Novel glycosylation-defective baby hamster kidney cells.

The plant lectin wheat germ agglutinin (WGA) has previously been used to select more than ten different glycosylation-defective phenotypes in a variety of mammalian somatic cells. Three WGA-resistant phenotypes have now been obtained spontaneously from baby hamster kidney (BHK) cells. These mutant BHK cells exhibit a pattern of cross resistance and sensitivity to multiple plant lectins, suggesting that the cell surface carbohydrates of these cells are altered. Two WGA-resistant BHK phenotypes appear similar to WGA-resistant CHO cells that lack terminal sialic acid and galactose residues on their cell surface carbohydrates. The third WGA-resistant BHK cell phenotype has not previously been seen in WGA-resistant mammalian cells.     

Mutator genes of baby hamster kidney cells.

Two mutator genes of mammalian cells were demonstrated. One was associated with the ribonucleoside diphosphate reductase, and the other was associated with an extreme adenosine sensitivity.     

Deoxycytidylate deaminase in pregnancy.

Deoxycytidylate (DCMP) deaminase was assayed at various times during and after normal and abnormal pregnancies. The level in amniotic fluid was assessed at induction and at caesarean section, and cord blood levels were estimated after normal delivery and at caesarean section. A rise occurred during labour and after hysterectomy and caesarean section--returning to normal after 2-3, and 12 days respectively. Levels above 4.8 X 10-minus 4 ml-minus 1 were found in cases of preeclamptic toxaemia and early intrauterine death and in twin pregnancies over 36 weeks'' gestation. It is suggested that because of its low incidence of false-negative and false-positive results this test is far superior to other enzyme tests in pregnancy, and a further trial is in progress to assess its role.     

Deoxycytidylate deaminase. Evidence for a new enzyme in cells infected by the virus of herpes simplex

The activities of dCMP deaminase and DNA polymerase I increased twofold and fivefold in BHK-21/C13 cells after infection by the virus of herpes simplex. The increases were greatly diminished, and under certain conditions prevented, by inclusion of actinomycin D or cycloheximide in the cell-virus system during the infective cycle. The dCMP deaminase purified from infected cells harvested 8h after infection differed from the deaminase purified from non-infected cells inasmuch as (a) it was more resistant to heating at 37 degrees C; (b) the substrate (dCMP) concentration at half-maximum velocity was lower; (c) maximum activation was achieved by a lower concentration of dCTP; (d) it was more resistant to inhibition by dTTP; and (e) it behaved differently when assayed in the presence of a herpes-virus-specific antiserum. The DNA polymerase activity in the infected cells was markedly decreased in the presence of the herpes-virus-specific antiserum.     

Thermal stability of ribosomal ribonucleic acid from baby hamster kidney cells

1. RNA has been prepared from baby hamster kidney cells by extraction with a phenol–EDTA mixture and further purified by passing through a column of Sephadex G-25 that had been equilibrated with water. 2. Aging of the total RNA extracts at 4° or heating at 95° followed by rapid cooling caused a conversion of 28s RNA into material sedimenting in sucrose gradients at approx. 18s. 3. When heated RNA was re-extracted with phenol the sedimentation profile was not returned to that of the unheated RNA. 4. The 28s and 18s RNA fractions were collected separately from sucrose gradients by precipitation with 2vol. of ethanol and passed through a Sephadex G-25 column equilibrated with water. 5. Heat treatment of purified 28s RNA at 95° caused the sedimentation coefficient to increase to approx. 40s, whereas similar treatment of 18s RNA caused no significant increase. If the RNA was heated before the Sephadex G-25 treatment the sedimentation coefficient of the 28s and 18s RNA decreased to approx. 12s and 8s. 6. Heating mixtures of purified 28s and 18s RNA at 95° caused some aggregation of 18s material with the 28s fraction.     

Isolation and characterization of norleucine-resistant baby hamster kidney cells

Variants resistant to low and high levels of the methionine analogue norleucine were isolated in baby hamster kidney cells and in two clonal sublines, B1 and TG2. Clones resistant to high levels of norleucine were observed only after chemical mutagenesis, whereas clones capable of growing in low concentrations of norleucine occurred with equal frequency spontaneously and after mutagenesis. The variants were characterized with respect to uptake of ¹⁴C-norleucine and ¹⁴C-methionine. Five clones were found to be deficient in ¹⁴C-norleucine uptake, and of these, four showed reduced ¹⁴C-methionine uptake. The variants were tested also for increased activity of N₅-methyl-tetrahydrofolate: homocysteine methyltransferase, the enzyme which catalyses the terminal reaction in methionine biosynthesis. In four clones, higher levels of the methyltransferase were present than in the wild-type cells, suggesting overproduction or stabilization of this enzyme.     

Release of the glucose-regulated protein 94 by baby hamster kidney cells

Glucose-regulated protein 94 (grp94) is a major component of the endoplasmic reticulum (ER) lumen of eukaryotic cells. We showed that grp94 is released from baby hamster kidney (BHK-21) cells into a serum-free medium. The exit of grp94 into the medium was not related to the protein discharge due to cell death and was independent of de novo protein synthesis. The treatment of cells with brefeldin A and monensin, the inhibitors of the classical pathway of protein secretion, did not decrease the extracellular level of grp94, indicating that the discharge of grp94 from cells does not occur through the ER/Golgi-dependent pathway. Exosomes, membrane vesicles secreted by several cell types, were not involved in the release of grp94 from cells. Methyl-β-cyclodextrin, a substance that disrupts the lipid raft organization, considerably reduced the extracellular level of grp94, indicating that lipid rafts are involved in the liberation of grp94 from BHK-21 cells. The results suggest that BHK-21 cells release grp94 into the serum-free medium via the nonclassical secretory pathway in which lipid rafts play an important role. Copyright ? 2012 John Wiley & Sons, Ltd.     

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.     

Differentiation and characterization of the cytoplasmic and nuclear deoxyribonucleic acid polymerases from baby hamster kidney cells.

Distinct DNA polymerase activities have been found in the cytoplasmic and nuclear fractions of a baby hamster kidney cell line. They were separated by chromatography on DEAE-cellulose and partially purified by ammonium sulfate fractionation, DNA - cellulose and linear sucrose gradients. The cytoplasmic DNA polymerase exhibited an S-coefficient of 6.95 S in 0.15 M NaCl and its activity was highly sensitive to inhibition by N-ethylmaleimide and elevated temperatures, regardless of the presence of DNA template or other cofactors. It was stimulated by monovalent salts in the order of NH4 Cl greater than KCl greater than NaCl greater than CsCl greater than LiCl (inhibitory). The DNA polymerase extracted from nuclei sedimented with an S-value of 3.47 S, was resistant to inactivation by N-ethylmaleimide, and maximally stimulated by NaCl, while also being inhibited by LiCl. For optimal activity, both DNA polymerase activities required a divalent cation, with MgCl2 being more effective than MnCl2. Although the optimal pH values for the two enzyme activities differed slightly, glycine - NaOH buffer induced an alkaline shift of 1.5 pH units in the optimum of both enzymes. This was accompanied by an increase in the effectiveness of MnCl2 relative to MgCl2 for the cytoplasmic DNA polymerase.     

Growth of baby hamster kidney cells in media containing neuraminidase

An established line of baby hamster kidney cells, BHK 21/C13 grows in media containing high concentrations of Vibrio cholerae neuraminidase at the same initial exponential rate as control cells grown in the absence of the enzyme. Glycoprotein fractions removed from the surface of cells grown with neuraminidase contain less than 4% of the sialic acid present in similar fractions removed from control cells. The significance of these results are discussed in relation to the role suggested for sial glycoproteins in growth control. A small but significant increase was observed in the density of confluent cells in media containing neuraminidase compared with control cultures.     

A quantitative analysis of the endocytic pathway in baby hamster kidney cells

A morphological analysis of the compartments of the endocytic pathway in baby hamster kidney (BHK) cells has been made using the fluid-phase marker horseradish peroxidase (HRP). The endocytic structures labeled after increasing times of endocytosis have been identified and their volume and surface densities measured. In the first 2 min of HRP uptake the volume density of the labeled structures increased rapidly and thereafter remained constant for the next 13-18 min. This plateau represents the volume density of endosome organelles and accounts for 0.65% of the cytoplasmic volume (or 6.8 microns 3 per cell). The labeled structures consist of tubular-cisternal elements which are frequently observed in continuity with 300-400 nm vesicles. After 15-20 min of internalization the volume density of HRP-labeled structures again increased rapidly and reached a second plateau between 30 and 60 min of labeling. This second increase corresponded to detectable levels of HRP reaching later, acid phosphatase (AcPase)-reactive compartments. These structures, comprising the prelysosomes and lysosomes, were mostly vesicular and collectively accounted for 3.5% of the cytoplasmic volume (or 37 microns 3 per cell). The absolute peripheral surface areas of the two classes of organelles (endosomes and prelysosomes/lysosomes) were estimated to be 430 and 370 microns 2 per cell, respectively. The volume of fluid internalized in the first 2 min of uptake was five- to sevenfold less than the volume of the compartment labeled in this time. To account for these results we propose that, after uptake from the cell surface, HRP is delivered to, and diluted in, endosomes that are preexisting organelles initially devoid of the marker. With increasing times of endocytosis the concentration of HRP in the early endosomes increases, as more of the marker enters this compartment. An elevation in HRP concentration in endosomes during the early time points was shown directly using anti- HRP antibodies and colloidal gold on cryosections. The stereological values given in the present study, in combination with earlier studies, provide a minimum estimate for both the total surface area of membranes and the rate of membrane synthesis in a BHK cell.     

Disruption of phosphatidylserine translocation to the mitochondria in baby hamster kidney cells

Phosphatidylserine synthase is found predominantly in the microsomal fraction, and phosphatidylserine decarboxylase is found predominantly in the mitochondrial fraction of baby hamster kidney (BHK-21) cells. This segregation of enzymes of phosphatidylserine metabolism allows serine metabolism to phosphatidylserine and phosphatidylethanolamine to be used as an indicator of the intracellular movement of phosphatidylserine. After BHK-21 cells were pulse-labeled with [3H]serine, phosphatidylserine was efficiently labeled, and subsequently 40-50% of this radiolabeled lipid turned over to form phosphatidylethanolamine during a 7.5-h chase. Treatment of cells with NaN3 plus NaF or cycloheximide at the end of the pulse labeling period markedly inhibited the rate and extent of phosphatidylserine turnover during the chase period. The inhibition of phosphatidylserine turnover could not be attributed to inhibition of either phosphatidylserine decarboxylase or phosphatidylserine exchange protein activity. Subcellular fractionation of the BHK-21 cells demonstrated that cells poisoned with NaN3 plus NaF accumulated phosphatidylserine in the microsomal fraction relative to unpoisoned cells. The results indicate that metabolic energy is required for the transport of phosphatidylserine to the mitochondria.     

1-Aminooxy-3-aminopropane reversibly prevents the proliferation of cultured baby hamster kidney cells by interfering with polyamine synthesis

The effects on cultured baby hamster kidney cells of 1-aminooxy-3-aminopropane, a potent new inhibitor of mammalian ornithine and S-adenosylmethionine decarboxylases and of spermidine synthase, were studied. At 0.5 mM concentration in the culture medium, the drug did not interfere with the transmethylation-transsulfuration pathway nor with the polyamine transport system, but it blocked the proliferation and macromolecule synthesis of the cells and reduced the cellular spermidine level to less than 10% of the control value at identical cell density. These changes were accompanied by a total cessation of the excretion of putrescine, spermidine, and acetylated polyamines into the culture medium, greatly increased activity of ornithine and S-adenosylmethionine decarboxylases, and an accumulation of both decarboxylated and intact S-adenosylmethionine. These effects were reversed by the removal of the inhibitor from the culture medium or by supplementing the medium with either 0.5 mM putrescine or 0.1 mM spermidine. In the former case, however, a lag period of 24 h was necessary for the cells to recover. The elevated concentration of decarboxylated S-adenosylmethionine normalized very slowly but apparently had no harmful effects on the cells. The clonigenic potential of the cells was only slightly reduced by prolonged treatment with 0.5 mM 1-aminooxy-3-aminopropane. Thus, the new drug is not toxic to the cells, but either directly or indirectly stops their proliferation by preventing the adequate formation of putrescine and spermidine.     

The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes.

The fluorescent probes pyrene, pyrene butyric acid and N-phenyl 1-naphthylamine have been used to investigate the changes that accompany in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe N-phenyl 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrene probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.     

Initial attachment of baby hamster kidney cells to an epoxy substratum. Ultrastructural analysis

In the presence of serum-containing medium, BHK cells attached and spread during a 1-h period onto a 3-5 nm thick serum layer absorbed on the substratum surface. The closest approach of the plasma membrane to the serum layer was observed to be about 9nm, which was determined by tilting the sectioned cells in a goniometer holder. Bundles of microfilaments or other cytoplasmic specializations were not observed in association with the regions of close contact. However, in the space between the plasma membrane and the adsorbed serum layer, a diffusely stained material could be visualized after fixation/staining by the tannic acid-glutaraldehyde technique. This technique also permitted increased clarity of visualization of trilaminar appearance of the plasma membrane. The distribution and mobility of anionic sites on the surfaces of attached and spreading cells was determined by labeling with polycationic ferritin. We observed movement of polycationic ferritin into large clusters on the cell surface, collapse of cell surface microextensions, and endocytosis, all of which were similar to our previous findings utilizing cells in suspension. However, the absolute amount of ferritin bound to the upper cell surface was less than that previously observed when suspended cells were put under similar labeling conditions. Also, polycationic ferritin did not appear to penetrate between the lower cell surface and the substratum.     

Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells

Incubation of cultured cells at 20 degrees C blocks the transport of newly synthesized plasma membrane proteins, and the proteins accumulate intracellularly in a terminally glycosylated form. When baby hamster kidney cells are infected with the ts O45 mutant of vesicular stomatitis virus, and incubated at 20 degrees C, the terminally glycosylated spike glycoprotein G of the virus accumulates in the membranes of a tubular network localized on the trans side of the Golgi cisternae, the trans-Golgi network (TGN). We have used the G protein of ts O45 as a marker for the TGN and isolated a TGN fraction using a combination of conventional cell fractionation techniques and immunoisolation. The TGN was separated from the bulk of the endoplasmic reticulum, mitochondria, lysosomes, plasma membrane, and endosomes, while the activity of trans-Golgi marker galactosyltransferase copurified with the G protein. Using G protein as the TGN marker we have determined that the TGN was enriched 25-fold in the final fraction relative to the total homogenate. Several polypeptides (Mr 75,000, 87,000, 92,000, and 120,000) copurified with the G protein in the isolated TGN fraction and most likely represent resident markers of the compartment.