To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative for primary open angle glaucoma (POAG), the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON and tTG-ASDON2 were significantly decreased as compared with that of the controls (P < 0.05). On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.
Cells, Cultured ; Glaucoma, Open-Angle/metabolism ; Oligonucleotides, Antisense/*pharmacology ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Trabecular Meshwork/cytology ; Trabecular Meshwork/*metabolism ; Transglutaminases/*biosynthesis ; Transglutaminases/genetics ; Transglutaminases/*pharmacology

To study whether cultured bovine trabecluar meshwork cells (BTMC) are capable of expressing tTG in protein and at mRNA level, BTMC were cultured in vitro and passaged three times, then the cells were transferred onto or cultured on sterile cover or submitted to isolation of RNA with Trizol, and the expression of tTG was detected by immunohistochemical technique and reverse transcription polymerase chain reaction (RT-PCR) respectively. Our results showed that tTG immunostaining was positive in the cytoplasm and rarely in the nucleus of cultured BTMC. No immunostaining was seen in the negative control. Moreover, a single RT-PCR amplified product whose sequence and size were in accordance with our known parameters was obtained. The expression of tTG in cultured BTMC was confirmed in protein and at mRNA level. BMTC is available more readily for the investigation of the relationship between tTG and primary open-angle glaucoma.
Cells, Cultured ; Glaucoma, Open-Angle/metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Trabecular Meshwork/*metabolism ; Transglutaminases/*biosynthesis ; Transglutaminases/genetics

Transglutaminase (TG) is a kind of calcium-dependent enzymes. The TGase family found in rodents and human contains 9 types, including TG1-7, blood coagulation factor XIIIa and erythrocyte membrane protein 4.2, with the former 8 types possessing catalytic activity. TG catalyzes various conversion reactions of glutamine, including transamination, deamination and esterification, and participates in post-transcriptional modification of proteins such as cross-linking peptides glutamine residue and lysyl-residue, stabilizing protein structure and catalyzing formation of protein aggregates. TGase has been found to contribute to a variety of important physiological and pathological processes and play a role in the pathogenesis of multiple diseases. Notably, neurodegenerative diseases such as Huntington's disease, spinocerebellar ataxia, Alzheimer's disease and Parkinson's disease, have a close connection with TGase's role in the human body.
Animals ; Brain ; enzymology ; Humans ; Neurodegenerative Diseases ; enzymology ; genetics ; Transglutaminases ; genetics ; metabolism

OBJECTIVETo assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes.

METHODSHepatocytes isolated from tissue transglutaminase gene knock-out mice and rats were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by (14)C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.

RESULTSTTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.

CONCLUSIONSIncreased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.

Animals ; Apoptosis ; Cell Nucleus ; metabolism ; Cytochrome c Group ; metabolism ; Hepatocytes ; cytology ; metabolism ; Male ; Mice ; Rats ; Signal Transduction ; Transglutaminases ; metabolism

OBJECTIVE: To investigate the expression of TGM3 in laryngeal carcinoma and analyze the relation of TGM3 and clinical factors. METHOD: We measured the expression of TGM3 protein in 50 laryngeal carcinoma fresh samples and 50 para-carcinoma tissues (to cutting margin > 0.5 cm) by Flow Cytometry (Epics-XL II), 20 normal laryngeal mucosa samples were also studied as controls. RESULT: 1) The quantitative and qualitative expression of TGM3 protein in laryngeal carcinoma tissues is obviously lower than those in para-carcinoma and normal laryngeal mucosa tissues respectively (P<0.05). There is no significant expression difference between the para-carcinoma and normal laryngeal mucosa tissues. 2) In laryngeal carcinoma, the expression of TGM3 protein isn't significantly related to patients' clinical classification, tumor size, patients' age and gender but related to lymphaden metastasis. differentiation and clinical stage. CONCLUSION The low expression of TGM3 may contribute to the carcinogenesis and development of laryngeal carcinoma. It is an important index of judging differentiation, infiltration, metastasis and staging of laryngeal carcinoma by measure the expression of TGM3 protein.
Adult ; Aged ; Female ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Staging ; Transglutaminases ; metabolism

The aberrant protein crosslinks formation during lung injury as results total body irradiation (TBI) and bone marrow transplantation (BMT) therapy has been examined as apossible contributory factor in organ or tissue pathogenesis. Female C3HeB/ FeJ mice were used for an experimental animal. Carbon monoxide uptake (V(CO)) was measured at 1, 2, 3, 4 and 5 months after TBI at respective doses of 12, 14, 16 and 18 Gy 16 h prior to syngeneic BMT. Also as a measure of aberrant protein crosslinking in the inured tissues, transglutaminase (TGase)-activities and crosslinked protein were examined along with thrombin, a protease known to activate TGases. Reductions of VCO were detected following TBI and BMT. Activities of thrombin and TGase 1, and crosslinked protein in bronchoalveolar lavage (BAL) fluid of the mice 1 wk after TBI at 12 Gy and BMT were identified and found to be elevated in the treated animals. These findings suggest that elevated levels of crosslinked proteins and TGase I in the bronchoalveolar larvage during the lung injury could have enhanced the organ pathogenesis following TBI and BMT.
Animals ; *Bone Marrow Transplantation ; Carbon Monoxide/metabolism ; Factor XIII/metabolism ; Female ; Lung/*metabolism/pathology/radiation effects ; *Lung Injury ; Mice ; Proteins/*metabolism ; Thrombin/metabolism ; Transglutaminases/metabolism ; *Whole-Body Irradiation

Direct secretory expression of active microbial transglutaminase (MTG) using heterologous hosts is a promising strategy, although its production level still needs to be improved for industrial production. Pichia pastoris is one of the most efficient expression systems developed in recent years. In this study, secretory expression of active MTG was successfully achieved by co-expressing the pro sequence and mature MTG genes in P. pastoris. Furthermore, we optimized the copy number of pro/MTG expression cassettes and the fermentation conditions. MTG production level reached 7.3 U/mL in 1-liter fermentor through high density fermentation, providing the feasiblity for industrial scale preparation of MTG.
Fermentation ; Genetic Vectors ; genetics ; Pichia ; enzymology ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Streptomyces ; enzymology ; Transglutaminases ; biosynthesis ; genetics

Transglutaminase enzymes (TGases) catalyze the calcium dependent formation of an isopeptide bond between protein-bound glutamine and lysine substrates. Previously we have shown that activated TGase 3 acquires two additional calcium ions at site two and three. The calcium ion at site three results in the opening of a channel. At this site, the channel opening and closing could modulate, depending on which metal is bound. Here we propose that the front of the channel could be used by the two substrates for enzyme reaction. We propose that the glutamine substrate is directed from Trp236 into the enzyme, shown by molecular docking. Then a lysine substrate approaches the opened active site to engage Trp327, leading to formation of the isopeptide bond. Further, direct comparisons of the structures of TGase 3 with other TGases have allowed us to identify several residues that might potentially be involved in generic and specific recognition of the glutamine and lysine substrates.
Animals ; Binding Sites ; Calcium/metabolism ; Calcium-Binding Proteins/*metabolism ; Glutamine/metabolism ; Human ; Lysine/metabolism ; Models, Chemical ; Models, Molecular ; Protein Binding ; Protein Structure, Tertiary ; Transglutaminases/*metabolism

The microbial transglutamunase (MTG) gene was amplified from the genomic DNA of Streptoverticillium mobaraensea by using PCR and inserted into pET vector to construct the expression plasmid called pET-MTG. The pET-MTG was transfected into E. coli (Rosetta DE3) and the MTG protein was found to be highly expressed as inclusion bodies. The inclusion bodies were isolated and subjected to denaturation and re-naturation, followed by strong cation ion-exchange chromatography to purify the expressed MTG. The specific activity of purified MTG was close to that of native MTG. Taken together, this study might provide a base for the industrial production of microbial transglutaminase.
Bacterial Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genes, Bacterial ; Inclusion Bodies ; enzymology ; Recombinant Proteins ; genetics ; metabolism ; Streptomycetaceae ; enzymology ; genetics ; Transglutaminases ; genetics ; metabolism

Carcinoma, Hepatocellular ; metabolism ; Cell Line, Tumor ; DNA, Complementary ; genetics ; GTP-Binding Proteins ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; RNA, Messenger ; genetics ; Transglutaminases ; metabolism