A rapid procedure for purification of EcoRI endonuclease.

A convenient and rapid procedure has been developed to purify restriction endonuclease Eco RI. The method involves sonication of cells at low ionic strength, precipitation of the endonuclease with Polymin P (a polyethyleneimine), elution of the enzyme from the Polymin P precipitate, ammonium sulfate precipitation and chromatography on phosphocellulose. The purified restriction endonuclease is free of exonuclease and other endonucleases.     

Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum     

The different actions of normal and supranormal calcium concentrations on the proliferation of BALB/c 3T3 mouse cells

Summary In the presence of a normal (1.25 to 1.80 mM) calcium concentration, addition of fresh bovine calf serum or completely changing the medium induces proliferatively quiescent BALB/c 3T3 mouse cells in dense cultures to start a growth division cycle and initiate DNA synthesis about 12 hr later. Fresh, low-calcium (0.02 mM physiologically available) medium also causes cells to start a growth-division cycle. However, the development of such stimulated, calcium-deprived cells stops just before the expected time of initiation of DNA synthesis, which can then be rapidly induced by restoration of the normal calcium concentration. Simply raising the calcium concentration to nonphysiologically high levels (without otherwise altering the medium) can mimic the action of fresh serum or fresh whole medium by inducing some of the cells in proliferatively quiescent confluent cultures to start a growth-division cycle and initiate DNA synthesis 22 hr later. Issued as NRCC No. 15371.     

Phosphoprotein phosphate activity at the outer surface of intact normal and transformed 3T3 fibroblasts

Using ³²P-labeled phosphocasein or phosphohistones as exogenous substrates it was possible to detect a phosphoprotein phosphate activity on the outer surface of intact normal and transformed 3T3 fibroblasts. Incubation of monolayers of intact cells in buffered salt solution with the radioactively labeled substrate resulted in the release of alkali-labile ³²P counts into the surrounding medium. The reaction was: (a) linear with time (at least up to 20 min); (b) proportional to the cell density; (c) dependent on the temperature and pH of the incubation medium; (d) stimulated by K⁺; and (e) inhibited by sodium fluoride, inorganic pyrophosphate, zinc chloride and relatively impermeant sulfhydryl reagents. Less than 2% of the externally located phosphoprotein phosphatase activity was detectable in pooled cell-free washings of the intact cell monolayer. Phosphocasein did not cause any detectable leakage of intracellular lactate dehydrogenase or soluble phosphoprotein phosphatase activity into the external medium; incubation of the cells with phosphohistones, on the other hand, resulted in appreaciable leakage of both these cytoplasmic activities. Neoplastic transformation was associated with a nearly two-fold decrease in the activity of the surface phosphoprotein phosphatase. Addition of serum to either non-transformed 3T3 or spontaneously transformed 3T6 cells resulted in a rapid and remarkable drop in the cell surface dephosphorylating activity. Acrylamide gel electrophoresis of the dephosphorylated casein or histone substrate revealed no proteolytic degradation or change in electrophoretic mobility. The intact cells showed no damage upon microscopic examination as a result of exposure to phosphocasein or phosphohistones.     

Efficient Establishment of Pig Embryonic Fibroblast Cell Lines with Conditional Expression of the Simian Vacuolating Virus 40 Large T Fragment

The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.     

The heparan sulfates of Swiss mouse 3T3 cells. The effect of transformation

Three major pools of heparan sulfate have been isolated from cultures of Swiss mouse 3T3 and SV40-transformed 3T3 cells: cell-surface, medium, and intracellular heparan sulfates. The cell-surface heparan sulfate is a high molecular weight proteogylcan which is partially degraded by pronase. Before pronase treatment, it has a peak molecular weight (as estimated by gel filtration) of appox. 7.2 · 10⁵ in contrast to only 2.4 · 10⁵ after pronase treatment. The medium heparan sulfate appears to be similar in structure to the cell-surface heparan sulfate, since they coelute on Bio-Gel A-15m and DEAE-cellulose, and are both proteoglycans. In contrast, the intracellular heparan sulfate has a low molecular weight (6.0 · 10³) and has little if any attached protein. Both the medium and intracellular heparan sulfate exhibit the transformation-associated change in structure reported earlier for cell-surface heparan sulfate (Underhill, C.B. and Keller, J.M. (1975) Biochem. Biophys. Res. Commun. 63, 448–454). This transformation-associated change, detected by DEAE-cellulose chromatography is not the result of changes in either molecular weight or protein core. Cellulose acetate electrophoresis of the cell-surface heparan sulfate at pH 1 suggests that the transformation-associated change in structure is due to a difference in sulfate content. Both types of heparan sulfate are produced in mixed cultures ot 3T3 and SV3T3 cells, indicating that neither serum factors in the culture medium nor secreted cell products are responsible for the transformation-associated change in heparan sulfate structure. The presented date are discussed with respect to the postulated role of heparan sulfate in cell social behavior.     

Dependence of interfacial properties of normal and transformed 3T3 cell membranes on treatment with factors modifying proliferation

Interfacial properties of the outer cell membrane of normal and transformed in vitro cultures of mouse 3T3 cells have been investigated. The contact angles of sessile drops on dried cell preparations were measured and the interfacial tensions derived using the thermodynamic approach introduced by Neumann. Interfacial tensions were found to be within an order of magnitude of those determined for other cell and model membranes. Treatment of cells with calf serum, a stimulant to proliferation, resulted in a decrease in the interfacial tension of normal and transformed cells, whereas use of concanavalin A and its succinylated derivative lead to an increase of interfacial tensions of both cell types. These and further results show a detailed correlation between the growth-regulating effects and the effects on interfacial properties of these proliferation-modifying factors. An inter-pretation of the results of serum depression of the interfacial tension in terms of a binding equilibrium dependent on the concentration of humoral growth factors in the medium is attempted.     

Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using BALB/c 3T3 mouse embryo fibroblasts

Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (¹²⁵I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with K _d =59.6 pM and an estimated 1.57×10⁵ receptors/cell. Half-maximal displacement of bound¹²⁵I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for¹²⁵I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that¹²⁵I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Apoptosis observed in BALB/3T3 cells having ingested Staphylococcus aureus.

Staphylococcus aureus was previously shown to be internalized by murine fibroblast. We examined the intracellular events of S. aureus ingested by BALB/3T3 cells. After uptake of strains A191 and A151, isolates from atopic lesion, and a laboratory strain, Cowan I, for 1 hr, BALB/3T3 cells were incubated with 1.25 microg/ml lysostaphin. Laddering of the DNA in multiples of approximately 180 bp occurred within 4 hr following bacterial addition in BALB/3T3 cells infected with A191 and within 18 hr in BALB/3T3 cells infected with A151: histochemical staining by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method revealed that the rate of the fragmentation of nucleic DNA in Cowan I-infected BALB/3T3 cells at 21 hr following bacterial addition was 0.52 +/- 0.25%, significantly higher than that in the control cells. Transmission electron micrographs of BALB/3T3 cells at 4 hr following A191 addition showed that the apoptotic features, including electron-dense nucleus and plasma membrane blebbing, occurred in some cells in which many staphylococci escaped the endosome and went on to cell division. At the same time, A151 organisms enclosed with endosome membrane were static in the intact BALB/3T3 cells. The significant increase of A191 was confirmed by counting intracellular live bacteria during 2- to 6-hr incubation. These results suggest that internalized S. aureus escapes the endosome, multiplies and induces apoptosis in the fibroblast cell.     

Mobile phone base station radiation does not affect neoplastic transformation in BALB/3T3 cells

A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields affect malignant transformation or other cellular stress responses. Our group previously reported that DNA strand breaks were not induced in human cells exposed to 2.1425 GHz Wideband Code Division Multiple Access (W-CDMA) radiation up to 800 mW/kg from mobile radio base stations employing the IMT-2000 cellular system. In the current study, BALB/3T3 cells were continuously exposed to 2.1425 GHz W-CDMA RF fields at specific absorption rates (SARs) of 80 and 800 mW/kg for 6 weeks and malignant cell transformation was assessed. In addition, 3-methylcholanthrene (MCA)-treated cells were exposed to RF fields in a similar fashion, to assess for effects on tumor promotion. Finally, the effect of RF fields on tumor co-promotion was assessed in BALB/3T3 cells initiated with MCA and co-exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). At the end of the incubation period, transformation dishes were fixed, stained with Giemsa, and scored for morphologically transformed foci. No significant differences in transformation frequency were observed between the test groups exposed to RF signals and the sham-exposed negative controls in the non-, MCA-, or MCA plus TPA-treated cells. Our studies found no evidence to support the hypothesis that RF fields may affect malignant transformation. Our results suggest that exposure to low-level RF radiation of up to 800 mW/kg does not induce cell transformation, which causes tumor formation.     

The influence of fluoro-phenyl-alanine on the synthesis of simian virus 40 DNA and T antigens.

Treatment of Simian Virus 40 (SV40) infected monkey cells with fluorophenylalanine (FPA) resulted in increased uptake of thymidine by the cells, and progressive inhibition of both viral and cellular DNA synthesis. Viral DNA synthesis was more sensitive to inhibition by FPA than cell DNA synthesis. Synthesis of SV40 T antigens was however unaffected by FPA, as judged from immunofluorescence assays. The M.W. of the major polypetides immunoprecipitated from cell extracts by antibodies from tumor bearing hamster sera was similarly unaffected. It is suggested that T antigen synthesized in the presence of FPA is non functional.     

Plate-Induced tumors of BALB/3T3 cells exhibiting foci of differentiation into pericytes,chondrocytes, and fibroblasts

The BALB/3T3 clone A31 mouse embryo cell line has been used by many investigators as a model “normal” “fibroblast” line for a variety of in vitro studies. It has been shown, however, that these cells are not “normal” because they will produce tumors within 2–4 months if 3 × 10⁴ cells are implanted subcutaneously in BALB/c mice attached to 0.2 × 5 × 10-mm plastic plates. Previous studies also suggested that these cells were not fibroblasts because they gave rise to tumors with the characteristics of vascular endothelium not fibroblasts. We now report that BALB/3T3 (clone A31), BALB/3T3-T, a proadipocyte subclone of clone A31 cells, and six recent subclones of BALB/3T3-T cells show additional differentiation patterns when tumors derived by implantation of these cells attached to plastic plates are examined. Differentiation into pericytes, chondrocytes, and fibroblasts was observed. We conclude that the BALB/3T3 clone A31 cell line and related lines are multipotent mesenchymal cells which are capable of differentiation into a variety of cell types.     

The role of calcium ions in the permeability changes produced by external ATP in transformed 3T3 cells

External ATP causes a rapid increase in passive permeability to nucleotides and phosphate esters in transformed cell lines, such as 3T6 mouse fibroblasts. However, untransformed lines, such as 3T3, do not show a similar sensitivity to external ATP. Ca²⁺ inhibits permeabilization, but only at concentrations approaching those of external ATP. In contrast, La³⁺ and Tb³⁺ inhibit ATP-dependent permeabilization at one-fifth the concentration of external ATP. Considering reports that lanthanides can substitute for calcium ion in many enzymatic reactions, often with a higher affinity, it would appear that Ca²⁺ plays a specific role in the maintenance of a passive membrane permeability barrier and in opposing the effects of external ATP.Other data suggest a regulatory role for the Ca²⁺-calmodulin complex in the permeabilization process. Trifluoperazine, chlorpromazine and W-7, compounds which inhibit cellular functions dependent on the Ca²⁺-calmodulin complex, are able to enhance the effect of external ATP. Thus, a dramatic stimulation of nucleotide permeability occurs with concentrations of external ATP and inhibitor that are ineffective when added alone. Calmodulin antagonists and low concentrations of external ATP increased membrane permeability to Na⁺ and K⁺ as was previously shown for permeabilization with ATP alone. Earlier studies have shown that energy inhibitors which reduce intracellular ATP levels greatly increase the sensitivity of transformed cells to external ATP. However, the Ca²⁺-calmodulin antagonists used in the present study exert their effects at concentrations which do not alter intracellular ATP levels.     

Elevated intracellular concentrations of cyclic AMP inhibited serum-stimulated, density-arrested BALB/c-3T3 cells in mid G1

The stimulation of DNA synthesis in quiescent, density-arrested BALB/c-3T3 cells by platelet-derived growth factor in plasma-supplemented medium was inhibited by the presence of isobutylmethylxanthine (IBMX) and cholera toxin, although neither IBMX or cholera toxin when used alone inhibited the stimulation of DNA synthesis. The cells were reversibly inhibited in mid G1 at a point 6 hr prior to the initiation of DNA synthesis. The inhibition of cell cycle traverse was associated with a 10-15 fold increase in cellular cyclic AMP concentration over basal levels. The reversal of this inhibition by removal of IBMX was correlated with a dramatic decrease in cyclic AMP levels. The traverse of G1 and the initiation of DNA synthesis after release from the cholera toxin and IBMX inhibition was dependent on the presence of plasma in the medium. Either somatomedin C (10-20 ng/ml) or insulin (10(-6)-10(-5) M) completely replaced the plasma requirement for late G1 progression and entry into S phase. Once the inhibited cells were released from the IBMX and cholera toxin block a subsequent increase in cyclic AMP did not prevent entry into S phase. The presence of cholera toxin alone inhibited the stimulation of human dermal fibroblasts. The elevation of intracellular cyclic AMP levels in the human dermal fibroblasts by cholera toxin was two to three fold greater than that found in the BALB/c-3T3 cells in the presence of cholera toxin and the IBMX.     

致死型夏氏疟原虫感染BALB/c小鼠CD4~＋ CD25~＋ Foxp3~＋调节性T细胞在Th2免疫应答极化中的作用研究

为探讨CD4＋ CD25＋ Foxp3＋调节性T细胞（Treg细胞）在疟疾感染过程中对Th2极化的调控作用,利用Treg细胞消除的致死型夏氏疟原虫（Plasmodium chabaudi chabaudi AS,P.c chabaudi AS）感染鼠疟模型进行研究。结果显示,对照组小鼠在感染后8 d原虫血症达到峰值40.5%,随后迅速下降,于感染后18 d小鼠自愈。相比,Treg细胞消除组于感染后10 d,原虫血症水平迅速上升至32%,随后小鼠相继死亡。在感染后8～10 d,Treg细胞消除小鼠脾脏CD4＋ CD25＋ Foxp3＋细胞占CD4＋细胞百分比含量明显低于对照组。同时,血清疟原虫特异性抗体IgG1和IgG2a水平均明显降低。结果提示,P.c chabaudi AS感染中CD4＋ CD25＋ Foxp3＋细胞参与调控Th2型免疫应答的极化,进而干预疟原虫清除。     

Sodium butyrate inhibits the synthesis of the transformation related protein p 53 in 3T6 mouse fibroblasts

Sodium butyrate, which blocks the cell cycle of many cell types in the G1 phase, strongly inhibits the synthesis of the transformation related, 53 kDa protein in 3T6 fibroblasts but much less so in SV 40 transformed mouse cells. By several criteria, this effect of the fatty acid appears to be indirect; p 53 synthesis takes place several hours after the butyrate-sensitive step in G1. The results are discussed in the light of a putative role of p 53 in growth control.     

野生及笼养猕猴SV40T抗原基因检测方法的建立

目的建立猴外周血单核细胞SV40DNA的PCR检测方法,对猕猴SV40T抗原基因进行检测。方法使用PCR方法对分别来自野外（云南宁蒗71只、景东60只）及笼养猕猴（64只）的SV40大T抗原基因进行检测,同时对扩增出的阳性结果进行测序。结果在195份样本中,有4只来自野生猴样本扩增出SV40大T抗原基因（3．1％,4／131）,1只来自笼养猴样本扩增出SV40大T抗原基因（1．6％,1／64）。测序结果显示：猴血样本扩增片段序列与GenBank中的SV40大T抗原C-末端的基因序列片段有15个核苷酸不同（3．3％差异）,与SV40．776标准株的序列基本一致,但SV40—776在nt3020处有一缺口。结论云南野生及笼养猕猴猴群均能检出SV40T抗原基因。因此建立SV40病毒的DNA检测技术,对用于科研及疫苗生产的实验猕猴的病毒学质量控制具有重要意义。     

Nucleotide sequence-directed mapping of the nucleosomes of SV40 chromatin

In our previous work we have shown by comparison of experimental and computational data that the positions of the histone octamers bound to the DNA molecule appear to be completely sequence-dependent. This provides a convenient and quick method for locating the nucleosomes along the DNA molecule, as soon as the nucleotide sequence is known. Using this computational approach, the complete nucleosomal map of the SV40 minichromosome has been constructed. The map consists of 25 nucleosomes, with their coordinates (centers) being specified with high accuracy. The map is found to be in remarkable agreement with available experimental data.