression of the contractile protein gene set (reviewed in reference 19), suggesting that growth factor signal transduction pathways control thesedevelopmental events. In this regard, several laboratories have shown that oncogenes such as ras, fos, and src inhibit skeletal muscle differentiation, presumably by interfering with the proper functioning of intracellular signalling pathways (see reference 1 for review ; 26,30,36,37) .The signal transduction pathways used by the various inducers and inhibitors of skeletal myogenesis remain unknown. Recently, our laboratory has shown that the differentiation of the mouse myogenic cell line, 23A2, is blocked © The Rockefeller University Press, 0021-9525/91/08/809/12 $2.00 TheJournal of Cell Biology, Volume 114, Number 4, August 1991809-820 is required for myoblast fusion, but not for the transcriptional activation of muscle-specific genes . In contrast, we found that the differentiation-defective 23A2 ras cells possess two-to threefold more PKC activity than wild-type myofibers and that reducing the PKC activity in these cultures does not reverse their nonmyogenic phenotype . On the other hand, if PKC activity is downregulated in 23A2 cells before the expression of activated ras p21, myogenesis is not inhibited. These results suggest that activated ras p21 relies on a PKC-dependent signal transduction pathway to initiate, but not to sustain, its negative effects on 23A2 skeletal myogenesis and undersc...

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An early and essential event in the protective immune response against most viruses and protozoa is the production of interferon-γ (IFN-γ). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-γ do not correlate with a protective response. We showed previously that African trypanosomes express a protein called T lymphocyte triggering factor (TLTF), which triggers CD8+ T lymphocytes to proliferate and to secrete IFN-γ. Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8+, but not CD4+, T cells to secrete IFN-γ. Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes. TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.

BackgroundLeishmania META1 has for long been a candidate molecule for involvement in virulence: META1 transcript and protein are up-regulated in metacyclic Leishmania. Yet, how META1 contributes to virulence remains unclear. We sought insights into the possible functions of META1 by studying its evolutionary origins.ResultsUsing multiple criteria including sequence similarity, nucleotide composition, phylogenetic analysis and selection pressure on gene sequence, we present evidence that META1 originated in trypanosomatids as a result of a lateral gene transfer of a bacterial heat-inducible protein, HslJ. Furthermore, within the Leishmania genome, META1 sequence is under negative selection pressure against change/substitution. Using homology modeling of Leishmania META1 based on solved NMR structure of HslJ, we show that META1 and HslJ share a similar structural fold. The best hit for other proteins with similar fold is MxiM, a protein involved in the type III secretion system in Shigella. The striking structural similarity shared by META1, HslJ and MxiM suggests a possibility of shared functions. Upon structural superposition with MxiM, we have observed a putative hydrophobic cavity in META1. Mutagenesis of select hydrophobic residues in this cavity affects the secretion of the secreted acid phosphatase (SAP), indicating META1's involvement in secretory processes in Leishmania.ConclusionsOverall, this work uses an evolutionary biology approach, 3D-modeling and site-directed mutagenesis to arrive at new insights into functions of Leishmania META1.

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