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PubMed1979

Non-random distribution of transduction termini in transductants from the integrated R plasmid, R100-1.

Tra+ and tra- derivatives of drug resistance plasmid, R100-1, were isolated by phage P1 from an Hfr donor with integrated R100-1 and then analyzed by complementation tests with tra- point mutants of Flac. Tra+ derivatives of R100-1 carrying tetracycline resistance alone and those carrying all six drug-resistrance genes could support transfer of tra- point mutants of Flac except Flac traJ, whereas all of tra- derivatives of R100-1 failed to complement any one of tra- point mutants of Flac. This suggests that these tra- derivatives of R100-1 carrying tetracycline resistance gene are deleted for all the transfer genes impaired in the Flac point mutants tested. We assume a "hot point", probably a specific base sequence similar to an IS element, at the left of the tetracycline gene (Fig. 1) becomes a transduction terminus in transduction of the integrated R100-1 by phage P1. Complementation analysis of tra- derivatives carrying five resistance genes except the tetracycline gene led us to a supposition that a gene(s), probably analogous to traJ of the F plasmid, is located on R100-1 near the tetracycline gene which plays an important regulatory role for self-transfer as well as for the complementation of tra- Flac mutants.

Base Sequence↗
PubMed2001

Transforming growth factor beta signal transduction in hepatic stellate cells via Smad2/3 phosphorylation, a pathway that is abrogated during in vitro progression to myofibroblasts. TGFbeta signal transduction during transdifferentiation of hepatic stellate cells.

To current knowledge, transforming growth factor beta (TGFbeta) signaling is mandatory to establish liver fibrosis and various molecular interventions designed to affect the TGFbeta system were successfully used to inhibit fibrogenesis. Activated hepatic stellate cells (HSC), which are one important source of TGFbeta, are the major producers of extracellular matrix proteins in liver injury. We have previously shown that the TGFbeta response of this cell type is modulated during the transdifferentiation process. This work delineates the activation of TGFbeta downstream mediators, the Smads, in quiescent HSC and transdifferentiated myofibroblasts (MFB). The expression level of all Smads remained largely unchanged during this process. The response of HSC to TGFbeta, leading to, e.g., induction of alpha2 (I) collagen expression, is mediated by phosphorylation of Smad2 and Smad3 and subsequent nuclear translocation of a Smad containing complex. Neither TGFbeta-dependent nor endogenously phosphorylated Smad2/3 was detectable in comparable amounts in transdifferentiated MFB, indicating loss of TGFbeta sensitivity. Ectopic expression of Smad7 in HSC led to inhibition of Smad2 phosphorylation and abrogated TGFbeta response. In transdifferentiated MFB, expression of a constitutively active TGFbeta receptor I, but not treatment with TGFbeta1, resulted in transcriptional activation of a TGFbeta responsive promoter, thereby demonstrating completely restored TGFbeta signal transduction. Our data indicate that in contrast to a postulated mechanism of enduring autocrine TGFbeta signal transduction, early and late stages of HSC activation have to be distinguished, which is of importance for antifibrotic therapies.

3T3 Cells↗
PubMed2003

Efficient transduction of murine primary T cells requires a combination of high viral titer, preferred tropism, and proper timing of transduction.

Retroviral vectors have been used exclusively for genetic modification of primary T cells. Most T cell infection protocols have been developed for human T cells, whereas systematic investigations of the optimal conditions for transduction of murine primary T cells are limited. In this study, ecotropic and 10A1-pseudotyped retroviral vectors were compared for their efficiency in infecting murine primary T cell cells, as well as T cell lines. Various factors that affect transduction efficiency were also explored, including virus titer, times of exposure, timing of infection, low-speed centrifugation, and use of fibronectin fragment. Our results showed that up to 80% of murine primary T cells could be infected after a single exposure. Successful infection required a combination of high virus titer (>10(7) CFU/ml), proper timing of infection (within 24 h after mitogen stimulation), and preferred tropism (ecotropic vectors). These optimization results may help to establish a standard protocol for infection of murine primary T cells and provide some insight into the obstacles to retroviral infection of T cells.

Animals↗
PubMed2001

Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo.

The protein transduction domain (PTD) embedded in the HIV TAT protein (amino acids 47-57) has been shown to successfully mediate the introduction of heterologous peptides and proteins in excess of Mr 100,000 into mammalian cells in vitro and in vivo. We report here that the modeled structure of the TAT PTD is a strong amphipathic helix. On the basis of this information, we synthesized a series of synthetic PTDs that strengthen the alpha-helical content and optimize the placement of arginine residues. Several PTD peptides possessed significantly enhanced protein transduction potential compared with TAT in vitro and in vivo. These optimized PTDs have the potential to deliver both existing and novel anticancer therapeutics.

Amino Acid Sequence↗
PubMed1977

The frequency of P1 transduction of the genes of Escherichia coli as a function of chromosomal position: preferential transduction of the origin of replication.

The frequencies with which the generalized transducing phage P1 transduced 26 selected markers on the E. coli. chromosome were measured. The frequencies were found to vary relative to argH+ = 1 from a maximum of 6.8 near the origin of replication to a minimum of 0.23 for a marker not far from the terminus. The low frequencies obtained for some markers were shown not to result from poor expression under the selective conditions employed. When plotted as a function of marker position on the chromosome the frequencies were found to exhibit a series of peaks and troughs which correspond to those in gene density noted by Bachmann et al. (1976). The possible relationship of these results to the structure of the E. coli chromosome and to the mechanism of generalized transduction are discussed.

Chromosome Mapping↗
PubMed1998

Heterologous desensitization in neoplastic thyroid cells: influence of the phospholipase C signal transduction system on the thyrotropin-adenylate cyclase signal transduction system.

Desensitization is defined as a decreased functional response after continuous or repetitive stimulation of a receptor with its agonist. Thyrotropin (TSH) increases cAMP levels in normal and neoplastic thyroid tissue. The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) activates protein kinase C (PKC). The aim was to determine whether TPA induces heterologous desensitization of the TSH-adenylate cyclase (AC) signal transduction system. Three human thyroid neoplasms in culture for 6 months or longer (one papillary carcinoma, one Hurthle cell carcinoma, one follicular adenoma) were incubated with TSH (10 mU/ml) and TPA (1.6 x 10(-8) M) separately and together for various time periods (from 10 minutes to 24 hours). The mixture was subsequently incubated for 30 minutes with TSH. TPA alone had no effect on cAMP levels, but co-incubation of TPA and TSH caused a significant reduction in cAMP response when compared to the cAMP response that resulted after stimulation with only TSH (p < 0.001). cAMP levels in response to TSH decreased by 31%, 44%, and 57% after preincubation with TSH for 10 minutes, 4 hours, and 24 hours, respectively (p < 0.01; ANOVA). Co-incubation of cells with TPA and staurosporine (10 ng/ml), a PKC inhibitor, prevented the effect of TPA on desensitization at 10 minutes and blunted the effect at 4 hours. This is the first demonstration in human neoplastic thyroid cells that TPA induced heterologous desensitization of the cAMP response to TSH. This TPA-induced effect appears to involve PKC activation, as it can be blocked by staurosporine.

Adenylyl Cyclases↗
PubMed2003

Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages.

Animals↗
PubMed1995

Interleukin-13 signal transduction in lymphohemopoietic cells. Similarities and differences in signal transduction with interleukin-4 and insulin.

Interleukin-13 (IL-13) and interleukin-4 (IL-4) are related in structure and function and are thought to share a common receptor component. We have investigated the signal transduction pathways activated by these two growth factors, as well as insulin, in cell-lines and primary cells of lymphohemopoietic origin. All three factors induced the tyrosine phosphorylation of a protein of 170 kDa (p170), which coimmunoprecipitated with the p85 subunit of P13'-kinase, via high affinity interactions mediated by the SH2 domains of p85. Antibodies raised against the entire insulin-receptor substrate-1 (IRS-1) protein immunoprecipitated p170 much less efficiently than they did IRS-1 from 3T3 cells. However, antibodies directed against the conserved pleckstrin homology domain of IRS-1 immunoprecipitated both p170 and IRS-1 with similar efficiency, suggesting they share structural similarities in this region. In lymphohemopoietic cells, IL-13, IL-4, and insulin failed to induce increased tyrosine phosphorylation of Shc, or its association with grb2, modification of Sos1, or activation of erk-1 and erk-2 mitogen-activated protein kinases, suggesting that p170 mediates downstream pathways distinct from those mediated by IRS-1. Both IL-13 and IL-4 induced low levels of tyrosine phosphorylation of Tyk-2 and Jak-1. IL-4 also activated the Jak-3-kinase, but, despite other similarities, IL-13 did not. Insulin failed to activate any of the known members of the Janus family of kinases. In that Jak-3 is reported to associate with the IL-2 gamma c chain, these data suggest that the IL-13 receptor does not utilize this subunit. However, both IL-13 and IL-4 induced tyrosine phosphorylation of the IL-4-140 kDa receptor chain, suggesting that this is a component of both receptors in these cells and accounts for the similarities in signaling pathways shared by IL-13 and IL-4.

3T3 Cells↗
PubMed1999

Association of the Ras to mitogen-activated protein kinase signal transduction pathway with microfilaments. Evidence for a p185(neu)-containing cell surface signal transduction particle linking the mitogenic pathway to a membrane-microfilament association site.

Microvilli of the aggressive 13762 ascites mammary adenocarcinoma contain a large, microfilament-associated signal transduction particle whose scaffolding is a stable glycoprotein complex (Li, Y., Hua, F., Carraway, K. L., and Carraway, C. A. C. (1999) J. Biol. Chem. 274, 25651-25658) associated with the growth factor receptor p185(neu). The receptor is constitutively tyrosine-phosphorylated in the cells and microvilli, predicting that it should recruit mitogenic pathway components to this membrane-microfilament interaction site. Immunoprecipitation of cell lysates with anti-phosphotyrosine and immunoblotting showed phosphorylated forms of the mitogenic pathway proteins Shc and MAPK in addition to p185(neu), suggesting that the Ras to MAPK mitogenic pathway is activated. Immunoblotting of p185(neu)-containing microvillar fractions revealed the presence in each of stably associated Shc, Grb-2, Sos, Ras, Raf, mitogen-activated protein kinase kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase, as well as the transcription factor-phosphorylating kinase Rsk. All of these pathway components co-immunoprecipitated with p185(neu) from cleared lysates of microvilli solubilized under microfilament-depolymerizing conditions. The recruitment of constitutively phosphorylated p185(neu) and the activated mitogenic pathway proteins to this membrane-microfilament interaction site provides a physical model for integrating the assembly of the mitogenic pathway with the transmission of growth factor signal to the cytoskeleton. This linkage is probably a requisite step in the global cytoskeleton remodeling accompanying mitogenesis.

Actin Cytoskeleton↗
PubMed2000

Fas- and tumor necrosis factor-mediated apoptosis uses the same binding surface of FADD to trigger signal transduction. A typical model for convergent signal transduction.

FADD is known to function as a common signaling conduit in Fas- and tumor necrosis factor (TNF)-mediated apoptosis. The convergent death signals from the Fas receptor and TNF receptor 1 are transferred to FADD by death domain interactions triggering the same cellular event, caspase-8 activation. In this work, we investigated whether the same binding surface of FADD is used for both signaling pathways by using FADD death domain mutants. Mutations in helices alpha2 and alpha3 of the FADD death domain, the interacting surface with the Fas death domain, affected TNF-mediated apoptosis to various extents. This indicated that TNF-mediated apoptosis uses the same binding surface of the FADD death domain as Fas-mediated apoptosis. The binding specificity is not the same, however. Some mutations affected the binding affinity of the Fas death domain for the FADD death domain, but did not influence TNF-mediated apoptosis and vice versa. Interestingly, all mutants tested that affected TNF-mediated apoptosis have structural perturbations, implying that the structural integrity, involving helices alpha2 and alpha3 in particular, is critical in TNF-mediated apoptosis. Our results suggest that different signaling molecules use a similar structural interaction to trigger the same cellular event, such as caspase-8 recruitment, which could be typical in convergent signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed2001

The TRANSPATH signal transduction database: a knowledge base on signal transduction networks.

UNLABELLED: TRANSPATH is an information system on gene-regulatory pathways, and an extension module to the TRANSFAC database system (Wingender et al., Nucleic Acids Res., 28, 316-319, 2000). It focuses on pathways involved in the regulation of transcription factors in different species, mainly human, mouse and rat. Elements of the relevant signal transduction pathways like complexes, signaling molecules, and their states are stored together with information about their interaction in an object-oriented database. The database interface provides clickable maps and automatically generated pathway cascades as additional ways to explore the data. All information is validated with references to the original publications. Also, references to other databases are provided (TRANSFAC, SWISS-PROT, EMBL, PubMed and others). AVAILABILITY: The database is available over (http://transpath.gbf.de) for interactive perusal. As an exchange format for the data, eXtensible Markup Language (XML) flatfiles and a Document Type Definition (DTD) are provided.

Algorithms↗
PubMed1997

Drosophila Jun relays the Jun amino-terminal kinase signal transduction pathway to the Decapentaplegic signal transduction pathway in regulating epithelial cell sheet movement.

We have characterized mutations in the Drosophila homolog of the mammalian proto-oncogene c-Jun gene (Djun). We demonstrate that DJUN in the embryo is a downstream target of the JNK signal transduction pathway during dorsal closure formation, and that the function of the JNK/DJUN pathway is to control the localized expression of decapentalegic (dpp), a member of the TGF-beta growth factor family. In contrast to previous observations, we find that both in the embryo and during photoreceptor cell determination, DJUN is not regulated by a pathway that involves MAPK.

Animals↗