Elektronenmikroskopische Autoradiographie mit H3-Thymidin an der Thymusrinde der Maus

Zusammenfassung Ultradünnschnitte durch die Thymusrinde erwachsener männlicher Mäuse wurden mit einer Einkristallschicht von Ilford-L 4-Photoemulsion überzogen. In elektronenmikroskopischen Autoradiogrammen können die mit H³-Thymidin markierten Zelltypen besser erkannt werden als in lichtmikroskopisch-autoradiographischen Untersuchungen am gleichen Gewebe.Den größten Anteil der freien Zellen stellen die kleinen Lymphocyten mit dichten, von wenigen Ausnahmen abgesehen, unmarkierten Kernen und schmalem Zytoplasmasaum. In der subkapsulären Zone finden sich gehäuft große Lymphozyten und ihre Vorstufen mit einem umfangreichen, nicht so dichten Kern und einem breiteren, freie Ribosomen enthaltenden Zytoplasmasaum. 83% der Kerne dieser Zellen sind deutlich markiert. Die epithelialen Retikulumzellen im Inneren der Rinde und das Randepithel haben unmarkierte Kerne. Nur vereinzelt werden markierte Retikulumzellen unmittelbar unter dem Randsaum gefunden. Plasmazellen sind in der Rinde nur selten zu finden. Ihre Kerne weisen keine Markierung auf. Die lichtmikroskopisch-autoradiographisch erhobenen Befunde von Hinrichsen (1963, 1965) werden elektronenmikroskopisch bestätigt: Bei der Maus stellt die unmittelbar unter der Kapsel liegende Randzone des Thymus die Keimschicht zur Bildung von Thymuslymphozyten dar.

---

Summary Sections of the thymus of adult male mice were covered with Ilford-L 4 photographic emulsion. In electron microscopic autoradiograms, the labelled cell types after H³-thymidine injection — can be more easily identified than with light microscopic autoradiographic methods.Most of the free cells are small lymphocytes with dense, mostly unlabelled nuclei and scanty cytoplasm. In the sub-capsular zone larger lymphocytes with a bigger, but not so condensed nucleus and more extensive cytoplasm containing free ribosomes can be found. 83% of their nuclei are labelled with tritiated thymidine. The nuclei of epithelial reticulum cells in the inner cortex and the surface epithelium are unlabelled. Only very few labelled reticulum cells can be found immediately under the connective tissue layer. Plasma cells are rarely found in the cortex. Their nuclei are not labelled. The light microscopic autoradiographic findings by Hinrichsen (1963, 1965) are electronmicroscopically confirmed: the lymphocytes of the mouse thymus originate in the immediately sub-capsular cortex of the thymus.

---

---

Herrn Professor Bargmann zum 60. Geburtstag gewidmet.

---

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Genetic identification of non-specific esterases of the mouse cauda epididymidis and description of esterase-28, a new carboxylesterase isoenzyme (EC 3.1.1.1)

Twenty-five (25) electrophoretic bands with esterase activity were distinguished in supernatants of cauda epididymidis of DBA/2J mice. Twenty (20) of these were assigned to 10 genetically defined esterases (ES-1, ES-2, ES-3, ES-6, ES-7, ES-11, ES-14, ES-17, ES-19, ES-22) which were already known from investigations of other mouse tissues. Furthermore, ES-10 was identified in cauda supernatants after isoelectric focussing. A hitherto genetically undefined esterase was assigned to locus Es-28 which was expressed solely in the epididymis. Three phenotypes were distinguished: ES-28A was present in the majority of the inbred strains examined. ES-28B was observed in AKR/Han mice and ES-28C was found in SEG/1 mice.     

Restoration of membrane incorporation of an Escherichia coli outer membrane protein (OmpA) defective in membrane insertion

The mechanism of sorting, to the outer membrane, of the 325-residue Escherichia coli protein OmpA has been investigated. It is thought to traverse the membrane eight times in antiparallel beta-strands, forming an amphiphilic beta-barrel which encompasses residues 1 to about 170; the COOH-terminal moiety is periplasmic. A mutant, carrying the substitutions Leu164----Pro and Val166----Asp within the last beta-strand (residues 160-170), has been described which was unable to assemble in the membrane (Klose, M., MacIntyre, S., Schwarz, H., and Henning, U. (1988) J. Biol. Chem. 263, 13297-13302). Linkers were inserted between the codons for residues 164 and 165 of the mutant protein. Of 13 different genes recovered, five encoded proteins which had regained the ability to assemble in the membrane. The properties of the mutant proteins, together with a structure prediction method, indicate the following rules for the final beta-strand to be compatible with, or possibly initiate, membrane insertion: (i) it must be amphiphilic or hydrophobic while its primary structure as such is fairly unimportant, (ii) it must extend over at least 9 residues, and (iii) it must not contain a proline residue around its center. One of the genes recovered coded for OmpA up to residue 164 and then followed by 10 linker-encoded residues. This 174-residue polypeptide was assembled in the membrane but did not, in contrast to all other proteins, expose sites sensitive to trypsin at the inner face of the membrane. This behavior agrees perfectly well with the OmpA model.     

Pituitary and ovarian responses of post-partum acyclic beef cows to continuous long-term GnRH and GnRH agonist treatment

Post-partum acyclic beef cows received continuous long-term treatment with GnRH (200 or 400 ng/kg body wt/h) or the GnRH agonist buserelin (5.5 or 11 ng/kg body wt/h) using s.c. osmotic minipumps which were designed to remain active for 28 days. All treatments increased circulating LH concentrations whereas FSH remained unchanged. Ovulation and corpus luteum (CL) formation as judged by progesterone concentrations greater than or equal to 1 ng/ml occurred in 0/5 control, 4/5 200 ng GnRH, 4/4 400 ng GnRH, 4/5 5.5 ng buserelin and 3/5 11 ng buserelin cows. The outstanding features of the progesterone profiles were the synchrony, both within and across groups, in values greater than or equal to 1 ng/ml around Day 6, and the fact that most CL were short-lived (4-6 days). Only 3 cows, one each from the 400 ng GnRH, 5.5 ng buserelin and 11 ng buserelin groups, showed evidence of extended CL function. Cows failed to show a second ovulation which was anticipated around Day 10 and this could have been due to insufficient FSH to stimulate early follicular development, or the absence of an endogenously driven LH surge. The highest LH concentrations for the respective groups were observed on Days 2 and 6 and by Day 10 LH was declining, although concentrations did remain higher than in controls up to Day 20.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brefeldin A affects the cellular distribution of endocytic receptors differentially.

The cell surface expression of three endocytic receptors was studied in human hepatoma Hep G2 cells treated with brefeldin A (BFA). Ligand binding and cell surface iodination revealed that BFA increased the number of mannose 6-phosphate/insulin-like growth factor II receptors twofold and decreased the amount of asialoglycoprotein and transferrin receptors by 40-60%. The altered expression of receptors at the cell surface was paralleled by changes in the respective ligand uptake. The implications of this finding on our understanding of intracellular trafficking are discussed.     

Effects of abscisic acid on somatic embryo maturation of hybrid larch

Somatic embryos of hybrid larch (Larixleptoeuropaea) whichhad been matured for 4 weeks on maturation medium, developednormally on medium supplemented with 60 µM ABA, but abnormallyon medium with no ABA. A comparative structural and histochemicalinvestigation was carried out on these two types of mature embryos.At the light microscope level, differences between both treatmentswere visible only after 2–3 weeks of maturation. At aroundthis time, abnormal development becomes evident macroscopically:ABA-minus embryos remain rather stubby as opposed to the morecylindrically shaped ABA-plus embryos. Whereas somatic embryosmatured with ABA consist of densely cytoplasmic cells showinga high rate of cell division, ABA-minus embryos are largelymade up of expanded and highly vacuolate cells, indicating thatgrowth in the latter is mainly due to cell expansion and notdivision. After 4 weeks of maturation, ABA-minus embryos beginto elongate in the hypocotyl region, and precocious germinationwas observed frequently. Again, these morphogenetic events werelargely due to abnormal timing of cell expansion. Histochemically,storage proteins were found only in somatic embryos maturedfor 4 weeks with ABA. This observation is in line with resultsobtained by total protein analysis, yielding significantly lowertotal protein contents in ABA-minus embryos both on a freshweight and a per embryo basis after 4–5 weeks of maturation.Deposition of starch grains mainly in the cortex tissue of thehypocotyl region was observed within 2 weeks of maturation invarying amounts regardless of ABA supply. Polyphenols, in particularcatechins and proanthocyanidins, were present in all embryosfrom the very onset of development. They were localized preferentiallyin the proximal suspensor cells and the basal region of theembryo. However, accumulation of polyphenols was generally muchmore pronounced in embryos matured without ABA, indicating alack of biochemical regulatory competence in those embryos. Key words: Abscisic acid, embryonal development, somatic embryo, storage protein, polyphenols