A 300,000-mol-wt intermediate filament-associated protein in baby hamster kidney (BHK-21) cells

Native intermediate filament (IF) preparations from the baby hamster kidney fibroblastic cell line (BHK-21) contain a number of minor polypeptides in addition to the IF structural subunit proteins desmin, a 54,000-mol-wt protein, and vimentin, a 55,000-mol-wt protein. A monoclonal antibody was produced that reached exclusively with a high molecular weight (300,000) protein representative of these minor proteins. Immunological methods and comparative peptide mapping techniques demonstrated that the 300,000-mol-wt species was biochemically distinct from the 54,000- and 55,000-mol-wt proteins. Double-label immunofluorescence observations on spread BHK cells using this monoclonal antibody and a rabbit polyclonal antibody directed against the 54,000- and 55,000-mol-wt proteins showed that the 300,000-mol-wt species co-distributed with IF in a fibrous pattern. In cells treated with colchicine or those in the early stages of spreading, double-labeling with these antibodies revealed the co-existence of the respective antigens in the juxtanuclear cap of IF that is characteristic of cells in these physiological states. After colchicine removal, or in the late stages of cell spreading, the 300,00-mol-wt species and the IF subunits redistributed to their normal, highly coincident cytoplasmic patterns. Ultrastructural localization by the immunogold technique using the monoclonal antibody supported the light microscopic findings in that the 300,000-mol-wt species was associated with IF in the several physiological and morphological cell states investigated. The gold particle pattern was less intimately associated with IF than that defined by anti-54/55 and was one of non-uniform distribution along IF, being clustered primarily at points of proximity between IF, where an amorphous, proteinaceous material was often the labeled element. Occasionally, "bridges" of label were seen extending outward from such clusters on IF. Gold particles were infrequently bound to microtubules, microfilaments, or other cellular organelles, and when so, IF were usually contiguous. During multiple cycles of in vitro disassembly/assembly of the IF from native preparations, the 300,000-mol-wt protein remained in the fraction containing the 54,000- and 55,000-mol-wt structural subunits, whether the latter were in the soluble state or pelleted as formed filaments. In keeping with the nomenclature developed for the microtubule-associated proteins (MAPs), the acronym IFAP-300K (intermediate filament associated protein) is proposed for this molecule.     

Isolation and characterization of baby hamster kidney (BHK-21) cell modulator protein.

A Ca2+-dependent modulator protein has been isolated from BHK-21 cells. The purification requires heat treatment, ion-exchange chromatography, and gel filtration. The protein appears homogenous on sodium dodecyl sulfate--polyacrylamide and isoelectric focusing gels. The protein comigrates with purified smooth muscle and brain modulators. BHK-21 modulator is characterized by a high content of aspartic and glutamic acids and by a high phenylalanine/tyrosine ratio. It lacks both cysteine and tryptophan. The protein is effective in activating brain-modulator-deficient phosphodiesterase. It can also be used in assay systems to generate Ca2+-sensitive actin activation of both BHK-21 and smooth muscle myosins. Therefore, it is proposed that the BHK-21 modulator protein is a component of the Ca2+-dependent mechanism involved in the regulation of actin--myosin interactions in BHK-21 cells.     

Rapid inhibition of pinocytosis in baby hamster kidney (BHK-21) cells following infection with vesicular stomatitis virus

Infection of baby hamster kidney cells with vesicular stomatitis virus (VSV) caused a reduced rate of pinocytosis (as judged by the uptake of horseradish peroxidase) after 1 h, and maximum inhibition (60-80%) was observed at 4-6 h. This inhibition occurred 2-3 h before release of virus or changes in cell morphology. Analytical cell fractionation of homogenates of VSV-infected cells indicated that the horseradish peroxidase taken up by pinocytosis was transferred to lysosomes. The inhibition of pinocytosis required viral gene expression: little or no inhibition was detected in cells infected with UV-irradiated virus, wild-type virus in the presence of cycloheximide, or a temperature- sensitive mutant which failed to synthesize viral proteins. When cells were infected with temperature-sensitive viruses with mutations in the five VSV genes, an inhibition of pinocytosis was observed only when the viral transmembrane glycoprotein was present on the surface of the cells.     

Genome modification in two lines of baby hamster kidney fibroblasts (BHK-21).

Reasons for the different levels of 5-methyl cytosine encountered in the DNA of two baby hamsters kidney fibroblast lines, BHK-21/C13 and BHK-21/PyY have been investigated. From enzymic studies it does not seem that there are large numbers of potentially methylatable cytosine residues in the C13 line DNA which contains a lower level of 5-methyl cytosine. Rather it is possible that the difference may be due to the reiteration in the PyY strain of certain sequences containing 5-methyl cytosine which simply occur less frequently in the other line.     

Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells

Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of approximately 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-nm filaments has been achieved by treatment of FC with 6 mM sodium- potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.     

Cytotoxicity of trichothecene mycotoxins isolated from Fusarium sporotrichioides (MC-72083) and Fusarium sambucinum in baby hamster kidney (BHK-21) cells

Twenty-six trichothecene mycotoxins produced by Fusarium sporotrichioides (MC-72083) and Fusarium sambucinum were screened for relative cytotoxicity in cultured baby hamster kidney (BHK-21) cells. The relative cytotoxicity was measured as LC₁₀₀. The most cytotoxic trichothecenes were T-2 toxin (5 ng/ml) and the recently isolated 4-propanoyl HT-2 (5 ng/ml) and 3-hydroxy T-2 toxin (5 ng/ml). T-2 tetraol (1 × 10⁴ ng/ml), 8--hydroxytrichothecene (1 × 10⁴ ng/ml), sporotrichiol (2 × 10⁴ ng/ml), 8-oxodiacetoxyscirpenol (6 × 10⁴ ng/ml) and 8-acetyl T-2 tetraol (1 × 10⁵ ng/ml) were the least toxic of the regular trichothecenes. None of the modified trichothecenes or the apotrichothecene were very cytotoxic: 8--hydroxysambucoin (2 × 10³ ng/ml), FS-1 (5 × 10³ ng/ml), 8--hydroxysambucoin (8 × 10⁴ ng/ml) and trichotriol (1 × 10⁵ ng/ml). The modified trichothecenes, FS-2 and FS-3, were not toxic even at 1 × 10⁵ ng/ml. The baby hamster kidney cell bioassay proved to be a very sensitive and reproducible means of screening new trichothecene mycotoxins for relative cytotoxicity.     

Uptake and excretion of polyamines from baby hamster kidney cells (BHK-21/C13) the effect of serum on confluent cell cultures

In confluent cultures of BHK-21/C13 cells there was little uptake oxogenous polyamines and only a low level of polyamine biosynthesis. These cultures continously excreted polyamines into the extracellular medium. Spermidine, in both the free and bound form, was the predominant excretion product, whereas the major intracellular polyamine was spermine implying that excretion of polyamines was specific. Reinitiation of growth by the addition of fresh serum immediately increased the uptake of exogenous putrescine, increased the biosynthesis of polyamines and decreased the excretion of polyamines. Thus, polyamine transport into and out of the cell appears to be regulated by the growth status of that cell.     

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.     

Baby hamster kidney (BHK-21/C13) cells can express striated muscle type proteins

When baby hamster kidney (BHK-21/C13) cell lines are subjected to low-serum medium, cell morphology changes from polygonal to elongated and occasionally fusion of cells is also observed. BHK-21 cells initially growing in Eagle's modified minimum essential medium (EMEM) containing 10% newborn bovine serum were induced to differentiate by changing the culture medium after the cells had grown to confluency. After this point the cells were grown in a low-serum medium (EMEM with 2% normal horse serum), for at least 4 days. The expression of different muscle-specific proteins (desmin, titin and skeletal muscle myosin) and of tropomyosins was studied in both polygonal and elongated BHK-21 cells using the indirect-immunofluorescence assay, two-dimensional (2D)-gel electrophoresis and immunoblotting. Filamentous staining was found with the desmin antisera in the polygonal cells and at all stages of BHK-cell elongation. While no reaction was seen with the titin and myosin antibodies in the polygonal cells, a punctate staining reaction for titin was detected 2 days after medium-change, although the cells had not yet elongated. After 4 days titin was found in a striated pattern. Filamentous staining was seen with the skeletal-muscle-specific myosin antibody at this stage. Confirmatory results were obtained from immunoblotting assays and 2D-gel electrophoresis of cytoskeletal preparations from undifferentiated and differentiated BHK cells. These latter experiments showed the initiation of tropomyosin expression only in the differentiated cells. The positive staining with antibodies to skeletal muscle myosin and titin indicates a striated-muscle nature of the (elongated) BHK-21/C13 cells.     

A model of the binding, entry, uncoating, and RNA synthesis of Semliki Forest virus in baby hamster kidney (BHK-21) cells

A quantitative understanding of viral trafficking would be useful in treating viral-mediated diseases, designing protocols for viral gene therapy, and optimizing heterologous protein production. In this article, a model for the trafficking of Semliki Forest virus and its RNA synthesis in baby hamster kidney (BHK-21) cells is presented. This model includes the various steps leading to infection such as attachment, endocytosis, and viral fusion in the endosome. The model estimates a mean fusion time of 4 to 6 min for the wild-type virus, and 38 min for Fus-1, an SFV mutant which requires a lower pH for fusion. These mean fusion times are consistent with the time-scale of endosomal acidification, suggesting viruses fuse almost instantaneously with the endosomal membrane as soon as the pH of the endosome drops below the pH threshold of the virus. Infection is most likely controlled at the level of viral uncoating, as shown by the close agreement between the efficiency of uncoating and the experimentally determined fraction of viruses that is infectious. The viral RNA synthesized per cell is best described by assuming that it depends on the number of uncoated viruses prior to the onset of replication according to a saturation-type expression. A Poisson distribution is used to determine the distribution of uncoated viruses among the cells. Because attachment is the rate-limiting step in the uncoating of the virus, increasing the attachment rate can lead to enhanced RNA synthesis and, hence, new virion production. Such an increase in the attachment rate may be obtained by lowering the medium pH or the addition of a polycation. (c) 1995 John Wiley & Sons, Inc.     

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.     

Ultrastructure of BHK-21 cells treated with dopamine

The ultrastructure of a suspension or of adhered BHK-21 cells exposed to dopamine has been studied. It was found that ultrastructural modifications were observed mainly in cellular regions enriched in actin, including in intercellular desmosome-like contacts, microvilli, and under the membrane cortical layer. Desmosome-like contacts are hypertrophied with increased electron density and fibrillar bridges that appear in specialized contacts. Microvilli fused with each other and with plasma membrane of neighboring cells or were split up and penetrated by fibrils. The cytoplasm is filled with randomly arranged F-actin filaments and has a blotchy appearance. The effect of dopamine is more pronounced in nonattached cells with undifferentiated actin cytoskeletons than in spread cells with stress fibers. The blockage of the D2 receptor with haloperidol does not affect the ultrastructure of BHK-21 or dopamine induced modifications. The data obtained suggest direct interactions between dopamine and the actin cytoskeleton.     

Adenosine modulates cell growth in baby hamster kidney (BHK) cells

Adenosine is known to modulate cell growth in a variety of mammalian cells either via the activation of receptors or through metabolism. We investigated the effect of adenosine on Baby Hamster Kidney (BHK) cell growth and attempted to determine its mechanism of modulation. In wild-type BHK cells, adenosine evoked a biphasic response in which a low concentration of adenosine (1-5 microM) produced an inhibition of colony formation but at higher concentrations (up to 50 microM) this inhibition was progressively reversed. However, no biphasic response was observed in an "adenosine kinase" deficient BHK mutant, "5a", which suggests that adenosine kinase plays an important role in the modulation of growth response to adenosine. Adenosine receptors did not appear to have a role in regulating cell growth of BHK cells. Specific A1 and A2 receptor antagonists were unable to reverse the effect of adenosine on cell growth. Even though a specific A3 adenosine receptor antagonist MRS-1220 partly reversed the inhibition in colony formation at 1 microM adenosine, it also affected the transport of adenosine. Thus adenosine transport and metabolism appears to play the major role in this modulation of cell growth as 5'-amino-5'-deoxyadenosine, an adenosine kinase inhibitor, reversed the inhibition of cell growth observed at 1 microM adenosine. These results, taken together, would suggest that adenosine modulates cell growth in BHK mainly through its transport and metabolism to adenine nucleotides.     

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.     

The transport of L-alanine by the hamster kidney cell line BHK-21-C13.

The uptake of L-alanine into BHK21-C13 cells in culture has been studied. This amino acid appears to be transported essentially via a relatively low affinity, high capacity, sodium ion dependent transport system. Inhibition studies using other amino acids or their analogues provided information about the specificity of this system. This alanine transport system was shown to exhibit a broad substrate specificity and appeared to be capable of transporting most naturally occurring neutral alpha-amino acids. Kinetic studies of the inhibition of L-alanine uptake also indicated the presence of a second neutral amino acid transport system capable of transporting this amino acid. However, it is unlikely that this second uptake system contributes greatly to L-alanine uptake. Inhibition of the uptake of L-leucine indicated that this transport system has a similar specificity to the "L"-system initially described for Ehrlich ascites carcinoma cells.     

The acidic amino acid transport system of the baby hamster kidney cell line BHK21-C13

The uptake of L-glutamate into BHK21-C13 cells in culture has been studied. This amino acid appears to be transported via a relatively high affinity, low capacity, Na+-dependent transport system capable of the rapid accumulation of substrate amino acids. Kinetic studies of the inhibition of L-glutamate uptake has provided information as to the substrate and the molecular configuration required for transport via the glutamate transport system. This system exhibited marked substrate specificity and was only capable of transporting L-glutamate and aspartate and certain closely related acidic amino acid analogues.     

Chromosome condensation may enhance X-ray-related cell lethality in a temperature-sensitive mutant (tsBN2) of baby hamster kidney cells (BHK21)

In the tsBN2 cell line, which has a temperature-sensitive defect in the regulatory mechanism for chromosome condensation, the lethal effect of X rays was enhanced by incubating the cells at a nonpermissive temperature (40 degrees C) following X irradiation. This enhancement was suppressed in the presence of cycloheximide, which inhibits induction of premature chromosome condensation. The findings obtained in the case of delayed incubation at 40 degrees C and in synchronized cells indicate that X-ray-related potentially lethal damage, which can be expressed by chromosome condensation, is produced in the cells at any stage of the cell cycle, but it is repairable for all cells except those at around the late G2-M phase, where chromosome condensation occurs at a permissive temperature (33.5 degrees C). These observations suggest that the high sensitivity of late G2-M cells to X rays is caused by the events associated with chromosome condensation.     

Purine and pyrimidine modification of growth and surface properties of baby hamster kidney cells (BHK21/C13)

Hypoxanthine or adenosine and thymidine added to soft agar medium stimulated the growth of non-transformed BHK21/C13 cells. Increases in concanavalin A agglutinability and synthesis of a plasminogen activator also occurred. Thymidine was required for colony formation in agar but not for increased lectin agglutinability or activator activity.     

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.