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Biomedical subjects

F Franĕk

Publications and source records attributed to F Franĕk.

At least 37 records · Page 2Linked to original sources
PubMed1985

Hybridoma cultivation in defined serum-free media: growth-supporting substances. I. Transferrin.

Effect of transferrin and effect of transferrin omission on growth of hybridomas in serum-free medium were studied. Transferrin at concentration 0.5 microgram/ml and higher concentrations stimulated growth of hybridoma cells. The optimum growth-supporting concentration of transferrin was 5 micrograms/ml. Omission of transferrin led to death of hybridoma T3-03 cells within 3-4 days. Omission of transferrin for a period of only 24 h damaged cells so severely, that they were unable to recover fully.

Animals↗
PubMed1984

Particulate protein-synthesis factors associated with translatable mRNA in mouse hybridoma cells.

Besides of mRNA, the postribosomal pellet of mouse hybridoma cells contains RNA species which become labeled more rapidly than rRNA. Their synthesis is inhibited by actinomycin D. Density-gradient centrifugation of the postribosomal pellet yielded fractions of approx. 55-60 and 90S, synthesizing after the addition of both ribosomal subunits and energy-sources light and heavy chains of immunoglobulin, as demonstrated by indirect immunoprecipitation. Analysis of translation products by electrophoresis indicated the presence of precursors of mRNAs for immunoglobulin chains in these particles. Postribosomal pellets thus apparently contain different particles composed of similar polypeptide chains and containing protein-synthesis factors associated with translatable mRNA.

Animals↗
PubMed1984

Isolation of Fc receptor shed from pig lymphocytes by a temperature shift.

Lymphocytes obtained from pig blood by gradient centrifugation were subjected to a temperature shift (4 to 37 degrees C). The proteins released from the plasma membrane were fractionated by affinity chromatography using immunoglobulin G immobilized on fine polyamide particles. The main component liberated from the adsorbent by diethylamine buffer (pH 11.5) exhibited an apparent Mr of 18000-20000 in SDS-polyacrylamide gel electrophoresis. This crude receptor preparation possessed a substantially higher affinity to immobilized immunoglobulin G than to immobilized Fab fragment and inhibited significantly the binding of labeled immunoglobulin G to pig lymphocytes.

Animals↗
PubMed1984

Purification of mRNA for immunoglobulin kappa-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA.

The principle of mRNA purification by hybridization to an immobilized DNA fragment was applied to the isolation of mRNA coding for immunoglobulin kappa-chains of mouse myeloma MOPC 21 and mouse hybridoma PTF-02. The DNA fragment comprising the 3'-untranslated region and a part of the constant region of the kappa-chain gene was covalently attached to diazobenzyloxymethyl-cellulose and used as an affinity adsorbent. A homogeneous 14S mRNA species was obtained by hybridization of total mRNA to the affinity adsorbent at 52 degrees C and by elution at 60 degrees C. Addition of the purified mRNA to a fractionated cell-free translation system resulted in a significant increase in the radioactivity immunoprecipitated by pig anti-mouse immunoglobulin antibodies. A single radioactive polypeptide of apparent Mr of 25,000, corresponding obviously to the kappa-chain, was identified as the only translation product.

Animals↗
PubMed1984

Serum-free medium for hybridoma and parental myeloma cell cultivation: a novel composition of growth-supporting substances.

Serum-free chemically defined medium for hybridoma and parental myeloma cultivation was developed on the basis of testing of individual substances supporting hybridoma growth under serum-free conditions. Optimized concentrations of transferrin, insulin, ethanolamine, linoleic acid, serum albumin, ascorbic acid, hydrocortisone, and trace elements could substitute serum. Developed serum-free hybridoma (SFH) medium differs from analogous previously described media mainly by a more complete combination of growth-supporting supplements and by the presence of ascorbic acid and hydrocortisone. Growth comparable with that in the medium supplemented with 10% bovine serum was achieved with four hybridomas and two myelomas. SFH medium was also suitable for long-term cultivation of hybridomas without cessation of monoclonal antibody production. Growth potency and the specific growth requirements of hybridomas in serum-free medium are, to a large degree, determined by parental myeloma.

Animals↗
PubMed1984

Limited enzymatic cleavage of pig immunoglobulin G and of specific antibodies. IV. Characteristics of the tFc' fragment obtained from the S-sulpho derivative of non-specific immunoglobulin G.

By tryptic cleavage of the S-sulpho derivative of non-specific pig IgG and subsequent separation by gel chromatography on Sephadex G-100 a fragment with molecular mass 11 900 +/- 500 was obtained. This fragment originates in the Fc part of the molecule because it precipitates with anti-Fc serum. It was therefore designated tFc'. From the pFc' fragment obtained by peptic cleavage it significantly differs in amino acid composition. tFc' fragment was subjected to ion-exchange chromatography on SE-Sephadex at pH 3.0 in a dissociating medium containing 8 M urea. Three major variants of tFc' fragment were purified by rechromatography and characterized by amino acid composition and by peptide maps. Their characteristic features are a high half-cystine content and a low content of arginine, histidine and methionine. The characteristics of all the three variants of tFc' fragment suggest that they most probably originate in the CH2 domain of immunoglobulin G.

Amino Acid Sequence↗
PubMed1983

Biosynthesis of rabbit serum albumin in a heterologous fractionated subcellular system.

As demonstrated by a simple procedure based on indirect immunoprecipitation, proteins retained on heparin-Sepharose 4B from postmitochondrial supernatants of rat liver and Zajdela hepatoma catalyse the translation of rabbit serum albumin mRNA in the presence of ribosomal subunits from rat liver, Zajdela hepatoma or rabbit reticulocytes. The albumin synthesis shows an optimum at 1.5 mM MgCl2 and 25 mM KCl and requires ATP and GTP. It is significantly stimulated by tRNA and proceeds for more than 2 h, suggesting a high rate of reinitiation. At the optimum ribosomes:mRNA ratio of 13:1, the immunoprecipitable radioactivity exceeds 15-20-times the blank values. Fluorography of polyacrylamide slabs after electrophoresis of immunoprecipitates revealed the presence of only complete full-size serum albumin without any smaller peptides resulting from premature terminations of polypeptide chains, demonstrating faithful translation. In stained gels only, both heavy and light chains of immunoglobulin G were found, indicating that the assay procedure is highly specific and reliable. The fractionated heterologous protein-synthesizing system described in this paper may be generally useful for studies on the synthesis of specific proteins and factors affecting their rates since, unlike comparable translation assays, a precise calculation of the balance of newly synthesized proteins is possible.

Animals↗
PubMed1983

Intramolecular motility in anti-Dnp antibodies and in their Fab fragments. EPR spectra of the complexes with a spin-labelled hapten in H2O-D2O mixtures at various temperatures.

To study intramolecular motility, the binding sites of the antibodies or Fab fragments were occupied by the spin-labelled hapten Dnp-NO. EPR spectra of the complexes were recorded under various conditions, in particular variable viscosity, and the correlation times of the antibody or its fragment were calculated. Intramolecular motility decreases, i.e. correlation time increases, with temperature in the range from 5 degree C to 30 degrees C. This anomalous dependence points to the important role of hydrophobic bonds in the interactions between domains of the antibody molecule. Substitution of a fraction of H2O by D2O (1 to 13%) is manifested by the increase in correlation time only at 5 degrees C, a maximal effect is already obtained at about 3% D2O. At 20 degrees C or in the presence of perturbants of the hydration shell such as 0.5 M sodium chloride, 1 M urea or 10% butanol, D2O has no appreciable effect. EPR spectra made it possible to study the gradual immobilization of the bound spin-labelled hapten at temperatures lower than 0 degrees C. Water in the vicinity of the bound spin-labelled hapten freezes in three discrete steps, -8 to -13 degrees C, -20 to -40 degrees C, and -40 to -80 degrees C. D2O influences the first and second step so that it shifts the transition to higher temperatures. The results contribute to our understanding of the fine structure of the hydration shell and water in the interdomain space of the antibody molecule.

Animals↗
PubMed1983

Limited enzymatic cleavage of pig immunoglobulin G and of specific antibodies. III. Large-scale isolation of tryptic fragments of antibodies and of non-specific immunoglobulin and characteristics of the Fc fragment.

Precipitating and non-precipitating anti-Dnp antibodies and S-sulpho non-specific IgG in gram quantities were subjected to limited cleavage by trypsin. Upon gel chromatography on Sephadex G-100 the fraction of Fab and Fc fragments was separated from incompletely split molecules and from tFc' fragments. The Fab and Fc fragments were separated from each other either by ion-exchange chromatography on QAE-Sephadex or by preparative electrophoresis in starch block. Both Fab and Fc fragments appeared to be heterogeneous as to electric charge. The Fc fragments were characterized by amino acid composition and N-terminal amino acids. The Fc fragment of non-specific IgG was cleaved by cyanogen bromide, and a C-terminal peptide containing 18 residues was isolated. Partial amino acid sequence of this peptide pointed to a high degree of homology with immunoglobulins of other animal species.

Amino Acids↗
PubMed1982

Complement-mediated fragmentation of soluble and insoluble immune complexes containing porcine anti-DNP antibodies.

Complement-mediated release of soluble immune complexes and immune precipitates containing DNP-PSA and precipitating or non-precipitating porcine anti-DNP antibodies was studied. A decrease in the average size of soluble immune complexes indicating their fragmentation was observed during incubation in excess human serum, the extent of the complex release was found to be in direct proportion to the time of incubation. The effect was complement-dependent. In the second part of the study, complement-dependent solubilization of the immune precipitates of the precipitating antibody preparation was compared to the solubilization of the precipitates of the non-precipitating antibody formed in the presence of PEG. Although, both types of precipitates activated complement in about the same extent, complexes of non-precipitating antibody were solubilized much slower than those of the precipitating one. As avidity of both antibody preparations to the antigen was high, the observed differences in the rate of the complex solubilization probably reflected differences in the structure of the two types of complexes.

Animals↗
PubMed1982

Monoclonal antibodies against transferrin. Precipitating mixtures and lack of inter-species cross-reactivity.

Five stable hybridoma lines were prepared using the myeloma cell line P3-X63-Ag.653 and spleen cells of mice hyperimmunized by pig transferrin. All hybridomas grew well in mouse peritoneal cavity and produced antibodies of the IgG1 subclass. Antibody preparations obtained from ascitic fluids tested for their capacity of antigen precipitation. No precipitation was obtained with single antibodies and with pairs of antibodies. Three out of 10 possible triads gave clear and sharp precipitation zones and rings in immunodiffusion tests performed in agar gel. All 5 antibodies were shown by quantitative enzyme-immunoassay to be specific for pig transferrin: no cross-reaction was obtained with mouse, human, horse and sheep transferrins.

Animals↗
PubMed1982

Conformational changes induced by hapten in pig antibodies to the dinitrophenyl group. Analysis by temperature-perturbation and solvent-perturbation spectroscopy.

Pig antibodies to the dinitrophenyl group and fragments derived from them by limited proteolysis were studied by temperature-perturbation and solvent-perturbation spectroscopy with particular attention to differences between the number of perturbed chromophores in free antibodies and in antibody-hapten complexes. The position of the maxima in the difference spectra show that solvent-perturbed chromophores are exposed to water, but thermally perturbed chromophores are located in a microenvironment the polarity of which corresponds to 25-50% ethylene glycol. A significant fraction of tyrosine residues (65-90%) and tryptophan residues (20-45%) is perturbed by temperature. Much lower fractions, i.e. 35-45% of tyrosine residues and less than 15% of tryptophan residues, are perturbed by 20% glycerol. The numbers of perturbed chromophores in fragments constituting the molecule are lower than or equal to the numbers in the original molecule. The effect of hapten binding is significant only with one of the antibody types, the precipitating antibody. The number of thermally perturbed tyrosine residues is by about 17% lower in the liganded antibody. The absence of an analogous effect in the Fab fragment suggests that the fine conformational mechanism of signal transfer from the binding sites operates only in intact antibody molecules.

Animals↗
PubMed1982

Limited enzymatic cleavage of pig immunoglobulin G and of specific antibodies. I. Different resistance of various antibody types to cleavage by pepsin.

Limited proteolysis by pepsin at pH 4.5 of non-specific pig IgG and of two types of pig anti-dinitrophenyl antibodies revealed striking differences in susceptibility to proteolytic cleavage. The digests were resolved on a column of Sephacryl S-200 and the elution profiles analysed using a computer programme. The individual fragments were characterized by immunoelectrophoresis, SDS-polyacrylamide gel electrophoresis and molecular mass determination. Whereas the non-precipitating antibodies and the non-specific IgG yielded F(ab')2 and pFc' fragments in reasonable quantities, the precipitating antibody proved to be stable even at the 70th h of digestion. The precipitating antibody became susceptible to peptic cleavage only when the pH was lowered to 4.0. It was concluded that the precipitating antibody represents a subclass of pig IgG with an anomalous resistance to proteolysis at acidic pH and that this subclass constitutes only a few per cent, at most, IgG of the healthy, non-immunized animals.

Animals↗
PubMed1982

Complexity of the genetic control of a structural marker in the CH1 homology region of pig IgG.

The occurrence of two variant peptides P1 and P2 originating in the vicinity of the heavy-light chain disulphide bridge in the CH1 homology region was examined in IgG samples prepared from 36 randomly selected pigs. The peptides labelled with 35S at the half-cystine residues were separated on two-dimensional peptide maps and their ratio was quantitatively evaluated by a non-destructive automated method using semiconductor detectors. Fourteen individuals were found to yield only the peptide P1, the remaining 22 individuals yielded both P1 and P2 with a mean ratio of 69.4: 30.6. A model of genetic determination of the amino acid interchange, serine-leucine, distinguishing the peptides P1 and P2 was suggested. It has been assumed that the gene for one gamma-chain subclass exists only in the form coding for the peptide P1 whereas the gene for another gamma-chain subclass exists in two allelic forms coding for either P1 or P2.

Animals↗
PubMed1981

Hapten-induced changes in pig anti-dansyl antibodies revealed by EPR spectra of spin-labelled antibodies.

Pig anti-Dns antibodies were labelled by 2, 2, 6, 6- tetramethyl-N1-oxylpiperidine-4-amino (N-dichlorotriazine) (label SL I) or by 4-amino-2,2,6,6-tetramethyl-piperidine-N1-oxyl (label SL II). SL I presumably reacted with Fab parts, whereas SL II was attached to the carbohydrate moiety of the Fc part of the antibody molecule. The new method of intramolecular mobility estimation based on viscosity-dependent correlation times of the N-O group of spin-labelled proteins was used, making it possible to separate the correlation times of the spin-label relative to the protein carrier (tauR) and of the labelled protein itself (tauM). The altered shape of the EPR spectrum observed upon binding of epsilon-Dns-lysine indicates that the relative fluctuations of the liganded antibody domains within the Fab part are reduced. The character of temperature dependence of the correlation times of the labelled antibody changed upon hapten binding as well, suggesting an altered interaction between antibody subunits. Evidence of hapten-induced changes of both tauR and tauM was obtained at 1 degrees C and 5 degree C using the label SL II bound to the Fc part. The character of the changes can be interpreted in terms of mobilization of the domains and of altered interaction between labelled carbohydrate and the protein moiety. At 5 degree C the addition of 3% of D2O to the antibody labelled by SL II brought about a substantial effect qualitatively similar to that induced by the hapten.

Animals↗
PubMed1981

General shape and hapten-induced conformational changes of pig antibody against dinitrophenyl. A small-angle scattering study.

Pig antibodies against dinitrophenyl were studied by neutron small-angle scattering and X-ray small-angle scattering with particular attention to the analysis of cross-section plots and determination of the radii of gyration of cross-section. The experimentally determined molecular parameters Rg (radius of gyration), Rq1 and Rq2 (two different radii of gyration of cross-section characterizing every antibody sample) show that the shapes of the two antibody types, precipitating and non-precipitating, are similar. The non-precipitating antibody is slightly more compact. The parameters Rg, Rq1 and Rq2 of complexes of antibodies with the hapten, 8-dinitrophenyl-5,8-aza-4-oxo-octanoic acid, are smaller than those of the free antibody. This indicates that a conformational change is induced by the binding of the hapten. The character of the change of parameters is consistent with a view that the observed contraction of the molecule proceeds via similarity transformation. In order to design a model of a pig antibody molecule, isolated building blocks of the molecule, the Fab and Fc fragments, were first studied. A comparison of the scattering curves with various models of fragments showed, however, that the isolated fragments acquire in solution elongated rod-like shapes. Over 300 tentative models of the intact antibody molecule, built of small identical spheres, were constructed before a good fit with the experimental data was achieved. The most probable models have a cavity in the Fc part and the Fab parts are either fully extended or slightly bent downwards to the Fc part.

Animals↗