New lines of spontaneously transformed cells obtained from "precrisis" cultures of embryonic rat cells

A new approach to selection of lines of spontaneously transformed cells from the rat embryo "precrisis" cultures is described and their phenotypes at the initial and advanced stages during a long-term cultivation are characterized. The new selective system, referred to as 2T7, differs from the well known 3T3, 2T6 and 3T12 systems (Todaro, Green, 1963; Aaronson, Todaro, 1968). It is based on the maintenance of cultures under maximum cell densities. Such an approach facilitated and accelerated the start of the "crisis" stage (up to 3-8 passages) with the following gradual death of almost the whole normal senescent cell population, the colony formation resulting from the proliferation of single clonogenic cells. The frequency of clonogenic cells was about 6 x 10(-6). Six lines of spontaneously transformed cells from embryos of noninbred white rats (LRec-1--LRec-6) and one line from the Wistar embryos (LRec-7) were established. All the lines are characterized as diploid or near-tetraploid, with 1-4 different marker chromosomes formed from chromosome 7, as was reported elsewhere (Artsybasheva et al., 1988). The values of saturation densities and the time of population doubling for all the 7 lines differed from those for the rat embryo primary cultures cells. LRec-1--LRec-6 cells were unable to form the colonies in soft agar, while LRec-7 cells were able to grow in agar. The lines LRec became oncogenic for 1-2 day old rats after different periods of cultivation in vitro--from 3 to 7 months. The line LRec-7 Wistar appeared to be highly oncogenic from the very beginning after its selection. The histological analysis revealed that the LRec-1 tumors could be classified as polymorphocellular sarcoma. Up to 20 passages the LRec-1 line had numerous clonogenic cells (50-60%) in sparse cultures independently on the serum content in the media. By a 3-step selection of LRec-1 cells, on cultivation in media with lower serum contents (1-0.1-0%), a semisuspension of LRec-1sf subline (serum free) was established. This line was highly oncogenic for 1-2 day old rats, was easily cryopreserved and proliferated in the serum-free media for unlimited time, forming small colonies in agar. Thus, the new approach allows to establish with high effectiveness spontaneous lines of rat embryo cells with differently transformed phenotypes, i.e. preneoplastic and oncogenic ones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cyclic adenosine monophosphate and the enzyme activity of its metabolism in L cells sensitive and resistant to the cytotoxic action of ethidium bromide

Using L-cells both sensitive and resistant to cytotoxic action of ethidium bromide (EB), a study was made of the intracellular level of cAMP, activities of adenylcyclase, phosphodiesterase and cAMP, liberated from cells into the surrounding medium. In EB resistant L-cells compared to EB sensitive ones, the higher level of cAMP with a decreased activity of adenylcyclase and an increased activity of the phosphodiesterase was shown to be associated with an impeded exit of cAMP from cells. It is suggested that the differences in cAMP levels in the EB sensitive and resistant cells are associated with the properties of cAMP-dependent protein kinases of these cells.     

Cellular energy functions of a subline of mouse fibroblasts resistent to the action of ethidium bromide]

The respiration of subline Leb-25 cells, resistant to ethidium bromide (EB, 25 g/ml), is 2.5 times slower than the respiration of parental L cells of mouse fibroblasts. The EB resistant cells have a normal level of ATP. Disturbances of mitochondrial functions can be observed such as a defect of the succinate dehydrogenase complex and the uncoupling of respiration and oxidative phosphorylation in mitochondria of Leb-25 cells.     

Cyclic adenosine monophosphate content and its regulatory characteristics in Chinese hamster cells with genetically determined changes in the cell surface

The intra- and extracellular concentrations of cyclic adenosine monophosphate (cAMP) in cell line CHO-K1, sensitive (clone 773) and resistant to cytotoxic action of ethidium bromide (EBr), colchicine (Cr) and actinomycin D (ADr), as well as the amount of cAMP in response to isoproterenol, 10% serum and ethidium bromide (EB) in these cells were studied. The increased level of cAMP in EBr- and, Cr-cells, and the decreased one--in ADr cells was found as compared with sensitive cells. The amount of cAMP extruded in the surrounding medium was lower for EBr- and Cr-cells and higher for ADr-cells, in comparison with sensitive cells. All the variants of resistant cells were characterized by a less intensive but a longer reaction for isoproterenol, as compared with sensitive cells. In all the investigated variants 10% serum induced a remarkable increase in the intracellular cAMP by the 2nd hour after their insignificant decrease. 1 mcg/ml concentration of EBr increased intracellular cAMP only in 773-cells. The rules changing the cAMP level to isoproterenol and EB2; are determined by differences in reaction of adenylate cyclase, as it has been demonstrated for the 773- and EBr-cells.     

Control of cell multiplication in vitro. IV. Isolation and characteristics of C3H10T1/2-line cells with decreased dependence for multiplication on blood serum

Mouse embryo fibroblasts of C3H10T1/2 cell line were selected in the medium with a low serum content. The frequency of clone occurrence was about 1.10(-5) (for 0.5% serum). The treatment of cells by N-methyl-N'-nitrosoguanidine significantly increased the frequency of occurrence of these clones. The obtained spontaneous variants were assayed for stability of growth characteristics. The phenotypic analysis of clones with low serum growth dependence discovered at least two cell types differing in some morphological and growth characteristics. The former did not differ from the parental line C3H10T1/2 in any phenotype characteristics but their serum growth dependence; whereas, the latter were poorly spread over the substrate and showed an enhanced saturation density and a decreased population doubling time. These cells also differed in their growth dependence on the homologous conditioned medium.     

Effect of the preparation baliz-2 on the integrative brain activity and biogenic amine metabolism in rats under stress

Effects of balis-2 on exploratory activity in the open field and elevated plus-maze, attention to sensory stimuli of different modalities, elaboration and retention of conditioned reflexes with food reinforcement, were studied in rats under stress. Prolonged treatment of rats with balis-2 seems to normalise integrative activity and metabolism of serotonine and dopamine in the animal brain.     

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H⁺ (mg Chl)^–1 h^–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal -CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO₃ ^– dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H⁺ (mg Chl)^–1 h^–1) were observed in the presence of detergents (Triton X-100 or n-octyl--D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H⁺ (mg Chl)^–1 h^–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effects of Carbonic Anhydrase Inhibitors on Proton Exchange and Photosynthesis in Pea Protoplasts

At concentrations of 100–200 M, ethoxyzolamide, a lipophilic inhibitor of carbonic anhydrase, considerably (by 60%) inhibited light-induced CO₂-dependent oxygen evolution in pea protoplasts at the optimum concentration of inorganic carbon (100 M CO₂) in the medium. At the same concentrations of the inhibitor, electron transport in isolated pea thylakoids was inhibited only by 6–9%. Acetazolamide, a water-soluble inhibitor of carbonic anhydrase, affected neither the rate of CO₂-dependent O₂evolution in protoplasts nor electron transport in thylakoid membranes. A light-dependent proton uptake by protoplasts was demonstrated. At pH 7.2, the induction kinetics and the rate of proton uptake were similar to those for CO₂-dependent O₂evolution. The rate of proton uptake was decreased twofold by 1 mM acetazolamide. This fact agrees with the notion that a membrane-bound carbonic anhydrase is operative in the plasma membrane of higher plant cells. A mechanism of its functioning is suggested. Possible functions of carbonic anhydrases in the cells of C₃-plants are discussed.     

Unusual signal peptide directs penicillin amidase from Escherichia coli to the Tat translocation machinery

The recently described Tat protein translocation system in Escherichia coli recognizes its protein substrates by the consensus twin arginine (SRRXFLK) motif in the signal peptide. The signal sequence of E. coli pre-pro-penicillin amidase bears two arginine residues separated by one aspargine and does not resemble the Tat-targeting motif but can nevertheless target the precursor to the Tat pathway. Mutational studies have shown that the hydrophobic core region acts in synergism with the positive charged N-terminal part of the signal peptide as a Tat recognition signal and contributes to the efficient Tat targeting of the pre-pro-penicillin amidase.     

Some observations on the effect of Daflon (micronized purified flavonoid fraction of Rutaceae aurantiae) in bancroftian filarial lymphoedema

BACKGROUND: Morbidity management is a core component of the global programme for the elimination of lymphatic filariasis. In a double-blind clinical trial, the tolerability and efficacy of Daflon (500 mg) + DEC (25 mg) or DEC (25 mg) alone, twice daily for 90 days, was studied in 26 patients with bancroftian filarial lymphoedema. RESULTS: None of the patients in either drug group reported any adverse reaction throughout the treatment period (90 days). Haematological and biochemical parameters were within normal limits and there was no significant difference between the pre-treatment (day 0) and post-treatment (day 90) values. The group receiving Daflon showed significant reduction in oedema volume from day 90 (140.6 PlusMinus; 18.8 ml) to day 360 (71.8 PlusMinus; 20.7 ml) compared to the pre-treatment (day 0, 198.4 PlusMinus; 16.5 ml) value. This accounted for a 63.8% reduction in oedema volume by day 360 (considering the pre-treatment (day 0) as 100%). In the DEC group, the changes in oedema volume (between day 1 and day 360) were not significant when compared to the pre-treatment (day 0) value. The percentage reduction at day 360 was only 9%, which was not significant (P > 0.05). CONCLUSION: This study has shown that Daflon (500 mg, twice a day for 90 days) is both safe and efficacious in reducing oedema volume in bancroftian filarial lymphoedema. Further clinical trials are essential for strengthening the evidence base on the role of this drug in the morbidity management of lymphatic filariasis.