Biomedical subjects

S T Jiang

Publications and source records attributed to S T Jiang.

8 recordsLinked to original sources

PubMed1999

Bcl-2 overexpression prevents apoptosis-induced Madin-Darby canine kidney simple epithelial cyst formation.

BACKGROUND: Madin-Darby canine kidney (MDCK) cells develop into simple epithelial cell cysts when cultured in type I collagen gel. We found that MDCK cells initially grow into multilayer cell aggregates and subsequently develop central lumen that contain apoptotic cells. We hypothesized that apoptosis might be essential for the formation of MDCK cysts. METHODS: Using MDCK cells cultured in collagen gel as the experimental model, we investigated how renal cells organize to form cysts. To delineate the role of apoptosis in the process of cyst formation, MDCK cells were transfected with the bcl-2 gene. Characterization of apoptosis was studied by morphological and biochemical methods. RESULTS: Bcl-2 overexpression conferred resistance to apoptosis. Cultured in collagen gel, Bcl-2 transfectants rarely formed a simple epithelial cyst, but instead remained as a multilayer cell aggregate containing central or multiple lumens, or even developing into branching structures. CONCLUSIONS: Because Bcl-2 overexpression averts cyst cavitation, these data clearly indicate that apoptosis is an essential initial event for renal cyst formation.

Animals↗

PubMed1999

Role of fibronectin deposition in cystogenesis of Madin-Darby canine kidney cells.

BACKGROUND: Madin-Darby canine kidney (MDCK) cells cultured within collagen I gel exhibit clonal growth and form spherical multicellular cysts. The cyst-lining epithelial cells are polarized with the basolateral surface in contact with the collagen gel and the apical surface facing the lumen. To understand whether MDCK cysts construct the basal lamina, we characterized the composition of the extracellular matrix deposited by MDCK cysts. The cyst-lining cells produced an apparently incomplete basal lamina containing a discontinuous laminin substratum. In addition, the basal cell surface of the cyst was surrounded by a thick layer of fibronectin. This study was conducted to delineate the role of fibronectin deposition in cystogenesis. METHODS: MDCK cells cultured in collagen gel were employed. We first used Arg-Gly-Asp (RGD) peptides containing disintegrin rhodostomin to disturb the interaction between fibronectin and the cell surface integrin. We then established several stable transfectants expressing the fibronectin antisense RNA and with which to directly examine the role of fibronectin in cystogenesis. RESULTS: Rhodostomin markedly decreased the growth rates of the MDCK cyst, suggesting the importance of a normal interaction between fibronectin and integrins. The stable transfectants overexpressing the fibronectin antisense RNA exhibited relatively lower levels of fibronectin and markedly lower cyst growth rates than the control clone. The lower growth rate was correlated with an increase in collagen gel-induced apoptosis. CONCLUSIONS: The results indicate that the deposition of fibronectin underlying the cyst-lining epithelium serves to prevent apoptosis induced by three-dimensional collagen gel cultures, and hence facilitates cyst growth of MDCK cells.

Animals↗

PubMed1998

Collagen gel overlay induces apoptosis of polarized cells in cultures: disoriented cell death.

In this study, we attempted to investigate the response of polarized cells to inappropriate interaction with the extracellular matrix. Cell lines of epithelial [Madin-Darby canine kidney (MDCK) and LLC-PK1], endothelial [bovine aortic endothelial cells (BAEC)], and mesenchymal (ESK-4 and NIH/3T3) origins were employed. With collagen gel overlay, MDCK cells underwent membrane remodeling and gradually developed lumen formation within 24 h. Apoptosis could also be observed following cell remodeling. The ratio of apoptosis was enhanced from 12.1 +/- 2.4% within 24 h to 58.4 +/- 9.8% at day 3, and finally the monolayer was disintegrated. Collagen gel overlay-induced apoptosis was not a result of physical stress, since agarose gel overlay did not induce any morphological alterations. All epithelial and endothelial cells examined developed apoptosis in response to collagen overlay. In contrast, collagen overlay did not affect growth of fibroblasts at all, although their growth under agarose gel was slightly hindered due to physical stress. Collagen overlay-induced apoptosis seems to be a unique phenomenon for polarized cells and thus is defined as "disoriented cell death." Furthermore, anti-alpha2-integrin antibody could abolish collagen overlay-induced morphological changes and apoptosis in MDCK cells, indicating that signals through alpha2-integrin on the apical membrane are required for disoriented cell death. Finally, Bcl-2 overexpression prolonged survival of MDCK cells in response to collagen overlay, but these cells eventually developed apoptosis due to downregulation of Bcl-2 protein. These findings indicate that inappropriate cell-matrix interaction results in apoptosis, which may account for cell death mechanisms during developmental processes or under pathological conditions.

3T3 Cells↗

PubMed1998

Effects of elution [correction of eution] conditions on the separation of calpastatin, mu- and m-calpain on DEAE-Sephacel chromatography.

A rapid stepwise measurement for the activities of calpastatin and mu- and m-calpains was developed by using 2-stage elution at pH 8.5 and then 7.0. The activities of calpastatin, mu-calpain and m-calpain can be rapidly assayed following the separation on DEAE-Sephacel chromatography by a 2 stage elution with 90 mM NaCl (pH 8.5), and then by 200 and 300 mM NaCl in elution buffer (pH 7.0). No significant differences in the recovery of these proteinases and inhibitor was observed between stepwise gradient and linear gradient methods.

Buffers↗

PubMed1998

Contribution of muscle proteinases to meat tenderization.

The exact mechanisms involved in the postmortem meat tenderization process and the nature of changes associated with improvement in tenderness are complex and not fully understood. Based on the relevant evidence thus far obtained, the focus of this review is on clarifying the factors affecting meat tenderness, particularly the toughening and tenderness phases, possible endogenous proteinases involved in meat tenderization and how these proteinases contribute to meat tenderization. Of the different biochemical and ultrastructural changes occurring in the meat tenderization process, myofibril disruption at the Z-disk and contractile proteins are discussed in detail. This myofibril disruption can perhaps be ascribed to the synergistic action of calcium-dependent proteinases (both mu- and m-calpains) and lysosomal proteinases, especially the cathepsins B and L.

Animals↗

PubMed1997

A continuous method for measuring calpain activity.

A simplified methodology for assaying the caseinolysis by calpains was developed. This methodology, including the incubation of calpain with casein and direct measurement of the absorbance at 500 nm, is based on the turbidity of the reaction mixture caused by the aggregation of hydrolysates during the reaction. Unlike the typical caseinolysis assay, this novel assay does not need to separate the substrate from hydrolysates and can be continuously monitored in visible wavelength range. The activity of calpain is expressed by the maximum reaction velocity (delta A500/min) at 25 degrees C).

Animals↗

PubMed1997

Inducible expression of bcl-2 by the lac operator/repressor system in MDCK cells.

The lac operator/repressor-inducible system was utilized to dissect the biological consequences of human bcl-2 gene expression in Madin-Darby canine kidney (MDCK) cells. Cells were made transgenic for a constitutively expressed lacI gene, encoding lac repressor, and the bcl-2 gene that had been inserted downstream of a simian virus 40 (SV40) promoter containing the lac operator sequence. The expression of the bcl-2 gene could therefore be repressed to basal level by binding of lac repressor to the lac operator sequence in proximity to this SV40 regulatory region and be specifically activated by administration of the lactose analog isopropyl-beta-D-thiogalactoside (IPTG). We showed that expression of bcl-2 gene could be induced by 0.01 mM IPTG, and the maximal induction was obtained at 1 mM. With the treatment of IPTG, the Bcl-2 protein could be induced within 6 h. Moreover, the IPTG-inducible expression of Bcl-2 protein is a reversible process. Finally, functional assays revealed that IPTG-induced expression of bcl-2 gene conferred partial or complete resistance to homeless cell death or confluent cell death, respectively. The inducible expression system should be particularly useful for dissecting the effect of bcl-2 in phenotypic or morphological changes of MDCK cells.

Animals↗

PubMed1996

L ferritin accumulation in macrophages infiltrating the lung during rat Angiostrongylus cantonensis infection.

A 22-kDa protein was increased quantitatively, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques, in lung microsomes prepared from Angiostrongylus cantonensis-infected rats. However, it was almost absent in normal rats. The protein was purified by sequential chromatography on Superdex 200 columns and identified chemically and immunologically as ferritin. Using isoelectric focusing and anion exchange chromatography, it was identified as L ferritin. Distribution of this 22-kDa protein in the lung tissue of A. cantonensis-infected rate was studied by immunocytochemistry. Positively stained cells were mainly infiltrated macrophages. Our results suggest that L ferritin accumulation in the macrophages may be related to the proliferation of connective tissue elements and the inflammatory response to A. cantonensis dwelling in the pulmonary arteries of the rat.

Angiostrongylus cantonensis↗