Identification of tgh-2, a filarial nematode homolog of Caenorhabditis elegans daf-7 and human transforming growth factor beta, expressed in microfilarial and adult stages of Brugia malayi.
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A novel member of the transforming growth factor beta (TGF-beta) family has been identified in the filarial nematode parasite Brugia malayi by searching the recently developed Expressed Sequence Tag (EST) database produced by the Filarial Genome Project. Designated tgh-2, this new gene shows most similarity to a key product regulating dauer larva formation in Caenorhabditis elegans (DAF-7) and to the human down-modulatory cytokine TGF-beta. Homology to DAF-7 extends throughout the length of the 349-amino-acid (aa) protein, which is divided into an N-terminal 237 aa, including a putative signal sequence, a 4-aa basic cleavage site, and a 108-aa C-terminal active domain. Similarity to human TGF-beta is restricted to the C-terminal domain, over which there is a 32% identity between TGH-2 and TGF-beta1, including every cysteine residue. Expression of tgh-2 mRNA has been measured over the filarial life cycle. It is maximal in the microfilarial stage, with lower levels of activity around the time of molting within the mammal, but continues to be expressed by mature adult male and female parasites. Expression in both the microfilaria, which is in a state of arrested development, and the adult, which is terminally differentiated, indicates that tgh-2 may play a role other than purely developmental. This is consistent with our observation that TGH-2 is secreted by adult worms in vitro. Recombinant TGH-2 expressed in baculovirus shows a low level of binding to TGF-beta-receptor bearing mink lung epithelial cells (MELCs), which is partially inhibited (16 to 39%) with human TGF-beta, and activates plasminogen activator inhibitor-1 transcription in MELCs, a marker for TGF-beta-mediated transduction. Further tests will be required to establish whether the major role of B. malayi TGH-2 (Bm-TGH-2) is to modulate the host immune response via the TGF-beta pathway. (+info)
Smad2 and Smad3 are downstream transforming growth factor-beta (TGF-beta) signaling molecules. Upon phosphorylation by its type I receptor, Smad2 or Smad3 forms a complex with Smad4 and translocates to the nucleus where the complex activates target gene transcription. In the present study, we report that Smad3 binds directly to the osteopontin (OPN) promoter and that Smad4 interacts with the Hox protein and displaces it from its cognate DNA binding site in response to TGF-beta stimulation. In gel shift assays, the glutathione S-transferase-Smad3 fusion protein was found to bind to a 50-base pair DNA element (-179 to -229) from the OPN promoter. Also, we found that both Hoxc-8 and Hoxa-9 bound to a Hox binding site adjacent to Smad3 binding sequence. Interestingly, Smad4, the common partner for both bone morphogenic protein and TGF-beta signaling pathways, inhibited the binding of Hox protein to DNA. FLAG-tagged Smad4 coimmunoprecipitated with HA-tagged Hoxa-9 from cotransfected COS-1 cells, demonstrating an interaction between Smad4 and Hoxa-9. Transfection studies showed that Hoxa-9 is a strong transcriptional repressor; it suppresses the transcription of the luciferase reporter gene driven by a 124-base pair OPN promoter fragment containing both Smad3 and Hox binding sites. Taken together, these data demonstrate a unique TGF-beta-induced transcription mechanism. Smad3 and Smad4 exhibit different functions in activation of OPN transcription. Smad3 binds directly to the OPN promoter as a sequence-specific activator, and Smad4 displaces the transcription repressor, Hoxa-9, by formation of Smad4/Hox complex as part of the transcription mechanism in response to TGF-beta stimulation. (+info)
Evaluation of R-Mix FreshCells in shell vials for detection of respiratory viruses.
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The performance of a mixture of mink lung and A549 cell lines in shell vials (MSVs) for the detection of respiratory viruses in 159 specimens was evaluated. MSVs, conventional culture, and direct immunofluorescence assay identified 96, 85, and 67% of the influenza A virus-positive specimens, respectively. MSVs provided both a high degree of sensitivity and rapid turnaround times for the detection of influenza A virus. (+info)
Common structure in panels of short ecological time-series.
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Typically, in many studies in ecology, epidemiology, biomedicine and others, we are confronted with panels of short time-series of which we are interested in obtaining a biologically meaningful grouping. Here, we propose a bootstrap approach to test whether the regression functions or the variances of the error terms in a family of stochastic regression models are the same. Our general setting includes panels of time-series models as a special case. We rigorously justify the use of the test by investigating its asymptotic properties, both theoretically and through simulations. The latter confirm that for finite sample size, bootstrap provides a better approximation than classical asymptotic theory. We then apply the proposed tests to the mink-muskrat data across 81 trapping regions in Canada. Ecologically interpretable groupings are obtained, which serve as a necessary first step before a fuller biological and statistical analysis of the food chain interaction. (+info)
Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer.
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Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbetaR-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbetaR-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta(1). These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect. (+info)
Inhibition of topoisomerase IIalpha expression by transforming growth factor-beta1 is abrogated by the papillomavirus E7 protein.
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Transforming growth factor-beta (TGF-beta) protects normal cells from etoposide-induced cell death, yet the mechanism has remained speculative. Studies have shown that etoposide modifies the activity of the topoisomerase IIalpha (topo IIalpha) enzyme, thereby causing DNA damage and inducing cell death. Expression of topo IIalpha is necessary for etoposide-induced cell death, and peak expression of topo IIalpha normally occurs during the G2 phase of the cell cycle. We predicted that by arresting growth in the G1 phase, TGF-beta1 would prevent the induction of topo IIalpha expression that normally occurs subsequent to the G1-S transition, thereby protecting cells from etoposide-induced cell death. Accordingly, we hypothesized that the inhibition of topo IIalpha expression by TGF-beta1 would be dependent on the ability of TGF-beta1 to arrest cell cycle progression in G1. Using mink lung epithelial cells (MvlLu), we found that TGF-beta1 decreases topo IIalpha mRNA expression, and the decrease occurs as cells begin to accumulate in the G1 phase of the cell cycle. Topo IIalpha protein expression decreases subsequent to the fall in mRNA expression. In contrast, topo IIalpha expression is not affected by TGF-beta1 in cells that fail to undergo G1 arrest because of inactivation of the retinoblastoma tumor suppressor protein (pRb) by the papillomavirus type 16 E7 protein. Our studies suggest that inhibition of topo IIalpha by TGF-beta1 is the principal mechanism that protects mink lung epithelial cells (Mv1Lu) from etoposide-induced toxicity. Furthermore, the inhibition of topo IIalpha protein expression by TGF-beta1 is dependent on pRb-mediated cell cycle arrest, suggesting that TGF-beta1 will not reduce the sensitivity of pRb-deficient cancers to etoposide. (+info)
Dynamics of connexin 43 levels and distribution in the mink (Mustela vison) anterior pituitary are associated with seasonal changes in anterior pituitary prolactin content.
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Because in mammals the anterior pituitary lacks innervation, we investigated whether gap junctions established between selected cells within the gland are part of an intrapituitary mechanism to ensure physiological synchronization of cells involved in the control of hormone secretion. We report here the dynamics of anterior pituitary connexin 43 (Cx43)-gap junctions throughout the mink (Mustela vison) annual reproductive cycle and its relationship with the anterior pituitary prolactin (PRL) content that parallels variations in serum PRL levels documented in the literature. We found that PRL anterior pituitary levels were maximal in spring and during lactation and that they were minimal in autumn and winter. Anterior pituitary Cx43 levels were maximal during periods of high PRL secretion. During these periods, Cx43-positive gap junctions localized to stellate-shaped cells occupying the center of anterior pituitary follicles and to the rounded cells occupying the remaining follicles. Connexin 43-positive gap junctions were also observed between adjacent follicles. During periods of low PRL pituitary content, Cx43-positive gap junctions localized to the stellate cells but not to the cells of the remaining follicles. Moreover, Cx43 labeling was undetected between adjacent follicles. To assess between which cells within the mink anterior pituitary the Cx43 gap junctions were established, the different anterior pituitary cell populations were separated by a discontinuous Percoll gradient, and Western blot analyses of each cell population using Cx43 antibodies were performed. The immunoblots showed a Cx43 immunoreactive band associated with the cell layer enriched in S-100-positive, stellate-shaped cells. The result was confirmed by fluorescence microscopy studies that showed that Cx43-mediated gap junctions were established preferentially between the cultured S-100-positive, elongated cells. The results show that in mink stellate cells, the junctional machinery associated with the Cx43 protein varies in synchrony with the anterior pituitary PRL content throughout the mink annual reproductive cycle. It is suggested that the Cx43 gap junctions on the stellate cells play an important role in the synchronization of cellular activity within selected follicles of the anterior pituitary, thus contributing to the control of PRL secretion during the annual reproductive cycle. (+info)
Transforming growth factor-beta1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism.
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Transforming growth factor-beta1 (TGF-beta) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-beta are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-beta-induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-beta do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-beta rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160(ROCK), by the expression of dominant-negative mutants, inhibited TGF-beta-mediated EMT. The data suggest that TGF-beta rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics. (+info)