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.     

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.     

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.

---

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.

---

---

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

---

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.     

Interchain hydrogen bonds via bound water molecules in the collagen triple helix

If the collagen triple helix is so built as to have one set of NH ? O hydrogen bonds of the type N₃H₃(A) ? O₂(B), then it is possible to have a linkage between N₁H₁(B) and O₁(A) through the intermediary of a water molecule with an oxygen O leading to the formation of the hydrogen bonds N₁(B) ? O and O (A). In the same configuration, another water molecule with an oxygen O can link two earbonyl oxygens of chains A and B forming the hydrogen bonds O O₁(A) and O O₀ (B). The two water oxygens also become receptors at the same time for CH ? O hydrogen bonds. Thus, the neighboring chains in the triple helix are held together by secondary valence bond linkages occurring regularly sit intervals of about 3 Å along the length of the protofibril. The additional water molecules occur on the periphery of the proto-fibril and will contribute their full share towards stabilizing the structure in the solid state. In solution, they will be disturbed by the medium unless they are protected by long side groups. It appears that this type of two-bonded structure, in which one NH ? O bond is to a water molecule, can explain several observations on the stability and hydrogen exchange properties of collagen itself and related synthetic polypeptides. The nature of the water bonds and their strength are found to be better in the one-bonded structure proposed from Madras than in the one having the coordinates of Rich and Crick.     

Skeletal muscle changes after endurance training at high altitude.

The effects of endurance training on the skeletal muscle of rats have been studied at sea level and simulated high altitude (4,000 m). Male Wistar rats were randomly assigned to one of four groups: exercise at sea level, exercise at simulated high altitude, sedentary at sea level, and sedentary at high altitude (n = 8 in each group). Training consisted of swimming for 1 h/day in water at 36 degrees C for 14 wk. Training and exposure to a high-altitude environment produced a decrease in body weight (P less than 0.001). There was a significant linear correlation between muscle mass and body weight in the animals of all groups (r = 0.89, P less than 0.001). High-altitude training enhanced the percentage of type IIa fibers in the extensor digitorum longus muscle (EDL, P less than 0.05) and deep portions of the plantaris muscle (dPLA, P less than 0.01). High-altitude training also increased the percentage of type IIab fibers in fast-twitch muscles. These muscles showed marked metabolic adaptations: training increased the activity levels of enzymes involved in the citric acid cycle (citrate synthase, CS) and the beta-oxidation of fatty acids (3 hydroxyacyl CoA dehydrogenase, HAD). This increase occurred mainly at high altitude (36 and 31% for HAD in EDL and PLA muscles; 24 and 31% for CS in EDL and PLA muscles). Training increased the activity of enzymes involved in glucose phosphorylation (hexokinase). High-altitude training decreased lactate dehydrogenase activity. Endurance training performed at high altitude and sea level increased the isozyme 1-to-total lactate dehydrogenase activity ratio to the same extent.(ABSTRACT TRUNCATED AT 250 WORDS)