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F Franĕk

Publications and source records attributed to F Franĕk.

At least 19 recordsLinked to original sources
PubMed1996

Purification of cholesterol-esterifying enzymes from rat liver cytosol by high-performance liquid chromatography.

Three enzymes esterifying cholesterol with long-chain fatty acids were purified approximately 31,000-fold to apparent homogeneity from the cytosol of normal rat liver. The enzymatic activity was tested by incubation of active fractions with tritiated cholesterol and separation of newly formed esters from non-reacted cholesterol by a passage through silica gel cartridges with subsequent assay for radioactivity by liquid scintillation. For the purification of enzymes, active proteins were precipitated by (NH4)2SO4 to 35% saturation. The bulk of inactive proteins was removed by size-exclusion chromatography on TSK G3000 SW. The active fraction was subsequently separated on Separon HEMA BIO 1000 DEAE in gradients of 0-500 mM KCl into three enzymatic activities differing in their retention and these proteins were finally purified by affinity HPLC on columns of cholesterol immobilized on HEMA BIO 1000 E-H. Final purified enzymes showed the same single band in polyacrylamide gel electrophoresis corresponding to 16.5 kDa. Combination of individual enzymes did not increase the overall yield of cholesteryl esters but the reaction-rate was significantly accelerated. These proteins are apparently subunits of a larger complex (M(r) 65,000) that can be demonstrated by electrophoresis in the absence of 2-mercaptoethanol. Results presented in this paper indicate that because of good and rapid separation of active proteins, HPLC may be a method of choice for enzyme purifications.

Animals↗
PubMed1996

Protection of B lymphocyte hybridoma against starvation-induced apoptosis: survival-signal role of some amino acids.

The phenomenon of starvation-induced apoptosis was studied in cultures of a mouse B lymphocyte hybridoma. In a continuous culture the limitation of nutrients was modelled by dilution of a protein-free medium with saline to 15%. Surprisingly, the hybridoma clone did not die out under extreme starvation conditions. A steady state was established in which the cells continued to grow at very low viable cell concentration, concomitantly with an enhanced rate of apoptotic death. Suppression of the death rate, and increase of steady-state viable cell concentration, could be achieved by additions of L-alanine, L-asparagine or L-glutamine, but not by addition of L-phenylalanine. This specificity pattern is in agreement with previous screening experiments that have identified a set of apoptosis-preventing amino acids (glycine, L-alanine, L-serine, L-threonine, L-proline, L-asparagine, L-glutamine, L-histidine). The analysis of amino acid consumption and production showed a consistent production of alanine and serine both in standard medium and in diluted media. When alanine was added at a final concentration of 2 mM to media diluted either to 40 or 20%, apoptosis was partly suppressed. A limited production of alanine was observed also in alanine-enriched diluted media. It is concluded that the apoptosis-preventing amino acids act as signal molecules, besides their nutritive function, and that the signal has a character of a survival factor. The observed phenomena are interpreted in terms of a survival-control mechanism that regulates the viable cell number of a lymphocyte clone in an adequate proportion with the level of available nutrients.

Amino Acids↗
PubMed1993

Kinetics of development of spontaneous apoptosis in B cell hybridoma cultures.

The kinetics of the development of apoptosis was studied in mouse B cell hybridoma batch cultures carried out in the iron-rich protein-free medium. One of the markers of apoptosis, the apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride, was found to rise significantly at 144 h, i.e., in the late stationary phase. At the decline of the culture (216 h) the value of the apoptotic index reached 29.1%. Analysis of another marker, the degree of DNA fragmentation determined on the basis of chromatographic resolution of isolated cellular DNA, revealed a significant increase as early as 96 h, i.e., at the end of the exponential phase. At 216 h the net value of the fragmented DNA fraction was about 30% of cellular DNA. The values of both markers were found to be very similar when the iron-rich protein-free supplement was replaced with conventional 10% foetal calf serum. This finding suggested that the growth factors present in the serum were not able to abolish the tendency of the hybridoma culture to undergo spontaneous apoptosis. The timing of the spontaneous onset of apoptosis in the exponential phase indicated that B cell hybridoma apoptosis was a process associated with cell proliferation and full metabolic activity rather than with the decline of cell vitality.

Animals↗
PubMed1992

Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures.

Markers of apoptosis were followed in batch hybridoma cultures carried out in protein-free medium. Samples were collected on day 0, representing early exponential phase (viability 91%), and on day 8, corresponding to late stationary phase (viability 8%). The apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride in the culture of day 8 (30%) exceeded markedly the index in the culture of day 0 (2.5%). A gel chromatography on Sepharose 2B was developed for quantitative evaluation of fragmented cellular DNA. This analysis, including a correction for nonspecific fragmentation, showed that on day 8 more than 30% of cellular DNA was fragmented, whereas on day 0 it was less than 5%. Control necrotic cells prepared by rapid killing in 1% sodium azide displayed a low apoptotic index (2.4%) and low DNA fragmentation. Electrophoretic patterns in agarose gel showed a typical "ladder" of fragments in the DNA sample of day 8. The demonstration of fragmented cellular DNA and of the high incidence of apoptotic bodies at late stationary phase adds substantial weight to the view that in hybridoma cultures apoptosis represents the prevalent mode of cell death.

Animals↗
PubMed1991

Nucleosomes occurring in protein-free hybridoma cell culture. Evidence for programmed cell death.

In addition to monoclonal immunoglobulin, two kinds of nucleoproteins, NP1 and NP2, were isolated from the supernatants of hybridoma cultures set up in a protein-free medium. As shown by SDS-electrophoresis the two nucleoproteins shared a set of proteins (apparent Mr 11,000 to 15,000), and differed in the DNA moiety (approximately 150 bp in NP1, approximately 350 bp in NP2). The amino acid composition of the protein moiety confirmed the nucleosomal origin of NP1 and NP2. The findings support the view that in hybridoma cultures the cells undergo death by apoptosis, i.e. a programmed process characterized by initial fragmentation of chromatin.

Amino Acids↗
PubMed1991

Hybridoma growth and monoclonal antibody production in iron-rich protein-free medium: effect of nutrient concentration.

The iron-rich (500 microM ferric citrate) protein-free supplement was added to six different basal media. Cell growth and monoclonal antibody production of a mouse-mouse hybridoma were investigated in 1.3 1 batch cultures performed in a laboratory bioreactor with automatic control of pH and dissolved oxygen concentration. RPMI 1640 served as the control medium. Fortification of the basal medium by balanced mixtures of amino acids and vitamins showed higher positive effect than daily supplementation by glucose and glutamine. Strongly fortified medium, based on RPMI 1640, was found superior to other basal media. The viability index increased by a factor of 3.04 and the total antibody production by a factor of 2.82, relative to the control.

Animals↗
PubMed1989

[Immunohistochemical detection of triiodothyronine in thyroid tumors using T3-01 monoclonal antibody].

Cells of the spleen of BALB/c mice immunized with a conjugate of triiodothyronine and bovine immunoglobulin were fused with cells of the murine myeloma line F O. Selection and cloning were used to look up the T3-01 hybridoma producing a monoclonal antibody of the IgG1 subclass which responded in a radioimmunoassay specifically to triiodothyronine. The T3-01 monoclonal antibody was used for the immunohistochemical demonstration of triiodothyronine on 19 tumour derivatives.

Antibodies, Monoclonal↗
PubMed1989

Growth-stimulating effect of transferrin on a hybridoma cell line: relation to transferrin iron-transporting function.

The relation of the growth-stimulating capacity of transferrin to its iron-transporting function was investigated in mouse hybridoma PLV-01 cells cultivated in a chemically defined medium. The cells were precultivated in protein-free medium supplemented either with ferric citrate (cells with a high intracellular iron level) or with iron-saturated transferrin (cells with a low intracellular iron level). Iron uptake was monitored after the application of 59Fe-labeled ferric citrate or pig transferrin. Cultivation of the cells at the optimum growth-stimulating concentration (500 microM) of ferric citrate resulted in an intracellular iron level about 100-fold higher than that of cells cultivated at the optimum transferrin concentration (5 micrograms/ml). Replacement of pig transferrin with bovine transferrin resulted in similar intracellular iron levels, but the growth-stimulating effect of bovine transferrin was more than one order of magnitude lower. Cells with a high intracellular iron level grew equally well when cultivated with iron-saturated transferrin or with apotransferrin + deferoxamine (2 micrograms/ml). On the other hand, cells with a low intracellular iron level required iron-saturated transferrin for further growth and apotransferrin + deferoxamine was ineffective. The results suggest that transferrin can act as a cell growth factor only in the iron-saturated form. However, several findings of this work indicate that supplying cells with iron cannot be accepted as the full explanation of the transferrin growth-stimulating effect.

Animals↗
PubMed1989

Isolation and characterization of pig lymphocyte Fc receptor.

Fc gamma Receptor contained in a mixture of plasma-membrane components released from pig peripheral blood lymphocytes following a 4----37 degrees C temperature shift was isolated by affinity chromatography on immobilized pig IgG. The main component of the receptor preparation exhibited an apparent molecular weight of 40-43 kDa in SDS polyacrylamide gel electrophoresis. Specificity of interaction of isolated Fc gamma receptor with immobilized pig IgG (Ka = 5.4 x 10(6) M-1) was investigated by competitive radioimmunoassay employing labelled Fc gamma receptor. The interaction was specifically inhibited by pig IgG, its Fc fragment, and less so by its pFc' fragment. Inhibition by peptides prepared from the pig IgG CH3 domain pointed to the heavy chain segment between residues 340 and 380 as the probable location of the binding site. In the competitive assay, bovine, human, mouse and guinea pig IgGs were as effective inhibitors as the homologous IgG, while rabbit IgG produced weaker inhibition. The amino acid composition of the pig lymphocyte Fc gamma receptor was determined. Comparison with the amino acid compositions of some other Fc receptors and other proteins revealed its structural relatedness to several Fc gamma receptors of lymphoid cells and to the poly-Ig receptor. In addition, the comparison of amino acid compositions suggested a structural relationship between the pig Fc gamma receptor and some seemingly unrelated proteins.

Amino Acids↗
PubMed1987

Limited enzymatic cleavage of pig immunoglobulin and of specific antibodies. V. Monoclonal antibodies to individual fragments and polypeptide chains.

Six hybridomas that produce monoclonal antibodies to different antigenic determinants of heterogeneous pig IgG and of pig anti-Dnp antibodies were obtained by fusion between spleen cells from BALB/c mice immunized with non-specific pig IgG and the myeloma line P3-X63-Ag8.653. Antigenic determinants which correspond to individual monoclonal antibodies were located in the individual domains of the IgG molecule by investigating the interaction of monoclonal antibodies with Fab, Fc, and pFc' fragments and with kappa, lambda, and gamma polypeptide chains. To evaluate the interaction, a quantitative equilibrium competitive radioimmunoassay was developed and [14C]formaldehyde-labelled non-specific pig IgG served as labelled antigen. Antibodies PGG-01, PGG-04, and PGG-06 were shown to be directed against determinants in the C lambda domain, antibody PGG-03 very probably against the determinant in the C chi domain, and antibodies PGG-02 and PGG-05 against determinants in the CH3 domain. Antibodies PGG-02 and PGG-05 are highly selective for IgG subpopulations; when combined, they interact with no more than 42% of the heterogeneous IgG population. These two antibodies can be used as a basis for preparing a complete set of reagents for identification of individual subclasses.

2,4-Dinitrophenol↗
PubMed1986

Correlation of the character of intramolecular melting with digestibility by pepsin in precipitating and non-precipitating pig anti-Dnp antibodies.

Precipitating and non-precipitating pig anti-2,4-dinitrophenyl group (Dnp) antibodies were investigated by differential adiabatic scanning microcalorimetry in the pH range 3.7-4.5. The partial heat capacity functions obtained revealed a notable difference between the two antibody types when the analysis was done at pH 4.5 or 4.0. The transition observed at temps around 50 degrees C in a non-precipitating antibody was absent in a precipitating antibody. Under analogous conditions, pH 4.5, the precipitating antibody was fully resistant to pepsin, while the non-precipitating antibody yielded appropriate F(ab')2 and pFc'fragments. At pH 3.7 no substantial difference in the partial heat capacity function could be observed between the two antibody types. Below pH 4.0 the precipitating antibody became susceptible to peptic cleavage and yielded fragments of the same general character as the non-precipitating antibody. This finding lends support to the view that the structural block in immunoglobulin G that melts first, i.e. at temps near 50 degrees C, is the CH2 domain or a part of it.

2,4-Dinitrophenol↗
PubMed1986

Immunoglobulin K chain genes in mouse hybridoma PTF-02 and parent myeloma. Insertion of hybridoma K gene into the plasmid pSV2-gpt.

The mouse myeloma line P3-X63-Ag8.653 currently used as the parent line for hybridoma construction contains only one (non-productive) gene for immunoglobulin K chains. The allelic gene is lost. In the mouse hybridoma PTF-02 two K genes can be found. One is identical with the gene of the myeloma parent line, the other originates in lymphocytes and is transcribed and translated in the K chain of the antibody secreted by the hybridoma. From the gene library of hybridoma PTF-02 in phage Charon 28 both K genes were isolated. Restriction endonuclease mapping and Southern blot hybridization demonstrated that the fragment comprising the lymphocyte gene was of the size (7.5 kb) sufficient to carry all exons, transcription and translation units. This gene was then recloned in the plasmid pBR322 and shuttle plasmid pSV2-gpt, which opens up possibilities for transfection of lymphoid cells and for study of the regulation of individual gene expression.

Animals↗
PubMed1986

The CH3 domain of pig immunoglobulin G. A study of structural heterogeneity and enzymic fragmentation.

The CH3 domain of pig immunoglobulin G has been isolated as the pFc' fragment by peptic hydrolysis at pH 4.5. By ion-exchange chromatography in a dissociating medium pH 3.0 the heterogeneous pFc' fragment could be resolved into three variants differing in electric charge. The variants were very similar in amino acid composition but differed mainly in the histidine content. The peptides obtained from the pFc' fragment by tryptic hydrolysis were separated by ion-exchange chromatography and characterized by amino acid composition. It appeared that the CH3 domain is split by tryptic hydrolysis to four large peptides of 20-30 amino acid residues in length and to a number of small peptides. The large peptides represent segments of the polypeptide chain suitable for study of the location of the binding site for the Fc receptor.

Amino Acid Sequence↗