G2 arrest, binucleation, and single-parameter DNA flow cytometric analysis

One important facet of flow cytometry involves the effects of pharmacological agents on cell cycle progression. Comparative G2 fraction perturbations were examined: effects of sodium butyrate on articular chondrocytes, effects of an antineoplastic agent (SOAZ) and an antirheumatic drug (D-penicillamine) on HeLa cells. Even though DNA flow cytometric analysis detects preferentially an induction of G2 arrest, the mode of action of these agents on the cell cycle is different. Sodium butyrate and D-penicillamine lead to an increase of binucleate cells due to cytokinesis perturbation. Because of similar fluorescence intensity, distinguishing G2 from binucleate GO/1 cells is not easily possible using DNA content measurement and reflects a failure of flow cytometry in the detection of binucleate cells. Rapid cell cycle analysis of single cells should contribute greatly to the study of pharmacological interactions, but DNA flow cytometric measurements obtained from cultured cells exposed to certain agents must be cautiously interpreted because those may interact on cytokinesis and induce artefacts in histogram interpretation.     

A theoretical study of anthracene and phenanthrene derivatives acting as A-T specific intercalators.

Theoretical computations are performed on the comparative A-T versus G-C binding selectivities of two DNA intercalating molecules recently synthesized by Wilson et al. These are derivatives of phenanthrene and anthracene with side chains containing an hydroxy group bound to its C alpha carbon and a cationic amino group bound to its C beta carbon. We have optimized the binding energies of these phenanthrene and anthracene derivatives (1 and 2, respectively) to the double-stranded tetramers d(ATAT)2 and d(GCGC)2, the intercalation occurring in the central pyrimidine (3'-5') purine sequence. The sum of the intercalator-oligonucleotide intermolecular interaction energy plus the conformational energy variation of the intercalator upon binding were computed by the SIBFA procedures, which use empirical formulas based on ab initio SCF computations. Both compounds are found to bind more favourably to the AT sequence than to the GC one. Moreover, the affinity of 1 for the AT oligomer is computed to be larger than that of 2, whereas conversely that of 2 is larger than that of 1 for the GC oligomer. The AT versus GC binding selectivity of 1 is significantly larger than that of 2. These results are in excellent agreement with the experimental findings of Wilson et al. However, contrary to the suggestion of these authors the alpha-hydroxy group of the side chain of the intercalators does not seem to play a decisive role in determining the A-T specificity.     

Rearrangement of tubulin, actin, and myosin in cultured ventricular cardiomyocytes of the adult rat

Antitubulin, phalloidin, and antimyosin were used to study the distribution of microtubules, microfilaments, and myofibrils in cultured adult cardiomyocytes. These cells undergo a stereotypic sequence of morphological change in which myotypic features are lost and then reconstructed during a period of polymorphic growth. Microtubules, though rearranged during these events in culture, are always present in an organized network. Myosin and actin structures, on the other hand, initially degenerate. This initial degeneration is reversed when a cell attaches to the culture substratum. Upon attachment, new microtubules are laid down as a cortical network adjacent to the sarcolemma and, subsequently, as a network in the basal part of the cell. Actin and then myosin filament bundles appear next, in a pattern corresponding to the pattern of the microtubules. Finally, striated myofibrils are formed, first in the central part of the cell, and subsequently in the outgrowing processes of the cell. A mechanism is suggested by which the eventual polymorphic shape of a cell is related to the shape of its initial area of contact with the culture substratum. Finally, a model of myofibrillogenesis is proposed in which microtubules participate in the insertion of myosin among previously formed actin filament bundles to produce myofibrils.     

Immunohistochemical localization of neuropeptide Y in the guinea pig medulla oblongata

Summary The immunohistochemical localization of neuropeptide Y (NPY) was correlated with those of dopamine--hydroxylase (DBH) and vasoactive intestinal polypeptide (VIP) by mapping serial 7 m paraffin sections at three levels of the guina pig lower brainstem: a) area postrema, b) dorsal motor nucleus of the vagus, and c) nucleus prepositus of the hypoglossal nerve. Based on differences in transmitter expression, three populations of NPY-immunoreactive (IR) neurons were distinguished: NPY-IR catecholaminergic cells (NPY/CA), NPY-IR VIP-ergic cells (NPY/VIP), and NPY-IR cells which were not reactive to either DBH or VIP. Within these populations, size differences among neurons in characteristic locations allowed differentiation among the following subpopulations: NPY/CA neurons in the lateral reticular nucleus — magnocellular part (mean neuronal size 538 m²) and parvocellular part (318 m²)-, in the vagus-solitarius complex (433 m²), and in the dorsal strip (348 m²); NPY/VIP neurons in the vagus-solitarius complex (368 m²) and in the nucleus ovalis (236 m²). Apart from scattered NPY-IR cell bodies in the regions listed above, NPY-IR cell bodies in the lateral portion of the nucleus solitarius and in the caudal part of the spinal nucleus of the trigeminal nerve did not exhibit IR to either DBH or VIP. NPY-IR neurons in the area postrema occurred too infrequently for co-localization studies. The differential distribution of heterogeneous NPY-IR cell subpopulations may reflect the involvement of NPY in a variety of neuronal functions.Supported by the Deutsche Forschungsgemeinschaft, grant He 919/6-1     

A modified method of UDS detection in vitro suitable for screening the DNA-damaging effects of chemicals

The UDS induced in cultured FL cells by exposure to chemicals was measured as hydroxyurea-resistant incorporation of 3H-TdR in the acid-insoluble fraction of the 14C-TdR-prelabelled cells synchronized by the combination of arginine starvation and pretreatment with hydroxyurea. The level of UDS is represented by the ratios of 3H/14C radioactivities which are measures of specific activities of 3H. Two direct-acting alkylating agents, MMS and MNNG, a cross-linking agent, mitomycin C, and 3 procarcinogens, B(a)P, AFB1 and cyclophosphamide elicited UDS in the absence or presence of the liver-metabolizing system. Three chemicals of unknown carcinogenicity were also able to induce UDS in this assay system, i.e., bis-(O,O-diethylphosphinothioyl)-disulphide, 4-chlorophenoxy acetic acid (sodium salt) and caramelized malt sugar. With the exception of 4-chlorophenoxy acetic acid, they were also active in the Ames test.     

Proliferation kinetics of rabbit articular chondrocytes in primary culture and at the first passage

The in-vitro proliferation kinetics of young rabbit articular chondrocytes were compared in primary culture and at the first passage. The growth curves labelling and mitotic indices, percentage labelled mitosis (PLM) curves and DNA content distributions by flow-microfluorometric analysis during a 7-day growth period were determined in both cases. The length of the cell cycle and the doubling time calculated from the exponential part of the growth curve were quite similar: Tc = 19 hr and Td = 20 hr for the primary culture, Tc = 17 X 3 hr and Td = 20 hr for the first passage. However, the growth curve and the DNA distribution during the 7-day period showed some differences. The duration of the lag period studied by the growth curve was longer in the primary culture than at the first passage. This phenomenon was also observed using the FCM analysis. The growth fraction determination on the second day of culture was in accordance with the lower proliferation capacity of the cells in primary culture. These data suggest that it would be better to study growth kinetics and drug modifications in articular chondrocytes at the first passage than in primary culture.     

Effects of batrachotoxin on membrane potential and conductance of squid giant axons

The effects of batrachotoxin (BTX) on the membrane potential and conductances of squid giant axons have been studied by means of intracellular microelectrode recording, internal perfusion, and voltage clamp techniques. BTX (550–1100 nM) caused a marked and irreversible depolarization of the nerve membrane, the membrane potential being eventually reversed in polarity by as much as 15 mv. The depolarization progressed more rapidly with internal application than with external application of BTX to the axon. External application of tetrodotoxin (1000 nM) completely restored the BTX depolarization. Removal or drastic reduction of external sodium caused a hyperpolarization of the BTX-poisoned membrane. However, no change in the resting membrane potential occurred when BTX was applied in the absence of sodium ions in both external and internal phases. These observations demonstrate that BTX specifically increases the resting sodium permeability of the squid axon membrane. Despite such an increase in resting sodium permeability, the BTX-poisoned membrane was still capable of undergoing a large sodium permeability increase of normal magnitude upon depolarizing stimulation provided that the membrane potential was brought back to the original or higher level. The possibility that a single sodium channel is operative for both the resting sodium, permeability and the sodium permeability increase upon stimulation is discussed.     

Elektronenmikroskopische Untersuchungen am Subcommissuralorgan des Meerschweinchens

Zusammenfassung Im Subcommissuralorgan des Meerschweinchens wird das mehrreihig hochprismatische Ependym von einem wechselnd breiten, gefäßführenden Hypendym unterlagert, das neben Astrocyten, Oligodendrocyten und Ependymfortsätzen Zellen mit den feinstrukturellen Merkmalen der Ependymzellen enthält.Die subcommissuralen Zellen in Ependym und Hypendym bilden verschiedene Arten von Sekret: Von den Cisternen des endoplasmatischen Reticulum schnüren sich helle Sekretsäckchen ab, die das Cytoplasma durchsetzen. Im apikalen Zellbereich konfluieren sie mitunter zu großen, unregelmäßig begrenzten Sekretarealen. Helle Sekretsäckchen liefern auch den Rohstoff für dichte Sekretgranula, die in manchen Zellen vom Golgi-Apparat gebildet werden; die in verschiedenen Varianten vorkommenden Granula sind apikal angehäuft. Vereinzelt anzutreffende Zellen sind mit Sekretvakuolen so dicht angefüllt, daß das Cytoplasma zu schmalen, dichten Stegen reduziert ist; der Zellkern ist pyknotisch. Die Sekretvakuolen enthalten sehr wenig flockiges Material. Im Hypendym konfluieren die Sekretvakuolen zu großen intracellulären Hohlräumen, in die gelegentlich Mikrovilli und Cilien hineinragen. Schließlich ist eine Art von apokriner Sekretion zu beobachten: Manche Ependymzellen besitzen nahezu homogene Protrusionen, die weit in den Ventrikel reichen; isoliert im Ventrikel werden organellenfreie Cytoplasmabereiche gefunden.Die Kapillaren besitzen ein unterschiedlich breites Endothel. Die Basalmembran ist an vielen Stellen aufgeweitet und umschließt kleine, helle Bezirke. Häufig ist ein echter perivasculärer Raum vorhanden; er ist mit ungeordnet liegenden Filamenten oder Kollagenfibrillen angefüllt und enthält gelegentlich Adventitiazellen. In schmalen perivasculären Spalträumen beobachtet man öfters ein Streifenmuster (Periode ca. 50 m); es handelt sich dabei um ausgedehnte, nicht fibrillär gegliederte Kollagenbereiche.Der entlang dem Recessus pinealis dünn ausgezogene supracommissurale Teil des Organs ist nur von Randbündeln der Commissura posterior unterlagert, die ein dünner Gliafilz von der Hirnoberfläche trennt.

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Electron microscope studies on the subcommissural organ of the guinea pig

Summary In the subcommissural organ of the guinea pig the ependyma is built up of several rows of prismatic cells. The hypendyma of varying width contains capillaries plus astrocytes, oligodendrocytes and ependymal cell processes as well as elements showing the structural characteristics of ependymal cells.The subcommissural cells in the ependyma and in the hypendyma form various types of secretory products: Light secretory sacs originating from the cisternae of the endoplasmic reticulum pile up in the cytoplasm. Sometimes they confluate to irregularly lined areas in the apical zone. In several cells the light secretory sacs deliver material for dense secretory granules which are produced by the Golgi apparatus; dense granules of varying shape are accumulated apically. Some cells are tightly filled with secretory vacuoles. The cytoplasm between the vacuoles is condensed and reduced to narrow rims; the nucleus is pyknotic. The secretory vacuoles contain very little fluffy material. In the hypendyma the secretory vacuoles confluate forming giant vacuoles, occasionally containing microvilli and cilia. Finally a kind of apocrine secretion is observed: Some ependymal cells have protrusions which possess a nearly homogeneous cytoplasm and extend far into the ventricular lumen. Isolated cytoplasmic areas lacking organelles are to be found within the ventricle.The endothelium of the capillaries varies in width. At some places the basement membrane is widened and encloses small areas of lower density. Often a true perivascular space is found, filled with disordered filaments or collagen fibrils; occasionally it contains adventitial cells. Sometimes a substance exhibiting a periodic pattern (period ca. 50 m) occurs in narrow perivascular spaces; this material consists of extended areas of non-fibrillar collagen.The thin supracommissural part of the organ extends along the recessus pinealis. The adjoining commissura posterior is flattened to only a few axon bundles which are separated from the cerebral surface by a thin felt of glial processes.

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Herrn Dozenten Dr. phil. Ernst Kinder zur Vollendung seines 60. Lebensjahres gewidmet.

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Die Arbeit wurde mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft ausgeführt. — Frau H. Asam danken wir für ausgezeichnete Mitarbeit bei der Präparation; ihr und Frl. C. Degen, Frl. I. Dürr und Frau B. Rottmann für die Ausführung der photographischen Arbeiten. Herrn Dr. med. A. Meinel gebührt unser Dank für wertvolle Diskussionen und Mithilfe bei der Fixierung der Objekte.