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M Stopa

Publications and source records attributed to M Stopa.

17 recordsLinked to original sources
PubMed2000

Participation of Smad2, Smad3, and Smad4 in transforming growth factor beta (TGF-beta)-induced activation of Smad7. THE TGF-beta response element of the promoter requires functional Smad binding element and E-box sequences for transcriptional regulation.

Smad7 has recently been identified as a player that antagonizes transforming growth factor beta (TGF-beta) signals by acting downstream of TGF-beta receptors. TGF-beta rapidly induces expression of Smad7 mRNA in a variety of cell types, suggesting participation in a negative feedback loop to control TGF-beta responses. We have previously described the genomic locus of rat Smad7 including the promoter region. Here we report polymerase chain reaction cloning of the corresponding promoter regions of human and murine Smad7 genes and functional characterization of the rat Smad7 promoter. Using transient transfection experiments of HepG2 cells, we identified the TGF-beta response element within a strongly conserved region, containing a perfect Smad binding element (SBE; GTCTAGAC). Performing electrophoretic mobility shift assay and cotransfection experiments, we were able to delineate DNA-binding complexes and identified Smad3, Smad4, and Smad2. Mutation of the SBE completely abolished TGF-beta inducibility of Smad7 in HepG2 cells, indicating that this sequence is necessary for TGF-beta-induced transcription. Furthermore, a 3-base pair adjacent E-box is additionally essential for TGF-beta-dependent promoter activation and an overlapping AP1 site is also involved. We conclude that regulation of Smad7 transcription by TGF-beta is mediated via a specific constellation of recognition motifs localized around the SBE, which is conserved in human, rat, and murine genes.

Animals↗
PubMed2000

Genomic locus and promoter region of rat Smad7, an important antagonist of TGFbeta signaling.

SMAD proteins are essential components of the intracellular signaling pathways utilized by members of the transforming growth factor beta (TGFbeta) superfamily of growth factors. Certain SMAD proteins (Smad1, 2, 3, and 5) can act as regulated transcriptional activators. This process involves phosphorylation of these proteins by activated TGFbeta receptors. Recently, Smad6 and Smad7 were identified; they antagonize TGFbeta signaling by preventing the activation of signal-transducing SMAD complexes. TGFbeta rapidly induces the expression of Smad7 mRNA, suggesting participation of Smad7 in a negative feedback loop to control TGFbeta responses. Similarly, epidermal growth factor (EGF) and interferon gamma (IFN-gamma) have been reported to induce Smad7 expression. In a rat model system of liver fibrosis, TGFbeta inducibility of Smad7 is abrogated during transformation of hepatic stellate cells (HSC), indicating an important switch in transcriptional regulation of the gene. With the detailed characterization of the rat Smad7 genomic organization including the promoter region, we present the first identified Smad7 gene so far. The gene is composed of four exons separated by three introns covering a DNA region of about 30 kilobases (kb) in total. The major transcription start site is conserved between rat and mouse, and two polyadenylation signals were detected. In the promoter region, a potential CAGA box, a signal transducer and activator of transcription (STAT) factor-related recognition site, and different AP1 sites were identified, which could be the targets of TGFbeta, IFN-gamma, and EGF-dependent Smad7 transcription initiation.

Animals↗
PubMed1998

Myoid cells and neuroendocrine markers in myasthenic thymuses.

We have studied myoid cells in normal and myasthenic thymuses as well as in thymomas. For the presence of neuroendocrine markers-producing cells and identification of synaptophysin (Syn) the immunohistochemical method and immunoblot analysis were used. Myoid cells can be demonstrated in the thymus of myasthenic patients in high number. These cells occur in the vicinity of Hassall's bodies but also within them. Some regenerated Hassall's bodies displayed majority of myoid cells with their concentric arrangement around the centrally situated lacunar-like cell with nuclei of monocytogenic origin. Such phenomenon may suggest cooperation of myoid cells and their epithelial transitional forms with monocytogenic cells in various thymic hormone production. It is likely that myoid cells are the source of some thymic epithelial cells. According to some authors, thymomatous epithelial cells and skeletal muscle share a common epitope defined by a monoclonal antibody (mAb), whereas thymic epithelial cells possess acetylocholine receptor (AChR) on their surface. The epithelial cells of some thymomas express also desmin. In normal thymuses of children, Syn and chromogranin A (Chg A) were demonstrated in some cells of Hassall's bodies by immunohistochemical method. In addition, antibodies to Syn stained nerve structures surrounding the thymic blood vessels. In myasthenic thymuses, Syn expression was in cortical and medullary epithelial cells, in myoid cells and only scanty and focal in keratinized epithelial cells of Hassall's bodies. The epithelial cells of some thymomas also express Syn. In some thymuses of all groups investigated in this study Chg A was seen in single cells of Hassall's bodies and focally in cortical epithelial cells. Our results show that in normal thymuses of cardiac surgery patients and in the adult myasthenic thymuses antibody raised against Syn recognized protein with molecular weight of 48,000 but not normal (38,000) Syn. It remains to be elucidated if the overexpression of synaptophysin-like protein in myasthenic thymuses is a compensatory phenomenon to the defect in normal synaptic function.

Adult↗
PubMed1997

Effect of sphingomyelin and its metabolites on the activity of human recombinant PLC delta 1.

In an attempt to obtain sufficient quantities of pure phospholipase C delta 1 (PLC delta 1) necessary for structural and kinetic studies, human fibroblast PLC delta 1 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6xHis) fusion protein and purified to homogeneity. From 11 of E. coli culture 21 mg of pure PLC delta 1 was obtained by a two-step purification procedure, which includes Ni(2+)-NAT agarose and Mono S cation exchange chromatography. Catalytic properties of recombinant PLC delta 1 with respect to activation by spermine and calcium ions and inhibition by sphingomyelin were similar to or identical to PLC delta 1 purified from rat liver. Calcium activation of PLC delta 1 was dependent on the presence of spermine. Half-maximal activity was attained at 250 and 170 nM of free Ca2+ in the presence and absence of spermine, respectively. Sphingomyelin and lysosphingomyelin were mixed type inhibitors with respect to PIP2. Ceramide inhibits PLC delta 1 very weakly. GM1, which is a ceramide bound glucosidically to the oligosaccharide moiety, was a strong non-competitive inhibitor of PLC delta 1. In the absence of spermine, sphingosine and phytosphingosine weakly activated PLC delta 1. The results indicate that the effect of sphingomyelin and its metabolites on PLC delta 1 activity depends on the presence of spermine. It is postulated that, among other factors, in vivo, activity of PLC delta 1 may depend on the turnover of sphingomyelin.

Animals↗
PubMed1992

[Mechanical properties of tissue generated as a result of fascia lata grafted into an articular cartilage defect of the knee joint (experimental observations)].

In 64 sheep knees crushing strength and elastic recovery of the tissue generated on the basis of fascia lata grafted into articular cartilage defect in the knee after active motion and load has been assessed. The newly generated tissue is characterized by increasing in time thickness, deformability and elastic recovery the greater the deeper the defect was. Primarily, the new tissue is able to carry smaller loads but after 26 weeks is capable of carrying much greater loads then the cartilage; it has also better plastic and elastic properties.

Animals↗