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Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-[beta] (TGF-[beta]) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-[beta] and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-[beta] signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-[beta] and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-[beta] signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-[beta] type I receptor (T[beta]R-I) more efficiently, and were more potent in repressing TGF-[beta] signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-[beta] receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-[beta] signaling.

Research was conducted to illustrate how the nuclear translocation of p42 and p44 mitogen-activated protein kinases (MAPK) can be triggered via the activation of the p42/p44 MAPK pathway. Nuclear accumulation was observed to be facilitated by the use of the chimera Raf-1:ER to directly activate the pathway. The neosynthesis of short-lived proteins are required for the nuclear accumulation of p42/p44 MAPK.

A study was conducted to demonstrate the proteolytic regulation of polypeptides intracellularly secreted by the bacterial pathogen Listeria monocytogenes. Three forms of polypeptides, which mediate bacterial cell-to-cell spread, were identified. These include two phospholipases of the C class (PLC), namely, phosphatidylinositol-specific PLC (PI-PLC) and broad spectrum PLC (PC-PLC). The third polypeptide form is a metalloprotease, which proteolytically activates PC-PLC as an inactive precursor (proPC-PLC).

Wnt family members are involved in the modulation of cell-cell interactions during the morphogenic movements required for the development of various tissues. Wnt-1 signalling is facilitated by its own cell surface receptor such as the hepatocyte growth factor and activin. The presence of an active receptor and signaling mechanism in the C57MG and AtT20 cells is confirmed by their response to Wnt-1 expression.

Neurons lacking nerve growth factor experience fragmentation of DNA, condensation of chromatin and membrane blebbing. Detection of DNA fragmentation by ethidium bromide staining of DNA is facilitated by the large number of cells that undergo fragmentation. Plasma membrane breakdown occurs while the neurons go through the entire apoptotic program.

Analysis of neurite formation in disintegrated neurons of cultured chick dorsal root ganglion elucidates the role of growth-associated protein- (GAP) -43 in neurite outgrowth, development and spreading of growth cones. GAP-43 is a possible candidate for effecting nodulation of axonal cortex and growth cone activity. This nodulation renders transduction of external signal into growth cones, and elicits its response in initiating neurite formation and guidance.

RNase protection studies and in situ hybridization reveal that scatter factor/hepatocyte growth factor (SF/HGF) and its receptor, c-met tyrosine kinase, influence epithelia and mesenchyme during growth of mouse. The expression of the genes encoding the c-met receptor tyrosine kinase and its particular ligand, SF/HGF, is examined during mouse embryogenesis, which indicates a paracrine manner of actions for the ligand and its receptor during the growth of epithelial organs.

The existence of the NRK fibroblast phenotype shows that the growth factor and adhesion/TGF-beta 1 requirements for cell cycle progression are genetically separable. The two major control points in the fibroblast cell cycle (G0/G1 and G1/S) are governed by distinct extracellular signals. The genes monitoring anchorage-independent growth are not essentially responsible for regulation of contact inhibition, focus formation or growth factor dependence. The synchronization of NRK ad NIH-3T3 fibroblasts immediately after an attachment-dependent transition governing anchorage-independent growth revealed that S-, G-2 and M-phase progression continue in the absence of attachment.

Epidermal homeostasis is partly governed by complex, reciprocally-induced paracrine acting factors and the interactions between cells and extracellular matrix impact. The impact of human dermal microvascular endothelial cells (DMEC) and fibroblasts on keratinocytes in conventional and organotypic cocultures when analysed showed that clonal keratinocyte growth was similarly stimulated in cocultures with irradiated DMEC and fibroblasts as feeder cells. In organotypic cocultures with keratinocytes developing on collagen cells, a normal epidermis was seen within 7 to 10 days, and keratinocyte growth was stimulated by dermal cells which also grew in the gel.

Immunohistochemical techniques were usedto determine whether the growth-associated protein GAP-43 has other functions aside from its role in axon motility and elongation. It was observed that GAP-43 is expressed in Schwann cell (SC) precursors and in nonmyelin-forming SCs in the mature peripheral nervous system. It was definitely established thatSCs deprived of axonal contact can synthesize GAP-43 in vitro. Whether GAP-43 is expressed in all nonmyelin-forming SCs is unknown, but it is suspected to have a role in cell membrane changes associated with cell-shape flexibility.

The G protein-coupled thrombin receptor can induce cellular responses in some systems by transactivating the epidermal growth factor (EGF) receptor. This is in part due to the stimulation of ectoproteases that generate EGF receptor ligands. We show here that this cannot account for the stimulation of proliferation or migration by thrombin of Swiss 3T3 cells. Thrombin has no direct effect on the activation state of the EGF receptor or of its downstream effectors. However, thrombin induces the subcellular clustering of the EGF receptor at filamentous actin-containing structures at the leading edge and actin arcs of migrating cells in association with other signaling molecules, including Shc and phospholipase C[Gamma]1. In these thrombin-primed cells, the subsequent migratory response to EGF is potentiated. Thrombin did not potentiate the EGF-stimulated EGF receptor phosphorylation. Thus, in Swiss 3T3 cells the G protein-coupled thrombin receptor can potentiate the EGF tyrosine kinase receptor response when activated by EGF, and this appears to be due to the subcellular concentration of the receptor with downstream effectors and not to the overall ability of EGF to induce receptor transphosphorylation. Thus, the EGF receptor subcellular localization which is altered by thrombin appears to be an important determinant of the efficacy of downstream EGF receptor signaling in cell migration. Key words: costimulation * rafts * G protein * actin arc * crosstalk

During differentiation, skeletal muscle cells withdraw from the cell cycle and fuse into multinucleated myotubes. Unlike quiescent cells, however, these cells cannot be induced to reenter S phase by means of growth factor stimulation. The studies reported here document that both the retinoblastoma protein (Rb) and the cyclin-dependent kinase (cdk) inhibitor p21 contribute to this unresponsiveness. We show that the inactivation of Rb and p21 through the binding of the adenovirus E1A protein leads to the induction of DNA replication in differentiated muscle cells. Moreover, inactivation of p21 by E1A results in the restoration of cyclin E--cdk2 activity, a kinase made nonfunctional by the binding of p21 and whose protein levels in differentiated muscle cells is relatively low in amount. We also show that restoration of kinase activity leads to the phosphorylation of Rb but that this in itself is not sufficient for allowing differentiated muscle cells to reenter the cell cycle. All the results obtained are consistent with the fact that Rb is functioning downstream of p21 and that the activities of these two proteins may be linked in sustaining the postmitotic state. Key words: C2C12 cells * E1A * cell cycle * p21 * DNA replication