A gelatin-specific protease from hamster lung-derived cell cultures

Summary Gelatin-specific protease activity from hamster lung fibroblasts and their culture media is described. The fibroblasts were derived from hamster lung explant cultures. The gelatin-specific protease activity is latent and seen only after dialysis of either cells or media. The enzyme activity shares many properties of previously reported gelatinases. The activity is inhibited by EDTA, cysteine, and dithioerythritol, whereas it is not inhibited byp-chloromecuribenzoate,N-ethyl maleimide, or phenylmethylsulfonyl fluoride. Of all substrates tested, activity was observed only against gelatin and not against other substrates tested. It was inactive toward collagen, elastin, and methemoglobin. This enzyme may have a role in the digestion of collagen that has been previously cleaved by mammalian collagenase. This research was supported by Program Project Grant HL-19717 from the National Heart, Lung, and Blood Institute, Grant AG 000-38-02 from the National Institute of Aging, and National Institute of Health Grant 5T32HL07035.     

Studies of a myosin-cleaving protease from dystrophic hamster heart

A myosin-cleaving protease was purified to homogeneity from the cardiac myofibrils of dystrophic hamsters. The biochemical properties of the enzyme was studied with both [³H] acetyl-casein and purified myosin as substrates. Steadily increasing levels of the enzyme correlated to the development of cardiomyopathy.     

Immunocytochemical localization of laminin in hamster tracheal epithelial cell cultures

EM examination of 28 day cultures of enzymatically dissociated hamster tracheal epithelial (HTE) cells grown on collagen coated millipore filters reveals that fragments of basal lamina may be present at the basal plasmalemma. Since the basal lamina consists of several major components including type IV collagen, heparan sulfate proteoglycans, entactin/nidogen, and laminin, questions naturally arise concerning the presence of such a structure in this cell culture system. When immunocytochemical procedures utilizing anti-laminin antibody and PAP techniques are carried out with paraffin sections of HTE culture at 1,2,3, and 4 weeks in vitro, LM analysis reveals that a thin, dense line of reaction product is present between the basal surface of the HTE cells and the underlying collagen substrate. Immunoblotting evaluation carried out with supernatants of 7d HTE cell homogenates and HTE cell conditioned media also indicate that laminin is being produced by the tracheal cells. Thus, the presence of basal lamina-like fragments, the immunocytochemical localization of laminin, and immunoblot identification of laminin in hamster tracheal epithelial cell cultures, suggest that, although basal lamina components may be produced by HTE cells, at the time points tested, they are not yet being organized into a complete basal lamina.     

Gestation stage-specific frequency of adipogenic cells in Syrian hamster cell cultures

High frequencies (up to 50%) of spontaneous adipocyte differentiation are observed in cultures of 9 day gestation Syrian hamster embryos (E9 cells) within six to eight population doublings after primary culture. This is in contrast to the absence of adipogenic cells in primary cultures derived from later gestation age Syrian hamster tissue. In addition, E9 primary cultures contain a transient subpopulation of presumptive mesenchymal stem or progenitor cells that lack density dependent inhibition of growth [contact-insensitive (CS-) cells]. Analysis of the temporal pattern of expression of the CS- and adipocyte phenotypes during the proliferative life span of E9 cells demonstrates that maximal expression of the CS- phenotype precedes maximal expression of adipocyte differentiation. In addition, lipid accumulation appears to occur primarily, if not exclusively, in the contact-sensitive (CS+) cells that are derived from CS- cells. These observations suggest that primary E9 cultures contain either adipoblasts or primordial mesenchymal cells that become determined to the adipocyte lineage early during the in vitro life span of the cultures, and that the CS- phenotype may be a marker for these earlier developmental cell stages.     

The development of ciliated and mucus cells from basal cells in hamster tracheal epithelial cell cultures

Hamster tracheal epithelia consist of three cell types: ciliated, mucus and basal cells. Autoradiographic data from several studies suggest that either basal or non-ciliated columnar cells may serve as stem cells for regeneration of lost or damaged ciliated and mucus cells. The objective of the present study was to examine the role of basal cells in the formation of ciliated and mucus cells in hamster tracheal epithelial (HTE) cell cultures via tritiated thymidine ([3H]-TdR) autoradiography. When 3 day cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (5 day total) label was present in the nuclei of basal and columnar epithelial cells suggesting that the labeled columnar cells may be derived from basal cells. However, the morphological reorganization occurring during this 2 day interval may create difficulties in this interpretation. Since these morphological changes are minimal during the 6 day to 8 day in vitro period, 6 day HTE cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (8 day total), and examined to further study the fate of labeled basal cells during this period. Analysis of these 8 day cultures revealed that labeled nuclei were present in both basal cells and adjacent ciliated and mucus cells. These results do not exclude the possibility of non-basal cell origin of ciliated and mucus cells in other systems but suggest that, at least in HTE cultures, undifferentiated basal cells have the ability to develop into ciliated and mucus cells.     

Membrane anchored protein production from spheroid, porous, and solid microcarrier chinese hamster ovary cell cultures

The influence of the microcarrier type on the performance of a controlled release process used to produce a recombinant glycosyl-phosphatidylinositol anchored protein was investigated. Chinese hamster ovary (CHO) cells expressing the human melanoma tumor antigen (p97) were cultured in 10% serum on Cultispher-GH porous microcarriers and then, for protein production, maintained in 2% serum. Cells were harvested every 48 h and p97 was recovered at 90 mug/mL and 40% purity. Harvested p97 concentrations were increased by harvestingfrom spheroid (241 mug/mL) and smaller porous microcarrier, Cultispher-G (167 mug/mL) cultures. The low total cell specific p97 production of cells cultured on Cultispher-GH was due to necrosis of cells within the beads, decreased p97 expression of the immobilized cells, dilution by the liquid (up to 40% volume) associated with settled beads, and incomplete recovery of p97 from within the beads. Cells cultured on solid microcarriers, Cytodex-1, had the highest cell viability and cell specific p97 production, It is recommended that a two-stage cyclic harvesting process of cells cultured on small Cultispher-G or on Cytodex-1 beads would minimize protein loss and maximize cell specific protein recovery. (c) 1995 John Wiley & Sons Inc.     

EGCG improves recombinant protein productivity in Chinese hamster ovary cell cultures via cell proliferation control

Chinese hamster ovary cell lines are good manufacturing practice-certified host cells and are widely used in the field of biotechnology to produce therapeutic antibodies. Recombinant protein productivity in cells is strongly associated with cell growth. To control cell proliferation, many approaches have previously been tested including: genetic engineering, chemical additives such as cell cycle inhibitors, and temperature shift of the culture. To be widely adopted in the biopharmaceutical industry, the culture methods should be simple, uniform and safe. To this end, we examined the use a natural compound to improve the production capacity. In this study, we focused on the antioxidants, catechin polyphenols, which are found in green tea, for cell proliferation control strategies. (–)-Epigallocatechin-3-gallate (EGCG), the major catechin that induces G0/G1 cell cycle arrest, was investigated for its effect on recombinant protein production. Adding EGCG to the cell culture media resulted in slower cellular growth and longer cell longevity, which improved the specific productivity and total yield of recombinant IgG1 in batch cultures by almost 50% for an extra 2 or 3 days of culture. A lower l-glutamine consumption rate was observed in cells cultured in EGCG-containing media, which may be suggesting that there was less stress in the culture environment. Additionally, EGCG did not affect the N-glycan quality of IgG1. Our results indicated that adding EGCG only on the first day of the culture enhanced the specific productivity and total amount of recombinant protein production in batch cultures. This approach may prove to be useful for biopharmaceutical production.     

Mammalian cell genetics. 3. Characterization of x-ray-induced forward mutations in Chinese hamster cell cultures

X-irradiation induces forward mutations from 8-azaguanine sensitvity to resistance in Chinese hamster cells in culture. At this locus the number of induced mutations increases non-linearly with X-ray exposure. The mutation rate increase from 4.2·10⁻⁷ per locus per R with 200 R to 1.8·10⁻⁶ per locus per R with 1200 R. Several factors including cell density markedly influence the mutational yield. Reversion tests using specific chemical mutagens on 72 randomly isolated, azaguanine-resistant mutants suggest that both point mutations and chromosome deletions might have occurred in the hamster cells after exposure to ionizing radiation.     

Degradation of argininosuccinate lyase by a protease synthesized in soybean cell suspension cultures

Suspension cultures of soybean (Glycine max L.) were shown to contain protease activity which could be inhibited by the addition of protease inhibitors such as p-hydroxymercuribenzoate and ethylenediaminetetraacetic acid. The use of these inhibitors, coupled with studies of the rate of degradation of argininosuccinate lyase (argininosuccinate-lyase = l-arginino-succinate arginine-lyase, EC 4.3.2.1) in extracts of cell cultures grown for 24 hours led to the hypothesis that a metal-dependent protease is synthesized by the cells after 24 hours of growth, to remove the lyase enzyme.     

Reduced protease activity in transformed rice cell suspension cultures expressing a proteinase inhibitor

In this study, we synthesized a synthetic serine proteinase inhibitor II gene (sPI-II) that harbored the chymotrypsin and trypsin inhibitor domains of the PI-II gene from Nicotiana alata. In an effort to reduce protease activity in a rice cell suspension culture, we first synthesized sPI-II using overlap PCR and then introduced the gene into a rice calli (Oryza sativa L. cv. Dongin) by particle bombardment-mediated transformation. The sPI-II gene was under the control of a rice alpha-amylase 3D promoter induced by sugar starvation. To verify the integration and expression of the sPI-II gene in the transformed rice cells, we employed genomic DNA PCR amplification and Northern blot analysis, respectively. The relative protease activity of the transformed cell suspension culture was reduced to approximately 23% when compared to the non-transformed culture. This indicates that a transformed suspension culture system expressing a proteinase inhibitor, may be a useful tool to protect against recombinant protein losses resulting from extracellular proteases.     

Characterization of deciduoma marker proteins in hamster uterus: detection in decidual cell cultures

The objective of this study was first, to identify the proteins associated with decidualization of the hamster uterus by comparing the protein maps of decidualized and nondecidualized endometrium in vivo, and second, to determine whether decidual cell cultures produced these characteristic proteins. Decidualization was induced in one uterine horn, and the contralateral horn was not stimulated (control tissue). Animals were ovariectomized and a subcutaneous progesterone implant was used to maintain decidualization. Uterine proteins from nuclear and cytosol fractions were analyzed by two-dimensional electrophoresis using a highly sensitive protein staining technique. Analysis of nuclear extract and cytosol from decidualized and nondecidualized endometrium from Days 6, 7, and 8 of pseudopregnancy demonstrated the presence of 11 nuclear and five cytosolic deciduoma-associated proteins. Serum and erythrocyte proteins were identified by two-dimensional electrophoresis, and none of the 16 deciduoma-associated proteins was a serum or erythrocyte contaminant. Forty-eight-hour cultures of decidual cells harvested from Day 5 of pseudopregnancy produced all 16 of the deciduoma-associated proteins found in whole tissue in situ. Culture conditions minimized serum and erthrocyte contamination, enhancing the detection of deciduomal cell proteins. Four nuclear and two cytosolic proteins were considered deciduoma specific, i.e., they were not associated with cellular proliferation, as evidenced by their absence from cultures of rapidly dividing fetal hamster fibroblasts. Thus, these studies show that the detection of deciduomal proteins may be a useful criterion for the assessment of decidualization in vitro and in vivo.