Stereoscopic back-scattered electron imaging of silver-stained proteins in nucleoli

By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the AgNOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz--anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a rope-like structure. In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripheraly, and the weakly staining region and the rope-like structure are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.     

A benzodiazepine antagonist inhibits the cerebral metabolic and respiratory depressant effects of fentanyl

It is reported that benzodiazepines such as diazepam will stimulate the opiate receptor system and that B-carboline drugs, which are benzodiazepine antagonists, may interact with opiate receptors directly. The ability of 3-hydroxymethyl-B-carboline (3-HMC) to antagonize several parameters of fentanyl anesthesia was tested here in rats. Fentanyl (25 and 100 micrograms/kg iv) produced dose dependent depression of cerebral blood flow (CBF), measured by radioactive microspheres, and cerebral oxygen consumption (CMRO2). These effects were significantly inhibited by 10 mg/kg 3-HMC iv. To test for the specificity of this effect, 3-HMC was also given to rats ventilated with inspire concentrations of 2% halothane. Halothane depressed CMRO2 equally in 3-HMC and vehicle treated rats, indicating no significant effect of the benzodiazepine antagonist. Blood pressure was increased in 3-HMC compared to vehicle treated animals during both fentanyl and halothane anesthesia. CBF was increased in 3-HMC vs vehicle treated rats during halothane anesthesia but this could be accounted for by the elevated blood pressure and lack of cerebral autoregulation rather than a direct cerebrovascular effect. 3-HMC decreased the sleep time and respiratory depressant effects of fentanyl but enhanced the analgesic effects of the opiate, as measured by time to respond to a hot plate stimulus. These results indicate that 3-HMC has the ability to specifically antagonize fentanyl anesthesia. These effects may be produced by an action of 3-HMC at the benzodiazepine receptor and/or by an action of the B-carboline at opioid receptors.     

Human inter-alpha-trypsin inhibitor: localization of the Kunitz-type domains in the N-terminal part of the molecule and their release by a trypsin-like proteinase

The N-terminal amino-acid sequence of human ITI has been found to be identical with that of the acid-stable human 30-kDa inhibitors (HI-30) from urine, serum, and those released from inter-alpha-trypsin inhibitor by trypsin or chymotrypsin. Serum HI-30 and HI-30 released by trypsin differ from the urinary inhibitor by an additional C-terminal arginine residue. Compared to these two inhibitors the inhibitor released by chymotryptic proteolysis is elongated C-terminally by an additional phenylalanine residue. These results strongly favour HI-30 as the N-terminus of the inter-alpha-trypsin inhibitor and its release from this inhibitor in vivo by cleavage of the Arg123-Phe124 peptide bond by trypsin-like proteinases.     

Isolation of acid-resistant urinary trypsin inhibitors by high performance liquid chromatography and their characterization by N-terminal amino-acid sequence determination

Two crude fractions of acid-resistant trypsin inhibitors (apparent molecular masses 44 and 20 kDa, respectively) were prepared from human urine by gel permeation chromatography. From both preparations the pure inhibitors were isolated by high performance liquid chromatography (HPLC). Their N-terminal amino-acid sequences were determined and compared with those of HI-30 and HI-14 as isolated by reversible binding to either immobilized trypsin or immobilized chymotrypsin. The N-terminal amino-acid sequence of the high-molecular mass inhibitor UI-I isolated by HPLC was identical with those of HI-30 and UI-C-I isolated via immobilized trypsin or chymotrypsin, respectively. The low-molecular mass inhibitors UI-II and UI-C-II differ from HI-14 by the N-terminal extension Glu-Val-Thr-Lys-when obtained by HPLC or by the extension Thr-Lys-when obtained via immobilized chymotrypsin, respectively. The comparison of these N-termini with the amino-acid sequence of HI-30 (Ala1-...-Val16-Thr-Glu-Val-Thr-Lys-HI-14) defines the low molecular urinary trypsin inhibitors as proteolytic degradation products of the high-molecular urinary inhibitor. Proteolysis may occur at different bonds. The existing discrepancies in molecular architecture and in molecular masses of the urinary trypsin inhibitors are discussed.     

Enterohepatic circulation of N-acetyl-leukotriene E4

N-Acetyl-leukotriene E₄, the end product of leukotriene C₄ metabolism in the mercapturic acid pathway, was rapidly eliminated from the blood circulation into the bile of rats. Part of the N-acethyl-leukotriene E₄ secreted from bile into the intestine undewent enterohepatic circulation. Leukotriene absorption occurred from the small intestine and from the colon. Biliary and urinary excretion within 5.5 h amounted to 15 and 2%, respectively, of the intraduodenally administered N-acetyl- H leukotriene E₄ in animals anesthetized with ketamine. HPLC analyses indicated that 35% of the biliary radioactivity corresponded to unchanged N-acetyl- H leukotriene E₄, while 65% in bile and 100% in urine were polar metabolites. Enterohepatic circulation extends the biological half-life of N-acetyl-leukotriene E₄.     

Methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A and its repressibility by serine

The methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A(k) was purified 100-fold. Because it is extremely labile, this enzyme required protection by 1 mm nicotinamide adenine dinucleotide phosphate (NADP(+)) during purification; 0.01 mm EADP(+) with 0.1% bovine plasma albumin stabilized the purified enzyme during storage at -20 C. Although the enzyme has properties of sulfhydryl enzymes, thiol compounds were not stabilizers. Oxidation of methylenetetrahydrofolate, catalyzed by the purified enzyme preparation, is NADP(+)-specific and yields methenyltetrahydrofolate and the reduced pyridine nucleotide. K(m) values for NADP(+) and for 5,10-methylenetetrahydrofolate (prepared as the formaldehyde adduct of biologically synthesized l,l-tetrahydrofolate) were calculated to be 0.021 and 0.026 mm, respectively. Neither purine bases and their derivatives nor serine inhibited the reaction. In growing cultures, the differential rate of synthesis of the methylenetetrahydrofolate dehydrogenase was dependent upon the composition of the medium. A medium which contained acid-hydrolyzed casein, and thus an exogenous source of serine, was repressive for this enzyme. In a serine-free, completely defined medium, the amount of folate added (for serine synthesis de novo) affected the duration of the initial exponential growth phase. At the termination of this phase, which primarily reflected the onset of a decreased rate of serine biosynthesis, synthesis of the methylenetetrahydrofolate dehydrogenase was derepressed. Exogenous serine in the completely defined medium prevented the derepression. Furthermore, physiological concentrations of l-serine were repressive not only for the dehydrogenase but also for the methenyltetrahydrofolate cyclohydrolase and the serine hydroxymethyl-transferase. Concomitantly, the differential rate of synthesis of the formyltetrahydrofolate synthetase of S. faecium var. durans A(k) was increased. Apparently, serine regulates the differential rates of syntheses of these enzymes.     

Über die Pseudopodien von Megakaryocyten und ihre Bedeutung für die Freisetzung von Thrombocyten

Zusammenfassung Im Knochenmark der Ratte werden große runde Megakaryocyten und Megakaryocyten mit zahlreichen Pseudopodien gefunden. Die prospektiven Plättchenfelder liegen in der Mitte der Pseudopodien und sind von der hyalinen Zone des Ektoplasmas umgeben. An der Spitze der Pseudopodien ist die hyaline Zone verbreitert. Die Pseudopodien ragen in Sinusoide und Kapillaren, werden aber auch extravasal gefunden. Gelegentlich läßt sich eine Kontinuität zwischen der Membran der Demarkationsbläschen und der Pseudopodienmembran beobachten. Eine intravasale Freisetzung von Thrombocyten aus Pseudopodien ist sehr wahrscheinlich.

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Pseudopodia of megakaryocytes and liberation of blood platelets

Summary In the bone marrow of rats large round megakaryocytes and megakaryocytes with numerous pseudopodia are to be found. The prospective fields of platelets are located in the central part of the pseudopodia. These fields are surrounded by the hyalin zone of ectoplasm. At the tip of a pseudopodium this zone is enlarged. Pseudopodia protrude into sinusoids and capillaries, but occur also extravasally. Continuity between the membrane of the vesicles for the demarcation of platelets and the cell membrane is occasionally observed. The intravasal liberation of thrombocytes from pseudopodia seems very likely.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Quantitative und qualitative elektronenmikroskopische Untersuchungen zur Struktur des Leberläppchens normaler Ratten

Zusammenfassung Untersucht wurden zufällig ausgewählte Leberstückchen normaler Wistarratten. 25 lückenlose Übersichten von periportalen und zentralen Arealen des Leberläppchens, die aus 2564 elektronenmikroskopischen Einzelaufnahmen (Vergrößerung 4600 x) zusammengesetzt waren, wurden bei einer Gesamtvergrößerung von 53000 x mit Hilfe eines Liniengitters nach der Methode. von Loud quantitativ ausgewertet.Die mittlere Cytoplasmafläche eines Hepatocyten, der im Läppchenzentrum liegt, ist größer als diejenige einer in der Peripherie des Läppchens gelegenen Leberzelle, die Zahl der Anschnitte von Mitochondrien pro Cytoplasmafläche im Zentrum geringfügig größer als in der Peripherie. Die Zahl der Lysosomen pro Cytoplasmafläche ist in der Peripherie des Lobulus dreimal höher als in seinem Zentrum.Der Flächenanteil der Mitochondrienanschnitte an der Cytoplasmafläche im Zentrum beträgt 12,3%, im periportalen Bereich 19,3%. Die mittlere Fläche eines Mitochondrienanschnittes ist im periportalen Gebiet doppelt so groß wie im Zentrum, die Membranprofildichte in Mitochondrien periportaler Zellen ist um etwa ein Drittel größer.Schüsselförmige und schlegeloder hantelförmige Mitochondrien mit parallel zur Längsachse ausgerichteten inneren Membranen wurden nur im Zentrum des Leberläppchens gefunden. Dasselbe gilt für Plasmaprotusionen der Hepatocyten in den Disseschen Raum. Glykogenablagerungen sind gleichmäßig über den Lobulus verteilt, auffallend ist jedoch die ungleichmäßige Verteilung auf die einzelnen Zellen.Die quantitativen Daten werden mit histochemischen Befunden und biochemisch ermittelten Enzymaktivitäten verglichen. Die morphologischen Beobachtungen werden im Zusammenhang mit ähnlichen Befunden, die an pathologisch veränderten Lebern erhoben wurden, diskutiert.

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Structure of the hepatic lobule of the rat

Summary Small randomly chosen pieces of liver tissue of normal Wistar rats were investigated with the electron microscope. 25 survey pictures, consisting of 2564 individual micrographs, were analyzed quantitatively at a final magnification of 53,000 x by a linear scanning method as described by Loud.The average area of cytoplasm is larger in the centrolobular than in the periportal area. The number of mitochondria per unit of cytoplasmic area was roughly the same throughout a liver lobule. However, in the periportal zone the mitochondria were about two times larger than in the center and displayed a 30% higher membrane profile concentration. It was found that the total mitochondrial area per unit of cytoplasm was 12,3% in the centrolobular region as compared to 19,3% in the periportal zone. The number of lysosomes per unit of cytoplasmic area was about three times higher in the periportal than in the centrolobular zone.Cup- and dumbbell-shaped mitochondria with densely packed and longitudinally arranged internal membranes were only found in the centrolobular area. Irregular protrusions of the liver cell into the space of Disse were also found exclusively in this area. The amount of glycogen varied considerably from cell to cell but no significant difference in this respect could be seen between the two zones of the liver lobule.The quantitative findings are discussed with reference to available bio- and histochemical data. The morphological observations are compared with similar observations made on pathologically altered liver cells.

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Herrn Prof. Dr. med. Helmut Ruska zum 60. Geburtstag gewidmet.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.