Initial theoretical framework and problem solving concerning the enigma of heredity.

The difference in formulation of the question of heredity on a different level of knowledge in Brno in the 1830s and after 1850 is discussed in this article. In order to solve the problem the most important source is foreshown in the new philosophy of plant physiology and in physics. Mendel was pleased to have met excellent teachers of both these fields. This explanation is an example of Mirko Grmek's thesis: 'l'histoire des sciences est le laboratoire de l'épistomologie'.

Differences in the DNA-stainability of spermatozoa from fertile and suspected infertile men.

The aim of this work was to determine whether it is possible to distinguish between fertile (control group, already fathers) and infertile men (suspected infertility), by comparing the fluorescence intensity of the sperm-DNA after incubation with appropriate dyes. First we examined two different DNA-specific dyes (DAPI and YOYO-1) using bull spermatozoa. Based on good results in immunohistochemical applications, YOYO-1 was chosen for further work. The fluorescence-intensity of 200 single, morphologically normal spermatozoa in each semen sample were measured in a cytophotometer, means + SD determined and histograms delineated. Of 20 samples from the control group, 17 had markedly higher fluorescence-intensity than did 7/15 of the suspected infertile men. It is concluded that the DNA of the latter seven samples was less accessible to the dye than was the DNA of the control group. There are cases of infertility known in which there is loss of one or more of the DNA-binding proteins, which in spermatozoa are mainly (85%) protamines. The relationship between the stainability of the sperm-DNA and the packaging with DNA-binding proteins is discussed. Two of the histograms showed abnormalities in the distribution of the fluorescence-intensities, one sample was extremely fragile and most of the sperm lysed during the staining-procedure. Five samples showed normal histograms in comparison with the control group.

gamma-Tubulin redistribution in taxol-treated mitotic cells probed by monoclonal antibodies.

Monoclonal antibodies were prepared against conserved synthetic peptide from the C-terminus of the gamma-tubulin and their specificity was confirmed by immunoblotting, competitive enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The antibodies decorated interphase centrosomes as well as half-spindles and midbodies in mitotic cells of various origin. The prepared antibodies were used to study the gamma-tubulin distribution in nocodazole and taxol-treated cells. In the cells recovering from the nocodazole treatment, gamma-tubulin was found in centers of all microtubule asters. Examination of relative location of gamma-tubulin and microtubule asters in taxol-treated mitotic cells 3T3, HeLa and PtK2 revealed that the number of taxol-induced microtubule asters exceeded the number of gamma-tubulin-positive spots. The gamma-tubulin was often found in the periphery of microtubule asters. Centrosomal phosphoprotein epitope detected by MPM-2 antibody colocalized with gamma-tubulin in taxol-treated mitotic cells. The presented data suggest that taxol-induced microtubule asters are in vivo nucleated independently of gamma-tubulin, and other minus-end nucleator(s) are necessary for formation of such asters. Alternatively, gamma-tubulin is present in subthreshold amounts undetectable by immunofluorescence.

Dawn of human genetics.

Several physicians of the 19th century had a fair knowledge of the transmission of some human hereditary traits. Critical examination of pedigrees culminated in formulation of a "law of heredity" (!) before the rediscovery of Mendel's classical paper in 1900.

Computer modeling of pole formation in cell division.

The regular position of spindle poles in bi- and multipolar cell divisions is interpreted as the result of expanding caps on the surface of cell nuclei first proposed by Mazia in 1986. In a computer model it is shown that expanding caps position their centers with the maximal distance between them, which is in complete accordance with findings in experimentally influenced cell divisions in which multiple poles are formed.

Nuclear migration and spindle formation in the fourth cleavage of sea urchin eggs under the influence of inhibitors.

Mitosis of sea urchin eggs was inhibited when exposed to 3 micrograms/ml aphidicolin from the 2-cell stage onwards. Nevertheless the nuclei migrated to the vegetal pole at the time of the fourth unequal division in control eggs. Two or four equal or unequal asters developed. Asters in proximity to the vegetal pole were always considerably smaller than those close to the center of the two blastomeres. In contrast to colchicine, cytokinesis but not migration of the nuclei in the vegetal half was prevented by treatments with 5 microM cytochalasin B or D. Various mitotic figures were formed in the vegetal blastomeres of eggs treated with 0.4 mM colchicine or 3 microM griseofulvin after the third cleavage. In some eggs a centrally localized monaster with chromosomes in sphere-like arrangement was formed in others a monopolar mitotic figure pushed the chromosomes in bowl-like arrangements to the most vegetal cortex. In anaphase one set of chromatids migrated to the monopole leaving the scattered sister-chromatids behind. The mechanism of migration of the nuclei and of chromosome arrangement in the metaphase plate is discussed.

Multipolar mitosis in procaine-treated polyspermic sea urchin eggs and in eggs fertilized with UV-irradiated spermatozoa with a computer model to simulate the positioning of centrosomes.

Procaine-treated eggs can be penetrated by more than one spermatozoon. Supernumerary male pronuclei can fuse with the female one giving raise to multipolar spindles or remain isolated within the egg's cytoplasm forming their own spindle. In all types of multiple mitotic figures (asters and spindles) the distribution of asters is equidistant either uniplanar or at maximum distance like at the apices of a polyhedron. Astral rays are not different from spindle fibers: they can attach to and attract chromosomes of "foreign" mitotic figures. When several mitotic figures are present in one egg, the partner asters are always of the same size, and microtubules of one aster never interdigitate with those of others. The hypothesis that positioning of centrosomes is brought about by spreading of a centrosome organizer in the form of an expanding calotte on the surface of the nucleus (Mazia, D., Int. Rev. Cytol. 100, 49-92 (1987)) is supported by a computer model.

Mitotic patterns and DNA synthesis in dinitrophenol-treated sea urchin eggs.

Eggs of Paracentrotus exposed to 1 mM 2,4-dinitrophenol (DNP) in sea water at 10-min intervals from 10 to 40 min after insemination and returned to normal sea water 60 min later, divide into two cells. If, however, such eggs are treated for 60 min beginning at 50 min after insemination, they cleave directly into four cells. Division centers can split and separate in the DNP solution though the asters appear very faint and the spindle considerably reduced in size. This means that DNP has an inhibitory effect on duplication of division centers but not on the splitting and separation of centers already duplicated. Cytophotometric comparison of the DNA content in di-, tri- and tetrapolar mitosis of DNP-treated and control eggs revealed no difference showing that chromosome cycle and centrosome cycle are independent events in the cell cycle.

Histochemical study of globules in accessory cells of sea-urchin ovary: in relation to jelly coat substance.

Accessory cells of the sea-urchin ovary were studied histologically and histochemically. Different types of globules in these cells were distinguished. Histochemical observations suggest that the accessory cells synthesize neutral mucosubstances, sulfo- and sialopolysaccharides, which might be precursors for jelly formation.

[Lethal neonatal deficiency of carbamyl phosphate synthetase (author's transl)]. / Carbamylphosphatsynthetasemangel bei einem Neugeborenen mit letalem Verlauf.

This is a report of a male neonate with hyperammonemia who showed progressive neurologic symptoms and pulmonary bleeding due to carbamyl phosphate synthetase deficiency. This newborn male died in coma after only 119 hours. His elder brothers died under similar circumstances in the first days of life, and a CPS-deficiency is not to be excluded. The pulmonary bleeding in the patient and in one brother caused by hyperammonemia is emphasized. In the pedigree repeated consanguinity could be detected.

Histochemical studies of jelly coat of sea-urchin eggs during oogenesis.

Jelly coat of sea-urchin eggs consists of polysaccharides and glycoproteins. Some properties of jelly coat have already been investigated, but not histochemically. The oogenesis in Paracentrotus lividus was studied histologically and the oocytes were classified into six different stages. The extracellular jelly appeared first around the growing oocytes II which remained attached to the germinal epithelium. The jelly became thicker when the oocyte approached maturation. Histochemical analysis revealed that the jelly consists of mucopolysaccharide-protein-complexes. The polysaccharide component is composed of both neutral and acid mucopolysaccharides. The former are amylase-resistant. The acid mucopolysaccharides contain both carboxyl and sulfate groups, which are in close proximity to vicinal hydroxyl groups. Sulfated mucopolysaccharide is hyaluronidase-resistant. Sialic acid could not be clearly demonstrated, because it seems to be resistant to neuraminidase. Pepsin digestion indicated the masking of acidic groups by proteins which compete with basic dyes (Alcian blue, Azure A, coriphosphine etc.). Proteolytic digestion enhanced dye-binding ability of jelly, but removed also some of the periodate-reactive mucosubstances. Also a protein component could be demonstrated histochemically. No histochemical difference between jelly coat of oocytes and that of eggs has been found. The possible molecular structure of jelly coat is discussed.

Transient recovery from trypsin-caused reduction in fertilizability of sea urchin eggs.

The effect of trypsin on the fertilizability of sea urchin eggs was studied withParacentrotus lividus andPseudocentrotus depressus. The main effects were two reductions of fertilizability, with a transient increase intervening. The first decrease was probably caused by degradation of sperm-binding sites at the vitelline sheet and the second by degradation of binding sites on the plasma membrane.

Effect of 5-bromodeoxyuridine on differentiation. II. The effect of early BUdR treatment on gastrulation of sea urchin embryos.

BUdR treatment of early cleavage stages is more effective on gastrulation the earlier the drug is administered before the 16-cell stage. The distribution of percentages of embryos gastrulating is in good agreement with the theoretical calculated values presented in part I and permit the assumption that BUdR may act by preventing normal transcription at, or shortly after, the 16-cell stage.

Effect of 5-bromodeoxyuridine on differentiation. I. Probability distribution of BUdR-containing DNA-strands in subsequent divisions.

The consequences on transcription of BUdR substitution of the normal thymidine in nuclear DNA are analyzed from a probabilistic point of view. The probability that at any stage subsequent to the administration of BUdR, a randomly chosen cell will be able to make good transcripts is calculated both under the assumption that incorporation of BUdR is complete and under the assumption that incorporation is partial. In the latter case both gradually increasing and decreasing incorporation rates are considered. The presented calculations may be of value for the investigation of any type of differentiating cells. Using the example of the sea urchin embryo it is shown how to calculate the probability that a particular group of cells, which in further development differentiate to a certain organ, will inherit as sufficient number of essential genes unaffected by BUdR in order to ensure a normal development. It is shown that the probability that the four macromeres (or micromeres) in the vegetal half of the 16-cell stage of the sea urchin embryo will contain at least one, two or four plus (or minus) strands of DNA uninfluenced by BUdR gradually decreases the earlier BUdR is administered. The theoretical calculations are in agreement with experimental observations on sea urchin embryos (described in part II).

Kinetics of RNA-synthesis in the 16-cell stage of the sea urchinParacentrotus lividus.

The concentration of RNA synthesised in the prolonged interphase of micromeres at the 16 cell stage is about 9 times higher per unit volume than in the other blastomeres. Since there are cytoplasmic connections between micromeres and macromeres, a transfer of the micromere RNA by simple diffusion is to be expected.