Effects of chloroquine and cytochalasin B on the infection of cells by Sindbis virus and vesicular stomatitis virus

The effects of cytochalasin B and chloroquine on the process of endocytosis of Sindbis virus particles and polystyrene spheres were determined by electron microscopy. The effects of these agents on the process of infection (attachment, penetration, and uncoating) of BHK-21 cells by Sindbis virus and vesicular stomatitis virus were also determined. Cytochalasin B completely blocked ingestion of Sindbis virus particles or latex spheres by BHK cells but had no effect on the ability of Sindbis virus or vesicular stomatitis virus to infect or replicate in BHK cells. Chloroquine did not inhibit the ingestion of either latex spheres or virus particles but greatly reduced the yields of virus produced. These data suggest that endocytosis is not essential for the infection of cultured cells by Sindbis virus or vesicular stomatitis virus.     

Aujeszky’s disease virus production in disposable bioreactor

A novel, disposable-bag bioreactor system that uses wave action for mixing and transferring oxygen was evaluated for BHK 21 C13 cell line growth and Aujeszky’s disease virus (ADV) production. Growth kinetics of BHK 21 C13 cells in the wave bioreactor during 3-day period were determined. At the end of the 3-day culture period and cell density of 1.82 × 10⁶ cells ml^-1, the reactor was inoculated with 9 ml of gE^- Bartha K-61 strain ADV suspension (10^5.9 TCID₅₀) with multiplicity of infection (MOI) of 0.01. After a 144 h incubation period, 400 ml of ADV harvest was obtained with titre of 10^7.0 TCID₅₀ ml⁻¹, which corresponds to 40,000 doses of vaccine against AD. In conclusion, the results obtained with the wave bioreactor using BHK 21 C13 cells showed that this system can be considered as suitable for ADV or BHK 21 C13 cell biomass production.     

Localization of vimentin and desmin in BHK21/C13 cells and in baby hamster kidney

Specific antibodies against the intermediate filament protein subunits, desmin and vimentin, were used to characterize the fibroblastic tissue culture cell line BHK21/C13 and the cells comprising baby hamster kidney (BHK). The BHK21/C13 cells have previously been shown to contain desmin and vimentin by biochemical techniques. The results from double immunofluorescence analysis show that both immunologically distinct intermediate filament subunit proteins are expressed simultaneously within the same BHK21/C13 cell, and that the filamentous patterns are very similar, if not superimposable even in cells treated with colchicine. There are some cells that may contain vimentin only. Double immunofluorescence on cryostat sections of BHKs and preparations of dissociated kidney cells demonstrate that the cells, most likely smooth muscle, comprising the blood vessel walls contain vimentin and desmin simultaneously. The simultaneous expression of vimentin and desmin is not a phenomenon which is restricted to tissue culture cells. Thus, the simultaneous presence of these two intermediate filament proteins within the BHK21/C13 cell may not be the result of growth in tissue culture.     

高通量miRNA活性谱检测发现BHK21细胞中miR-206高活性

用我们建立的活细胞miRNA活性谱检测技术测定BHK21、HEK293和Vero三种肾来源细胞系中58种miRNA活性谱,发现miR-206在BHK21细胞中具有特征性高活性.鉴于miR-206是典型的横纹肌特征性miRNA,进一步以小鼠成肌细胞C2Cl2及人胚肾细胞HEK293为对照,检测了BHK21细胞中miR-206的活性及表达水平.之后,用马血清诱导培养BHK21细胞,检测诱导前后BHK21细胞中骨骼肌肌球蛋白重链(Slow skeletal myosin heavy chain,MHC)表达情况,miR-206活性和表达水平以及受miR-206负调控的Connexin43(Cx43)的表达水平.结果发现,miR-206在BHK21细胞中活性和表达水平都明显高于C2C12细胞;马血清诱导后,BHK21细胞中MHC表达水平升高,miR-206活性和表达水平都升高,而Cx43表达水平下降.结果提示BHK21细胞具有成肌细胞特性.本研究首次发现BHK21细胞中miR-206的高活性,从miRNA角度证实了BHK21细胞来源于肾间质细胞,而不是肾实质细胞.研究结果还提示BHK21细胞有可能作为一种体外模型用于miR-206的功能研究.     

Fibroblast Growth Factor stimulates anchorage independent growth in agar of BHK21/13 and SV3T3 cells

The effect of Fibroblast Growth Factor (FGF) on the growth in suspension in soft agar of secondary baby hamster kidney fibroblasts, the partially transformed cell line BHK21/13 and its virally transformed derivative SV28 was studied. Only BHK21/13 cells could be induced by FGF to form colonies in soft agar. Secondary baby hamster kidney fibroblasts did not grow in suspension even with FGF, whereas SV28 grew independently of the presence of FGF. Thus viral transformation of BHK21/13 was not a prerequisite for growth in agar if FGF was present. Epidermal Growth Factor (EGF) was ineffective at promoting growth in agar of secondary baby hamster kidney fibroblasts or BHK21/13 cells. Balb/c 3T3 behaves like the secondary baby hamster kidney and SV3T3 like BHK21/13. We conclude there are three degrees of transformation with different potentials for growth in agar.     

Modifications of sialylation in BHK 21/C13 cells after in vitro transfection by c-Ha-ras human oncogene

A Hamster kidney fibroblast cell line (BHK 21/C13) has been transfected by c-Ha-ras human oncogene. The expression of the oncogene significantly modified the global sialytransferase activity of the cell extract. This activity is enhanced on an average 2.5 fold whatever the acceptor. In addition, the proportion of cell-surface associated N-glycolylneuraminic acid is enhanced in transfectants, the ratio N-acetylneuraminic acid/N-glycolylneuraminic acid decreases from 7.10 to 2.80. These results suggest that a tight relationship exists, in c-Ha-ras transfected BHK cells, between the expression of the oncogene and the neuraminic acid metabolism as well as a gene regulation of glycoconjugate sialylation.     

Immunofluorescent evidence for a transient association of actin with phagosomes in BHK 21 cells

The association of actin with latex phagosomes in BHK 21 cells, a normal fibroblastic cell line, was studied using fluorescent antibody to anti-DNase I. DNase I binds specifically to actin and in fixed, acetone-extracted BHK cells much of the immunofluorescent staining is restricted to microfilament bundles. A concentrated rim of actin specific fluorescence is observed about newly formed phagosomes. After long ingestion periods or a chase, most latex phagosomes appear as black holes devoid of any actin specific fluorescence. The data indicate that actin polymerization about phagosomes in fibroblasts is a transient event persisting for approximately 2 min.     

Engineering the microstructure and permeability of thin multilayer latex biocatalytic coatings containing E. coli.

The microstructure and permeability of rehydrated 20-100 microm thick partially coalesced (vinyl-actetate acrylic copolymer) SF091 latex coatings and a 118 microm thick model trilayer biocatalytic coating consisting of two sealant SF091 layers containing a middle layer of viable E. coli HB101 + latex were studied as delaminated films in a diffusion apparatus with KNO(3) as the diffussant. The permeability of the hydrated coatings is due to diffusive transport through the pore space between the partially coalesced SF091 latex particles. Coating microstructure was visualized by fast freeze cryogenic scanning electron microscopy (cryo-SEM). The effective diffusion coefficient of SF091 latex coatings (diffusive permeability/film thickness) was determined as the ratio of the effective diffusivity of KNO(3) to its diffusivity in water (D(eff)/D). Polymer particle coalescence was arrested by two methods to increase coating permeability. The first used glycerol with coating drying at 4 degrees C, near the glass transition temperature (T(g)). The second method used sucrose or trehalose as a filler to arrest coalescence; the filler was then dissolved away. D(eff)/D was measured as a function of film thickness; content of glycerol, sucrose, and trehalose; drying time; and rehydration time. D(eff)/D varied from 3 x 10(-4) for unmodified SF091 coatings to 6.8 x 10(-2) for coatings containing sucrose. D(eff)/D was reduced by the flattening of latex particles against the surface of the solid substrate, as well as by the presence of the colloid stabilizer hydroxyethylcellulose (HEC). When corrected for the flattened particle layer, D(eff)/D of HEC-free coatings was as high as 0.20, which agreed with the value predicted from analysis of cryo-SEM images of the coat surface. D(eff)/D decreased by one-half in approximately 5 days in rehydrated SF091 coatings, indicating that significant wet coalescence occurs after glycerol, sucrose, or trehalose are leached from the films. D(eff)/D of SF091 latex trilayer coatings containing viable E. coli HB101 cells decreased as cell loading was increased from 2.2 x 10(-2) for 64 g dry cell weight per liter of coat volume to 5 x 10(-3) for 151 g DCW/L of coat volume. The reduction in coating permeability with increasing cell loading is predicted by Maxwell's equation for D(eff)/D in periodic composites.     

Regulation of S-adenosyl-L-methionine decarboxylase by 1-aminooxy-3-aminopropane: enzyme kinetics and effects on the enzyme activity in cultured cells

The kinetics of inactivation of adenosylmethionine decarboxylase of rat liver and of baby hamster kidney cells (BHK21/C31) by 1-aminooxy-3-aminopropane was studied. The apparent dissociation constants (Ki) for the hepatic and BHK21/C13 enzymes were 1.5 and 2.0 mM and the times of half-inactivation at infinite concentration of the inhibitor (tau 1/2) were 1.2 and 3.8 min, respectively. Treatment of BHK21/C13 with 0.5 mM 1-aminooxy-3-aminopropane prevented cell growth and depleted the cells of putrescine and spermidine within 1 day. The depletion of spermidine resulted in increased activity of S-adenosylmethionine decarboxylase which was due, at least partly, to the increase in the half-life of the enzyme activity. Because spermine levels were not significantly affected, it appears that spermidine is the principal feedback regulator of S-adenosylmethionine decarboxylase. So, 1-aminooxy-3-aminopropane is a very weak inhibitor of S-adenosylmethionine decarboxylase and the cellular effects can be correlated primarily with its inhibitory effects on ornithine decarboxylase and spermidine synthase. In cell-free systems, however, 1-aminooxy-3-aminopropane is likely to find use in unraveling the reaction mechanism of S-adenosylmethionine decarboxylase.     

Hexokinase activity as marker to assess time of harvest of foot-and-mouth disease virus in BHK21 Cl13 cell cultures

Hexokinase (B.C. 2.7.1.1) activity as a marker enzyme during FMD viral infection has been observed spectrophotometrically in a system coupled with glucose-6-phosphate dehydrogenase, in supernatants of BHK(21)Cl(13) suspension as well as anchored cell culture at a minimum of 10(4) infective virus particles/ml. Specific activity increased with virus concentration in culture supernatants and abruptly decreased with a fall in virus titer, as has been noted by TCID/50,146 S concentration, and enzyme-linked immunosorbent assay (ELISA) readings.     

The lateral mobility of cell membrane components is not altered following cell fusion induced by Sendai virus

The interaction of Sendai virus glycoproteins with cell membranes was proposed to increase the lateral mobility of membrane proteins, enabling membrane fusion and the aggregation of intramembrane particles by thermotropic separation (Volsky, DJ & Loyter, A, Biochim biophys acta 514 (1978) 213 [13]; Maeda, T et al. Exp cell res 123 (1979) 333 [15]; and Kim, J & Okada, Y, Exp cell res 132 (1981) 125 [44]). In order to test this hypothesis, we employed fluorescence photobleaching recovery to investigate the effects of Sendai virus-induced fusion on the lateral mobility of membrane proteins and lipids in a variety of cell types (human erythrocytes, BHK21, HeLa, 3T3 NIH, and mouse spleen lymphocytes). The results of the lateral diffusion measurements demonstrate that no significant alterations occur in the lateral motion of membrane proteins or a fluorescent phospholipid on all the cell types examined, including cells which revealed high susceptibility to the virally mediated fusion (human erythrocytes and BHK21 cells). These findings suggest that a permanent increase in the lateral mobility of cell surface components does not generally occur during Sendai virus-induced cell fusion, and thus cannot play a role in the fusion mechanism. The possible involvement of transient alterations in the lateral mobility of membrane components in the fusion mechanism is discussed.     

The detection of smooth muscle desmin-like protein in BHK21/C13 fibroblasts.

Three distinct proteins, actin (42,000 daltons), the principal form of fibroblast 10 nm filament protein (55,000 daltons), and a protein with a molecular weight of 52,000 and a pI of 5.8 were detected in nonionic detergent-insoluble cytoskeletal and 10 nm filament preparations of control BHK21/C13 and line 9 hamster fibroblasts. Cytoskeletal preparations of other hamster fibroblast cell types, such as NIL8 and primary embryo fibroblasts, contained the 55,000-dalton component but lacked the 52,000-dalton protein. A Rous sarcoma virus transformant of the BHK21/C13 line and an adenovirus transformant of line 9, resembled the NIL8 and other fibroblast types in that they contained only the 55,000- and 42,000-dalton polypeptides. The identity of the 52,000-dalton protein in BHK21/C13 cells was studied. This protein co-electrophoreses on both one- and two-dimensional polyacrylamide gels with the predominant muscle form of 10 nm filament protein. Further, one-dimensional peptide maps of the hamster smooth muscle 10 nm filament protein and the hamster fibroblast 52,000-dalton protein are identical to one another and distinct from the peptide maps of both the 42,000- and the 55,000-dalton components of the fibroblast cytoskeletal preparations. We conclude that BHK21/C13 cells contain both the fibroblast and the muscle form of 10 nm filament protein.     

Glycopeptides derived from individual membrane glycoproteins from control and Rous sarcoma virus-transformed hamster fibroblasts.

The membrane glycoproteins from control (BHK21/C13) and Rous sarcoma virus-transformed (C13/B4) baby hamster kidney cells grown in medium containing [14C]- or D-[3H]glucosamine have been separated into two distinct classes: a phenol-soluble fraction and an aqueous fraction. The membrane glycoproteins from both BHK21/C13 and C13/B4 partitioned similarly into these two fractions. The phenol and aquesous-soluble glycoproteins differed in their sodium dodecyl sulfate-polyacrylamide gel profiles, polyacrylamide isoelectric focusing profiles, and glycopeptide distribution on Sephadex G-50. A number of aqueous and phenol-soluble glycoproteins from BHK21/C13 and C13/B4 cells were purified to near homogeneity by means of polyacrylamide electrophoresis and gel electrofocusing. These glycoproteins range in molecular weight from 179,000 to 31,000 and have isoelectric points of 7.5 to 3.0. Our results show that the pronase glycopeptides of 20 out of 24 homologous membrane glycoproteins of equivalent molecular weight and isoelectric point from BHK21/C13 and C13/B4 cells are dissimilar as measured by Sephadex G-50 gel filtration.     

Studies on the function of cell surface glycoproteins. II. Possible role of surface glycoproteins in the control of cytoskeletal organization and surface morphology

Immunoglobulin from goat antiserum directed against purified surface membranes from transformed BHK21/C13 cells (anti-M) has been shown to cause both control and transformed hamster cells to round and detach from the substrate (see accompanying paper). This paper documents the effects of the antiserum on the cytoskeletal organization and cell surface morphology of control BHK21/C13 cells examined by scanning and transmission electron microscopy. As a result of antiserum-induced rounding, the normally smooth cell surface becomes covered with filopodia and blebs, and the organization of all three components of the filamentous cytoskeleton is altered. In terms of cell surface morphology and cytoskeletal organization, the cells resemble rounded, postmitotic or trypsinized BHK cells rather than cells treated with either anticytoskeletal drugs or lectins. Immunocytochemical and radioimmune assay experiments support the suggestion that the rounding reaction induced by anti-M serum results from the specific interaction of antibodies with molecules on the cell surface. It is suggested that anti-M serum induces alterations in cytoskeletal organization via a transmembrane signal and that cytoskeletal reorganization is a fundamental part of the rounding and detachment process.     

Chikungunya Virus Infection of Cell Monolayers by Cell-to-Cell and Extracellular Transmission

Both cell-to-cell and extracellular transmission of chikungunya virus infection was demonstrated in BHK21/C13 cell monolayers. The mode of virus infection may depend on the cell line. Virus transmission in L-929 and guinea pig lung cell lines was extracellular.     

Isolation of a human X chromosome-linked gene essential for progression from G1 to S phase of the cell cycle

The tsBN462 cell line, a temperature-sensitive (ts) mutant isolated from the hamster cell line, BHK21/13, cannot progress into S phase at 39.5 degrees C, following the release from isoleucine deprivation. The mutant cells were transfected with high molecular weight (HMW) DNA from human KB cells, and several human DNA bands were found to be conserved through three cycles of ts+ transformation. Conserved human DNA was isolated from the cosmid library of the secondary ts+ transformant (K-1-1), using 32P-labelled total human DNA as a probe. The isolated human DNA covers about 70 kb of human DNA flanked with hamster DNA, and originates from the human X chromosome. The middle part (56 kb) of the isolated human DNA was conserved through the primary, secondary and tertiary ts+ transformation, without gross rearrangement.     

The apparent infection of NA-C1300 cell cultures by nucleocapsid material of the Canadian Arctic strain of rabies virus

Murine neuroblastoma (NA-C1300) and baby hamster kidney (BHK-21/C13) cell cultures were infected with the Canadian Arctic strain of rabies virus. Subcultures were passed following incubation for 3 to 4 days at 35 degrees C. The supernatant fluids from the BHK cultures demonstrated increasing infectivity in both NA and BHK cells concomitantly with an increase in the number of parent cells staining with an anti-glycoprotein stain. On the other hand, the supernatant fluids from the NA cultures initially showed higher infectivity in NA cells than in BHK cells. This feature was related to a low production of glycoprotein-staining cells in the parent NA cultures. The reduction of infectivity in NA cells of some NA supernatant fluids (and brain suspensions) by anti-nucleoprotein antibodies suggests that nucleocapsid material is, in some manner, capable of infecting NA cells. Infectivity of this virus strain in experimental mice is also related to the production of glycoprotein and may not be correlated with the degree of infection in NA cell cultures.     

An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells

Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.     

Toxoplasma gondii: virulence of tachyzoites in serum free media at different temperatures

Highly virulent Toxoplasma gondii tachyzoite multiplication was recorded on the 4th and 5th days post cultivation (dpc) in seven selected cell lines either with or without fetal calf serum (FCS) in the maintenance media. The multiplication rate was slightly lower in the absence of FCS. The cell line mono-layers collapsed dying by the 6th day of infection both in presence or absence of FCS at 37 degrees C. Carcinoma of human larynx (Hep2) and Madian Darby Bovine Kidney (MDBK) cell lines were the most suitable for in vitro multiplication, followed by that of African green monkey kidney cells (VERO), pooled kidney from 1-day-old hamster (BHK), rabbit kidney cells (RK13) and human rhabdomyosarcoma (RDA), while Chicken embryo cells (CER) were the least suitable. In absence of FCS, CER, BHK, Hep2, RDA and MDBK were able to maintain virulent tachyzoites at +4 degrees C for 14 days. The infectivity of the tachyzoites was however lower, killing 40% of the inoculated mice. Tachyzoites survived at room temperature, in the dark, for 14 days in Hep2, RDA and MDBK. However, Hep2 was the only one able to keep virulent tachyzoites until 21 dpc at room temperature and at +4 degrees C. Hep2 propagated tachyzoites were still alive but with low infectivity up to 28 dpc. The cell-lines failed to support the development of tachyzoites after 7 dpc at 37 degrees C and after the 35 dpc at lower temperatures.     

The role of 5-S RNA in temperature-sensitive ribosomal RNA maturation.

The synthesis of 5-S RNA was found to be unchanged at both the permissive (33.5 degrees C) and non-permissive (38.5 degrees C) temperatures in a temperature-sensitive Baby Hamster Kidney cell line (BHK 21 ts 422 E) as measured relative to synthesis of 18-S rRNA. The 5-S RNA is shown to be associated with nucleolar ribonucleoprotein particles even though rRNA processing does not yield a functional 28-S rRNA at the non-permissive temperature. The amount of 5-S RNA found associated with the 80-S ribonucleoprotein particles was the same at the permissive and non-permissive temperatures, indicating that an aberrant 5-S RNA contribution to rRNA processing is not a primary cause for the temperature-sensitive lesion of rRNA maturation in this mutant cell line. The amount of 5-S RNA in nucleolar 80-S RNA particles indicated that the association of 5-S RNA with the rRNA precursor particle occurs before the cleavage step at which 32-S precursor RNA is produced.