Localization and characterization of N-ethylmaleimide sensitive inhibitor(s) of thiol cathepsin activity from cultured nil and polyoma virus-transformed nil hamster cells

Exposure of cultured Nil (a stable line of fibroblast cells from Syrian hamsters) or polyoma virus-transformed (PyNil) hamster fibroblasts to 0.5 mM N-ethylmaleimide for 5 minutes resulted in striking increases in thiol cathepsin activity in unfractionated cell-free lysates. The paradoxical increase in activity of the normally N-ethylmaleimide-sensitive cathepsins apparently occurred as the result of the protective compartmentalization of the cathepsins in the lysosomes (20,000 X g sedimented fraction) and the unprotected localization of an inhibitor(s) in the soluble cytoplasm (175,000 X g supernatant fraction). Under continuous exposure of the cells to N-ethylmaleimide, a rapid increase in cathepsin activity (seen in the first 5 minutes) was followed by a steady decrease in activity (half inactivation time, 90 minutes). The relative difference in rates of N-ethylmaleimide inactivation of thiol cathepsins and thiol cathepsin inhibitors provides a means for estimating lysosomal cathepsin activity in whole cell extracts without the need for more time-consuming fractionation procedure. In reciprocal inhibition tests, it was found that, regardless of the source of cathepsins, the Nil and PyNil cathepsin inhibitor(s) inactivated the cathepsins to approximately the same extent. The inhibitors were heat stable (90-100 degrees C for 15 minutes) at pH 4, but were totally inactivated when boiled at pH 8.5. On a calibrated Sephadex G-100 column, the relative molecular weight (Mr) of the inhibitor(s) was 13,000 daltons. On the same column, the Mr of the cathepsins was 24,000 daltons. Compared with the cathepsin activity from Nil cells, there was about five times less cathepsin activity recoverable from the PyNil cells.     

Characterization of a CMP-sialic acid:lactosylceramide sialyltransferase activity in cultured hamster cells

Previous studies have shown a strong correlation between reduced levels of GM3 ganglioside and an increase in the oncogenic transformation of cultured cells. CMP-sialic acid:lactosylceramide sialyltransferase, which catalyzes GM3 synthesis, was characterized in cultured hamster fibroblasts (NIL-8) with respect to substrate binding, pH optimum, detergent requirements, metal ion requirements, activity during cell cycle phases and activity during cell growth phases. The apparent Km values for CMP-sialic acid and lactosylceramide were 0.16 and 0.11 mM, respectively. The enzyme required Mn2+ (15 mM) for maximal, but Mg2+ and Ca2+ were able to substitute to a lesser extent. Triton CF-54 (0.3%, w/v) compared to other nonionic detergents gave the greatest enzyme activation, while ionic detergents inhibited the enzyme. A broad pH optimum (4.5-8.0) was obtained, with maximum activity at pH 6.5 in cacodylate-HCl buffer. No buffer effects on enzyme activity were seen. Sialyltransferase activity was found to be highest in the M and G1 phases of the cell cycle and in the contact-inhibited phase of cell growth.     

Characterization of two poly(A) polymerases from cultured hamster fibroblasts.

The enzymatic and physiochemical properties of poly(A) polymerases IIA and IIB from cultured hamster fibroblasts were investigated. The enzymes show an absolute requirement for Mn2+ as the divalent ion. Although Mg2+ alone is inactive, maximum activity is observed in the presence of both Mn2+ and Mg2+. An optimal pH of approx. 8 is found for polymerases IIA and IIB. The enzymes, however, differ somewhat in the pH curves as well as in the Mn2+ and Mg2+ concentration curves. Poly(A) polymerases IIA and IIB have an isoelectric point of about 6 and a sedimentation coefficient of 3.5--4 S. The molecular weights, obtained by gel filtration chromatography, are 145 000 and 155 000 for enzymes IIA and IIB, respectively. Poly(A) polymerases IIA and IIB can utilize a variety of natural and synthetic RNAs as well as DNA as primers. Poly(A) polymerase IIA is saturated at much lower concentrations of primer than enzyme IIB. On the other hand, the chain length of the product synthesized by polymerase IIA is independent of the primer concentration, whereas, with polymerase IIB, the length of the product decreases when the concentration of RNA is increased.     

Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells

Erythropoietin (Epo), a glycoprotein that regulates the formation of erythrocytes in mammals, was produced in cultured tobacco BY2 cells (Nicotiana tabacum L. cv. Bright Yellow 2) by introducing human Epo cDNA via Agrobacterium tumefaciens-mediated gene transfer. Epo was correctly processed and subsequently penetrated the plasma membrane of tobacco cells. However, it remained attached to the cell wall and was not released into the culture medium. Although Epo produced by tobacco cells was glycosylated with N-linked oligosaccharides, these carbohydrates were smaller than those of the recombinant Epo produced in mammalian cells. Epo produced in tobacco exhibited in vitro biological activities by inducing the differentiation and proliferation of erythroid cells. However, it had no in vivo biological activities. A lectin-binding assay indicated the lack of sialic acid residues in the N-linked oligosaccharides of Epo, suggesting that Epo was removed from the circulation before it reached erythropoietic tissues.     

Hexose transport regulation in cultured hamster cells

Hamster (nil) cells maintained overnight in culture medium containing cycloheximide and either glucose or fructose exhibit strikingly different rates of hexose transport and metabolism (i.e., uptake). Pretreatment of cultures with sulfhydryl reagents makes it possible to determine initial transport rates for a physiological sugar such as galactose which is a catabolite in hamster cells. Using galactose transport as a model, hexose uptake enhancements can now be shown to be due almost entirely to increase in the rate of the transport step. The transport regulation can best be accounted for by a model comprised of 2 antagonizing mechanisms. This model involves turnover of transport carriers as well as inhibitory units (“regulators”). The experimental as well as the theoretical model may also apply to the well-known uptake enhancements observed in oncogenically transformed cells.     

Mutagenic and clastogenic activity of nitracrine analogues in cultured V79 Chinese hamster cells

Nitracrine is used clinically as an antitumour agent, and analogues are actively being developed in some laboratories. The mutagenic activity of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-nitro (nitracrine), 2-, 3- and 4-nitro derivatives was evaluated at the 6-thioguanine and ouabain resistance loci in cultured Chinese hamster fibroblasts (V79-171b cell line). The des-nitro, 2- and 3-nitro caused no statistically significant mutagenic activity at either locus. Each of these 3 compounds weakly increased (approximately 2-fold) the incidence of micronuclei in the same cell line when tested at cytotoxic doses. Both the 1- and 4-nitro compounds increased the incidence of 6-thioguanine resistant cells from around 1 in 10(-6) to approximately 1 in 10(-4). The former compound significantly increased the frequency of ouabain-resistant cells. Both of these compounds were potent inducers of micronuclei in V79-171b cells, indicating high clastogenic activity. It would appear prudent to regard both of these compounds as potential human carcinogens.     

5-bromo-2''-deoxyuridine-stimulated calcium ion- or magnesium ion-dependent ecto-(adenosine triphosphatase) activity of cultured hamster cardiac cells.

1. Treatment of hamster heart cells in primary culture with 5-bromo-2'-deoxyuridine resulted in the greatly increased activity of a particulate Ca2+- or Mg2+-dependent ATPase (adenosine triphosphatase). 2. 5-Bromo-2'-deoxyuridine exerted these effects only when it was incorporated into cellular DNA, and then in a concentration-dependent manner. 3. Serially replated cells contained less of the activity (expressed as a function of total cell protein) than did the primary cultures, but the stimulation caused by 5-bromo-2'-deoxyuridine addition was much greater. 4. The affected enzyme was apparently localized in the plasma membrane of the cells with its active centre exposed to the outer environment [ecto-(ATPase) dependent on Ca2+ or Mg2+].5. The activity was unaffected by treatment with p-chloromercuriphenylsulphonate, ouabain andverapamil. 6. Ecto (5'-nucleotidase) activity was not increased by 5-bromo-2'-deoxyuridine treatment of cells, and ecto-(p-nitrophenyl phosphatase) activity was only slightly enhanced.     

Persistent infection with bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) in cultured hamster cells

Summary Bovine herpesvirus-1 infection in hamster embryo cells was found to be dependent upon input multiplicity; productive infection was achieved at input multiplicities greater than one, while persistent infection was established when input multiplicities were about 0.5. This persistence was characterized by a noncyclic, minimal degree of cytopathic effect with a low level of released virus. Maintenance of the persistently infected cultures did not require external supportive measures. Subcultivation of the persistently infected cultures led to virus replication followed by CPE and then cell regrowth. Within 3 to 4 weeks after subcultivation a persistent infection was re-established. The possible mechanism for the bovine herpesvirus persistence in hamster cells is discussed. This work was supported in part by Public Health Service Research Contract FDA 233-74-1035 and by Research Grant AI-08648 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.     

Agmatine uptake by cultured hamster kidney cells

Agmatine, the product of arginine decarboxylation, has been recently found in a wide variety of animal tissues. In spite of the emergent interest on agmatine in animals, the mechanism of agmatine uptake in mammalian cells has been scarcely studied. An analysis of radiolabeled agmatine uptake was carried out by using a classical, kinetic approach with BHK-21 hamster kidney cells in culture. A high affinity, temperature- and energy-dependent agmatine transport system in BHK-21 kidney cells is here kinetically characterized which seems to be a "general" transporter shared by di- and triamines and different to a highly specific carrier for the tetraamine spermine.     

Induction of chromosomal aberrations in cultured Chinese hamster cells in a superoxide-generating system

The induction of chromosomal aberrations in a superoxide-generating system using xanthine oxidase and hypoxanthine was investigated in cultured Chinese hamster cells. The production of chromosomal aberations in this system was inhibited by the addition of cytochrome C. This finding indicates that the generation of superoxide was the primary requirement for induction of chromosomal aberrations. On the other hand, superoxide dismutase showed no effect on the frequency of chromosomal aberrations, whereas catalase was effective in preventing the aberrations. It is conceivable, therefore, that the induction of chromosomal aberrations in the superoxide-generating system may be directly or indirectly due to hydrogen peroxide formed in the cultured medium as a result of the spontaneous dismutation reaction of superoxide.