The effect of ZIMET 3164 and ZIMET 3393 on phagocytosis in Tetrahymena pyriformis GL.

Tetrahymena pyriformis GL is a suitable model to study the influence on phagocytotic activity of two membrane-stabilizing benzimidazole nitrogen mustard derivative, ZIMET 3164 and ZIMET 3393 (cytostasan). Treatment by both compounds causes a gradual inhibition of food vacuole formation dependent on the concentrations used. Complete cessation of phagocytosis is observed by 5 micrograms/ml ZIMET 3164 and 50 micrograms/ml ZIMET 3393. Additionally, disturbances of ciliary movement including immobilization and changes of cell shape are induced. The cells resume food vacuole formation and ciliary movement after being rinsed with control solution. The results correspond well with those described recently in mammalian mononuclear phagocytes.

Interaction of Lotus-tetragonolobus Lectin (LTL) and an MIF-like factor with guinea-pig macrophages. I. Effects on macrophage migration inhibition and receptor-binding studies.

Purified Lotus-tetragonolobus lectin (LTL) was studied for its interaction with guinea-pig peritoneal macrophages in the migration-inhibition and spreading-inhibition assay. LTL was found capable of inhibiting macrophage migration in a dose-response relationship similar to that of an MIF-life factor present in rat Zajdela-hepatoma ascites, whereas macrophage spreading was less affected. Mitogenic activity of the fucolectin could be excluded. Both the LTL and MIF-like factor seem to come into action by means of alpha-L-fucopyranosyl residues-containing macrophage surface receptors. Both substances act synergistically in inhibiting migration of macrophages. Since binding studies with 125I-LTL demonstrated competition with the MIF-like factor, it is suggested that the latter and LTL share a common surface receptor, and that optimal occupation densities are required for the realization of the inhibitory effect. Possibly, LTL acts in a monomeric form in the migration inhibition assay.

Immunoelectron microscopy of Bacillus subtilis cells secreting human interferon alpha 1 or staphylokinase.

Post-embedding labelling techniques with colloidal gold-IgG or -protein A complexes were used to determine the subcellular location of IFN alpha 1 and staphylokinase secreted from Bacillus subtilis GB500 cells. Both proteins were present in the cytoplasma and the cell envelope pointing to a posttranslational mode of translocation across the cytoplasmic membrane. 5- to 10-fold higher concentrations of gold particles per 0.1 micron 2 were found on the cell envelope and clustering was observed suggesting preferential regions for secretion sites. Several control experiments ensured the specificity of the labelling data.

Expression and subcellular location of native and mutant hTNF alpha proteins in Escherichia coli.

Several mutant hTNF alpha genes were constructed by deletion and stepwise reconstitution of regions coding for C-terminal sequences. The mutant hTNF alpha proteins behaved differently from native hTNF alpha when expressed in Escherichia coli. They were either sensitive to proteolytic degradation or formed insoluble aggregates depending on the strains and conditions used for expression. By contrast, native hTNF alpha was always present in a soluble form and had a tendency to associate with the cytoplasmic membrane. It was even transported to the periplasmic space in E. coli as shown by both cell fractionation and immunoelectron microscopy. The different behaviour of mutant hTNF alpha proteins probably results from a disturbance of protein folding.

Streptococcal erythrogenic toxin type C is not a phosphorylated protein. Description of two different purification procedures and investigation of its phosphorylation state.

Erythrogenic toxin type C (ETC) from different streptococcal group A strains was successively purified by absorption on phenylsepharose, acidic dialysis of the eluate at 40% saturated ammonium sulphate solution, CM-Sepharose chromatography, finally by immunoaffinity chromatography on monoclonal antibodies. Second, after growing of bacteria in the presence of [32P]orthophosphate to phosphorylate ETC, the ETC was purified with phenylsepharose following immunoaffinity chromatography. The occurrence of phosphoamino acids in the purified ETC was investigated by an immunoassay. No phosphoamino acids could be detected in the ETC molecule. Also after radiolabelling with 32P it was not possible to demonstrate a radioactive signal. The treatment with alkaline phosphatase has no influence on the mitogenicity or position of ETC in isoelectric focusing. The results obtained led to the conclusion that in contrast to the literature, ETC is not a phosphorylated protein.

The distribution of the tail moments in single cell gel electrophoresis (comet assay) obeys a chi-square (chi2) not a gaussian distribution.

The parameter tail moment in single cell gel electrophoresis (comet assay) is calculated as the product of the two values: the percentage of DNA in the comet tail and the tail length in microm. Experiments were performed with cultured mammalian cells: B-Lymphoblasts, epithelial cells of a kidney tissue and a plate-epithelial cell line of a human carcinoma. They were irradiated in suspension with UV A at lambda = 343 nm, generated by an excimer laser-pumped dye laser. DNA migration was assessed and analysed. It is demonstrated that the distribution of the tail moments can be fitted by a chi2 (chi-square) distribution, whereas the factors of the product tail moment tend to be normally distributed. From this result, consequences for the statistical evaluation of the results can arise, especially for the computation of the confidence limits and for the valuation of the parameter tail moment from other comet assay experiments.

Methodological approaches to the study of carbohydrate surface receptore on macrophages and tumor cells.

Ultracytochemical visualization of specific carbohydrate receptors by means of lectins can provide valuable topological information about the functional status of cell surfaces in normal and transformed cells. In addition to the concanavalin A (Con A) rection and precoupling of lectin--peroxidase (PO) a new method is suggested using precoupling of the marker enzyme (PO) with an appropriated glycoprotein (glycopeptide) containing a lectin-specific carbohydrate. Furthermore, quantitative estimations of Con A reaction by means of automatic umage analysis are performed on peritoneal macrophages which point out a cluster formation, but give no hints for a loss of glycocalyx during the preparation process contrary to tumor cells.

Effect of a membrane-stabilizing compound on calcium binding to the plasma membrane of Acanthamoeba castellanii.

Binding of calcium ions at the plasma membrane was studied in Acanthamoeba cells pretreated with ZIMET 3164, a benzimidazole nitrogen mustard derivative, which is known to show a potent immunosuppressive action combined with a membrane-stabilizing effect in mice. For reference, 2 compounds were applied: ZIMET 3393 (Cytostasan¿), another benzimidazole mustard derivative, which exerts only a moderate membrane effect and acts as a strong cytostatic, and ZIMET 176/68, a barbituric acid derivative, which acts as an inhibitor of humoral immune responses but without membrane-stabilizing effect. Application of any of the 3 compounds does not reduce the appearance of calcium binding sites, visualized by means of ultracytochemical reaction, notwithstanding their different action in the mammalian organism. On the contrary, it was estimated by morphometric analysis that the number of Ca-dependent deposits was augmented after treatment with low doses of any of the 3 compounds, what seems to be connected with the induced metabolic disturbances in low molecular phosphates level. High doses and/or prolongation of treatment of the cells resulted in diminution of the number of deposits and induces profound disturbances in cell ultrastructure, probably due to the toxic action of the applied doses. In these cases, band-like structures crosslinking the two leaflets of the plasma membrane may be observed; it is suggested that they represent integral membrane proteins.

Production and partial characterization of monoclonal antibodies against erythrogenic toxins type A and C from Streptococcus pyogenes.

Hybridoma cell lines producing monoclonal antibodies against streptococcal erythrogenic toxins type A and C were established from fusions of immunized BALB/c mice splenocytes with P3X63-Ag8.653 myeloma cells. Six MAbs recognize ETA and 11 MAbs bind to ETC. Two MAbs (designated ETA-2 and ETC-10) were produced in ascitic fluid and further characterized. ETA-2 (IgG2a) binds to ETA with an affinity constant of 1.8 x 10(10) M-1 and ETC-10 (IgG1) binds to ETC with an affinity constant of 3.5 x 10(9) M-1. The specificities of the MAbs were evaluated by ELISA and immunoblotting. Both MAbs ETA-2 and ETC-10 are useful in developing specific double-sandwich ELISAs, in which the MAbs were used as solid-phase capture antibodies for the quantitative determinations of ETA and ETC.

Down-regulation of H tau 40 protein expression by minor groove binders.

The DNA minor groove binders SN6999, SN6570, and SN6113, structurally related to netropsin and distamycin, were investigated for sequence-specific interactions with the 154 base pair cDNA fragment of the human tau 40 protein, involved in pathology of Alzheimer's disease. Footprinting results indicated that both the former compounds displayed a pronounced AT-preference, while the latter SN-derivative bound to DNA in a non-sequence specific manner. The influence of these ligands on the protein synthesis was analysed using monoclonal antibodies against h tau protein. Both sequence specific binders markedly impeded protein synthesis. The non-specific binder, however, did not affect protein biosynthesis.

Critical point drying by use of distilled CO2.

For avoiding specimen contamination with particles or liquid residues from CO2-supply cylinders during critical point drying the carbon dioxide is taken from the gaseous phase. Condensation takes place in the water cooled pipe between supply cylinder and CPD-apparatus. A sufficient flow of liquid CO2 is guaranteed, all solid and nonvolatile liquid contaminations are eliminated.

The use of a simple method to avoid cell shrinkage during SEM preparation.

In the course of preparing specimens for scanning electron microscopy, both glutaraldehyde and OsO4-fixed cells exhibit a considerable shrinkage with a reduction of the mean cellular diameter of about 45% after critical point drying. However, if cells are successively treated with glutaraldehyde, OsO4, tannic acid and uranyl acetate solutions, cellular shrinkage of only 5% is observed.

Antigen spot test for the detection of specific antibodies in the nanogram range by means of the PAP-technique.

A modification of a method is described for detection of antigens and antibodies by means of nitrocellulose paper discs as high-capacity solid phase antigen carrier and mouse PAP. Mouse IgG spotted on nitrocellulose paper may be easily detected at a level of 1 ng. Detection of specific antibodies is possible down to a concentration of 80 ng per ml. This method is of practical importance for the screening of hybridoma supernatants.

[Effect of preparation procedures on the calculation of average cell volumes from stereological data]. / Einfluss der Präparation auf die Berechnung des mittleren Zellvolumens aus stereologischen Daten.

The shrinkage of peritoneal cell during the electron microscopical preparation was investigated by means of a special device. Independently of the procedure of fixation after critical point drying the cells exhibit only 54 to 59% of the original diameter (residual volume about 18%). The reduction of cell diameter after Epon embedding is about 25%, corresponding to a residual volume of 40%.

Effect of several fixatives on the density of concanavalin A binding sites of murine peritoneal macrophages determined by fluorescence microscopy and X-ray microanalysis.

The influence of various fixatives (glutaraldehyde, formaldehyde, OsO4) on the retention of antigenicity of peritoneal macrophages was studied by means of fluorescence microscopy, scanning electron microscopy, and X-ray microanalysis. After fixation with formaldehyde the Concanavalin A (Con A) binding sites were found to be highest, whereas the fixation with OsO4 results in loss of many binding sites. The fluorescence microscopic investigations of glutaraldehyde fixed cells is impaired by fixative induced fluorescence. Therefore X-ray microanalytical determinations show better results for these cells than fluorescence microscopic measurements. On contrary to direct labelling method the number of unspecific Con A binding sites on peritoneal macrophages labelled with the sandwich method is negligible.

Early phase karyotype analysis of chromosome segregation after formation of mouse-mouse hybridomas with chromosome painting probes.

FISH analysis with chromosome painting probes allows, better than karyotyping after Giemsa banding, the study of chromosome segregation after hybridoma formation. FISH is particularly useful for intraspecies hybrids and allows visualization of small chromosome fragments. Cell hybrids were constructed between P3 x 63Ag8.653 mouse myeloma cells and lymphocytes from BALB/c mice by PEG fusion and by selection in hypoxanthine azaserine medium. Three hybridomas (A4, D8, F10) were selected and, after cloning, the cells were cultivated in vitro over a period of 28 days. During this time in culture, air-dried metaphase spreads were prepared by standard methods. For FISH chromosome painting, digoxigenin- and biotin-labeled mouse chromosome painting probes and rhodamine-antidigoxigenin antibodies and fluorescein-avidin were used for dual color detection. Total chromosome numbers and the numbers of mouse chromosomes 1, X, 6 and 12 were estimated as function of days in culture. Mean chromosome numbers of 78 (D8), 82 (F10) and 150 (A4) were observed. The major rearrangements of chromosome numbers occured in the first 28 days in culture and did not change significantly between day 28 and day 56. Mouse chromosome #12, which had the largest chromosome fragments in the parent myeloma, remained stable while the number of X chromosomes, which were significantly fragmented already in the parent myeloma, decreased by approximately 50%.

[Inhibition of concanavalin A and phytohemagglutinine P mediated agglutination of L 1210 cells by different azomethines and possible relations to their cytostatic activity (author's transl)]. / Hemmung der durch Concanavalin A und Phytohämagglutinin P vermittelten Agglutination von L 1210-Zellen mit verschiedenen Azomethinen und mögliche Beziehungen zu deren zytostatischer Wirksamkeit.

Compounds of a special azomethine type have been tested on their activity against concanavalin A and phytohemagglutinine P mediated agglutination of leukemia L 1210 cells. With exception of two compounds possessing a bis-(beta-hydroxyethyl) amino group at the phenyl moiety in para-position to the azomethine group all substances considerable inhibited agglutination of L 1210 cells. In vitro active concentrations are also obtained in animal experiments at least for a short time at the site of injection (i.p.) The importance of cellular reactions for effectivity in vivo is discussed.

Kinetics and regulation of erythrogenic toxins type A and C during growth of Streptococcus pyogenes.

The production of erythrogenic toxins type A (ETA) and C (ETC) is described as a function of growth kinetics. Group A streptococcal strains C 203 S and NY 5 were cultivated in yeast-peptone extract, Todd-Hewitt medium and a synthetic medium. Two main growth phases occurred during growth: a first logarithmic phase and a second linear phase. These phases were separated by a short stationary interphase caused by limitation of the amino acids L-serine and L-leucine. Maximum production of ETC was observed during the logarithmic phase, it was correlated to a high level of viable cells. ETA was produced mainly during the short stationary interphase. The production of ETC is regulated by L-isoleucine. A stagnation or reduction of the concentration of viable cells was observed during the interphase. The phosphate limitation caused during streptococcal growth induced expression of the extracellular protein phosphatase and surprisingly, of a serine proteinase activity. The association between these results and the pathogenicity of streptococci is discussed.

Analysis of the migration behaviour of single microtubules in electric fields.

By video contrast microscopy, individual microtubules formed from pure tubulin in the presence of taxol were studied in constant electric fields. At nearly physiological conditions, i.e., in a buffer at pH 6.8 and 120 mM ionic strength, suspended microtubules moved towards the anode with an electrophoretic mobility of approximately 2.6 x 10(-4) cm(2)/V s, corresponding to an unbalanced negative charge of 0.19 electron charges per tubulin dimer. Strikingly, this value is lower by a factor of at least 50 than that calculated from crystallographic data for the non-assembled tubulin dimer. Moreover, the taxol-stabilized microtubules had an isoelectric point of about pH 4.2 which is significantly lower than that known for the tubulin monomers. This indicates that microtubule formation is accompanied by substantial changes of charge distribution within the tubulin subunits. Constant electric fields were shown to affect also the orientation of microtubules gliding across a kinesin-coated surface at pH 6.8.