Biomedical subjects

S T Jiang

Publications and source records attributed to S T Jiang.

16 recordsLinked to original sources

PubMed2001

High-level production of recombinant chicken cystatin by Pichia pastoris and its application in mackerel surimi.

A high level of the secreted form of recombinant chicken cystatin was expressed in Pichia pastoris X-33 by chromosomal integration of multiple copies of an expression cassette containing chicken cystatin under the control of glyceraldehyde-3-phosphate dehydrogenase promoter. The inhibition ability of the recombinant for papain-like proteinase was found to correspond to those of natural chicken cystatin. The recombinant cystatin substantially inhibited the proteolysis of myosin and gel softening, which consequently improved the gel properties of mackerel surimi.

Animals↗

PubMed2001

Expression of soluble form carp (Cyprinus carpio) ovarian cystatin in Escherichia coli and its purification.

A DNA encoding thioredoxin-mature carp ovarian cystatin (trx-cystatin) fusion protein was ligated into a pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3) expression host. After induction by isopropyl beta-D-thiogalactopyranoside, a high level of the soluble form of recombinant trx-cystatin was expressed in the cytoplasm of E. coli. The recombinant trx-cystatin could be purified by Ni(2+)-NTA agarose affinity chromatography. The molecular mass (M) of the recombinant trx-cystatin was approximately 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa). Both recombinant trx-fused and mature carp ovarian cystatins were stable at pH 6-11. No obvious decrease in activity was observed even after 5 min of incubation at 60 degrees C. They exhibited papain-like protease inhibition activity comparable to that of the mature carp ovarian cystatin, which could inhibit papain and mackerel cathepsins L and L-like, but not cathepsin B.

Animals↗

PubMed2001

Role of alpha(3)beta(1) integrin in tubulogenesis of Madin-Darby canine kidney cells.

BACKGROUND: We isolated several Madin-Darby canine kidney (MDCK) subclones that exhibit different degrees of branching tubulogenesis in lower concentrations of collagen gel. The M634 clone formed cell aggregates in 0.3% collagen gel, but developed branching tubules vigorously in 0.1% collagen gel. In contrast, the Y224 clone formed cysts in 0.3% collagen gel and displayed fewer branching structures in 0.1% collagen gel. Morphologically, M634 cells exhibited higher levels of cell scattering as well as collagen-induced cell migration than Y224. We conducted this study to delineate the underlying mechanism of branching tubulogenesis in M634 cells. METHODS: Components of the focal contact machinery were analyzed in both cell lines, including the extracellular matrix glycoproteins fibronectin, laminin, and vitronectin; cytoskeleton-associated elements alpha-actinin, talin, and vinculin; and receptors for extracellular matrix and alpha(2), alpha(3), alpha(5), alpha(v), beta(1), and beta(3) integrins. Furthermore, we established several stable transfectants of alpha(3) integrin antisense RNA in M634 cells to examine the role of alpha(3)beta(1) integrin in branching morphogenesis directly. RESULTS: There were no obvious differences in levels of the focal adhesion complex proteins between M634 and Y224 cells, except that the content of the alpha(3) and beta1 integrins were 1.2- and 0.6-fold higher in M634 cells, respectively. The expression of alpha(3) integrin antisense RNA significantly lowered the levels of alpha(3) integrin mRNA and protein. The potential of cell scattering, migration, and branching tubulogenesis in M634 cells was inhibited according to the decrease in alpha(3) integrin expression. CONCLUSION: Our data indicate that expression of alpha(3)beta(1) integrin regulates cell scattering, migration, and branching tubulogenesis of MDCK cells, possibly via adhesion to or serving as a signaling molecule for type I collagen.

Animals↗

PubMed2000

Involvement of focal adhesion kinase in hepatocyte growth factor-induced scatter of Madin-Darby canine kidney cells.

Focal adhesion kinase (FAK) has been implicated to play a critical role in integrin-mediated control of cell behavior. However, it is unclear whether FAK also participates in the regulation of growth factor-elicited cellular functions. In this study, we have demonstrated that although overexpression of FAK in Madin-Dardy canine kidney cells did not alter their growth property or ability to form tubules within collagen gel upon hepatocyte growth factor (HGF) stimulation, it apparently enhanced HGF-induced cell scattering. This enhancement was largely because of an increase in the third phase (i.e. cell migration) of cell scattering rather than the first two phases (i.e. cell spreading and cell-cell dissociation). Conversely, the expression of FAK-related nonkinase significantly ( approximately 60%) inhibited HGF-induced cell migration. Moreover, we have found that the effect of FAK on promoting HGF-induced cell motility was greatly dependent on cell-matrix interactions. We showed that HGF treatment selectively increased the expression of integrins alpha(2) and, to a lesser extent, alpha(3) in Madin-Dardy canine kidney cells and that a monoclonal antibody against integrin alpha(2) efficiently blocked HGF-enhanced cell migration on collagen. In our efforts to determine the mechanism by which FAK promotes HGF-induced cell migration, we found that FAK mutants deficient in phosphatidylinositol 3-kinase or p130(Cas) binding failed to promote HGF-induced cell migration. Interestingly, cells expressing a FAK mutant defective in Grb2 binding exhibited a rate of migration approximately 50% lower than that of cells expressing wild type FAK in response to HGF stimulation. Taken together, our results suggest a link between HGF-increased integrin expression, FAK activation, and enhanced cell motility and implicate a role for FAK in the facilitation of growth factor-induced cell motility.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification.

A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.

Animals↗

PubMed2000

Purification and characterization of calpastatin from grass prawn muscle (Penaeus monodon).

Calpastatin, a specific calpain inhibitor was purified to electrophoretical homogeneity from grass prawn (Penaeus monodon) muscle by 100 degrees C heat-treatment, DEAE-Sephacel, and Q-Sepharose chromatographs. No significant change in the inhibitory activity of crude calpastatin was observed even after 20 min incubation at 100 degrees C, pH 7.0. The purified prawn calpastatin had a molecular weight (M(r)) of 80 and 88.7 kDa determined by SDS-PAGE and Sephacryl S-200 HR gel filtration, respectively. According to the active site titration, the purified calpastatin revealed four beef mu-calpain and two beef m-calpain binding domains, respectively. It was stable during 1 h of incubation at 30 degrees C under pH 4.5-10.0 and shown to be a highly specific inhibitor for calpain.

Animals↗

PubMed2000

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

BACKGROUND: Madin-Darby canine kidney (MDCK) epithelial cells grown in collagen gels in the presence of hepatocyte growth factor (HGF) form branching tubules. The tubule-lining epithelial cells are polarized with the basolateral surface in contact with the collagen gel and the apical surface facing the lumen. To delineate whether MDCK branching tubules construct the basal lamina, we characterized the composition of the extracellular matrix deposited by MDCK tubules. The tubule-lining cells produced an apparently incomplete basal lamina containing a discontinuous laminin substratum. In addition, a thick layer of fibronectin surrounded the basal cell surface of the branching tubule. In an attempt to delineate the role of fibronectin deposition in branching morphogenesis, we conducted this study. METHODS: MDCK cells cultured in collagen gel were employed. We first used arginine-glycine-aspartate peptides containing disintegrin rhodostomin to disturb the interactions between fibronectin and cell surface integrins. Furthermore, we established several stable transfectants expressing fibronectin antisense RNA to examine the role of fibronectin in branching morphogenesis directly. RESULTS: Rhodostomin inhibited the formation of branching tubules. The transfectants expressing fibronectin antisense RNA exhibited relatively lower levels of synthesized fibronectin and markedly diminished growth rates of branching tubules than the control clone. An inhibition of branching morphogenesis induced by the overexpression of fibronectin antisense RNA was manifested by the decrease in cell growth rates and cell migration. CONCLUSION: These results indicate that the deposition of fibronectin underlying the tubule-lining epithelium serves to enhance cell proliferation and migration, and hence facilitates the branching tubulogenesis of MDCK cells.

Animals↗

PubMed2000

Age effect of type I collagen on morphogenesis of Mardin-Darby canine kidney cells.

BACKGROUND: Mardin-Darby canine kidney (MDCK) cells cultured in hydrated collagen gels develop simple epithelial cysts or branching tubules, depending on the presence of hepatocyte growth factor (HGF). Constituents of extracellular matrix can modulate the morphogenesis of MDCK cells. Collagen is one of the few well-defined structural entities that display gross structural changes with aging. This study was conducted to delineate the effects of age-induced changes of collagen on the morphogenesis of MDCK cells cultured in collagen gel. METHODS: We employed Y224 and MDCK clone II 3B5 cells to study cystogenesis and branching tubulogenesis, respectively. Cells were cultured in three-dimensional collagen gels prepared from 1-, 4-, 8-, and 16-month-old rat tail tendons, and their capacity to develop cysts or branching tubules was assessed. We also analyzed the compositions and physical structures of collagen of various ages. RESULTS: Y224 cells developed generally larger spherical cysts in collagen gels prepared from rats that were more than four months old. The ratio of apoptosis of cells cultured in one-month-old collagen gel was markedly higher than in the gel of older ages. The results were consistent with the observations that collagen gel overlay-induced apoptosis of Y224 cells in one-month-old collagen was higher than that in older collagen. On the other hand, 3B5 cells exhibited a remarkable scattering morphology when cultured in one- or four-month-old collagen gel with HGF. In contrast, 3B5 cells exhibited more intercellular adhesion and were organized into branching tubule structures only in the collagen gel that was more than eight months old. The differences in morphogenesis could be explained by the observations that collagen of younger ages exerted markedly higher HGF-triggered migration capability than collagen of older ages. CONCLUSIONS: Age-related alterations in collagen influence epithelial cell morphogenesis via regulation of cell apoptosis, proliferation, and/or motility.

Acids↗

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↗