CORRELATED MORPHOMETRIC AND BIOCHEMICAL STUDIES ON THE LIVER CELL : II. Effects of Phenobarbital on Rat Hepatocytes

The changes occurring in rat hepatocytes during a 5 day period of treatment with phenobarbital were determined by morphometric and biochemical methods, particular attention being paid to the endoplasmic reticulum. The hepatocytic cytoplasm played an overwhelming part in the liver hypertrophy, while the hepatocytic nuclei contributed to only a moderate extent. The endoplasmic reticulum accounted for more than half of the increase in cytoplasmic volume. The increase in the volume and number of hepatocytic nuclei in the course of phenobarbital treatment was associated with changes in the ploidy pattern. Until the 2nd day of treatment both the rough-surfaced endoplasmic reticulum (RER) and the smooth-surfaced endoplasmic reticulum (SER) participated in the increase in volume and surface of the whole endoplasmic reticulum (ER). Subsequently, the values for RER fell again to control levels, whereas those for SER continued to increase, with the result that by the 5th day of treatment the SER constituted the dominant cytoplasmic element. The specific volume of mitochondria and microbodies (peroxisomes) remained constant throughout the duration of the experiment, while that of the dense bodies increased. The specific number of mitochondria and microbodies displayed a significant increase, associated with a decrease in their mean volume. The phenobarbital-induced increase in the phospholipid and cytochrome P-450 content of the microsomes, as well as in the activities of microsomal reduced nicotinamide-adenine dinucleotide phosphate-cytochrome c reductase and N-demethylase, was correlated with the morphometric data on the endoplasmic reticulum.     

Elektronenmikroskopische Untersuchungen an spezifischen Organellen von Endothelzellen des Frosches (Rana temporaria)

Zusammenfassung Endothelzellen der Wirbeltiere enthalten spezifische Organellen, deren Funktion unbekannt ist. Diese Organellen werden beim Frosch (Rana temporaria) nach unterschiedlichen Fixierungen elektronenmikroskopisch untersucht. Die Organellen sind walzenförmig mit mannigfachen Abweichungen, bis zu 2 lang und 0,1 bis 0,5 dick. Ihre oft unterbrochene Außenmembran ist dicker als zytoplasmatische Membranen. Das Innere der Organellen besteht aus Tubuli, die in eine elektronendichte Matrix eingebettet sind. Die Dichte dieser Matrix zeigt deutliche Abstufungen. Die Tubuli sind möglicherweise aus einer spiralförmigen Molekülkette aufgebaut. Das Verteilungsmuster der Organellen wird mit stereologischen Methoden untersucht. Die größte Volumendichte weisen die Aortae thoracicae mit 8% auf. Die Volumendichte der Organellen im Zytoplasma der Endothelzellen scheint mehr von der Entfernung der betreffenden Gefäßstrecke zum Herzen abzuhängen als von der Gefäßgröße. Es werden Verbindungen der Organellen zu zytoplasmatischen Membransystemen aufgezeigt. Auf Besonderheiten des Endothels, darunter Aggregationen von Ribosomen, wird hingewiesen.

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Electron microscopic studies on specific organelles of endothelial cells in the frog (Rana temporaria)

Summary Endothelial cells of vertebrates contain specific organelles of unknown function. These organelles are studied by electron microscopy with different fixations. The organelles are rod-shaped with many variations, up to 2 in length and 0.1 to 0.5 in thickness. Their outer membrane, which is often discontinuous, is thicker than cytoplasmic membranes. The density of the matrix shows distinct gradations. The organelles contain tubules, possibly built up by a spiral molecular chain. The distribution of the organelles is investigated with stereological methods. Their volume density in endothelial cell cytoplasm appears to depend more on the distance from the heart than on vessel size, the thoracic aortae showing the highest organellae content of 8%. Connections between organelles and cytoplasmic membranes are demonstrated. Particularities of endothelium, among them aggregations of ribosomes, are pointed out.

Herrn Prof. Dr. med. F. Hammersen und Fräulein Dr. med. M. Lewerenz danke ich herzlich für Anregungen, Unterstützung und Kritik. Die Arbeit entstand während eines vom Deutschen Akademischen Austauschdienst dankenswerterweise gewährten Stipendiums; sie wurde unterstützt vom Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung, Kredit-Nr. 3952.     

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Transfer of amino acids and nitrate from the roots into the xylem of Ricinus communis seedlings

The loading of amino acids and nitrate into the xylem was investigated by collection and analysis of root-pressure exudate from the cut hypocotyl stumps of seedlings of Ricinus communis L. Glutamine was found to be the dominant amino acid in the exudate and also to be the amino acid which is transferred to the xylem most rapidly and accumulated to the greatest extent. The comparison between uptake and xylem loading showed significant differences in specificity between these two transport reactions, indicating a different set of transport systems. Nitrate is transferred to the xylem at a higher relative rate than any amino acid despite the great nitrate-storage capacity of the root system. Thus the supply of nitrate to Ricinus plants leads to enhanced nitrogen allocation to the shoots.     

In vitro production of proteoglycans in the articular-epiphyseal cartilage of growing pigs

The failure of cartilage mineralization in osteochondrotic cartilage may be due to an impaired proteoglycan production. Thein vitro production of proteoglycans was therefore studied in the joint cartilage of growing pigs, aged 9–18 weeks, after incubation of cartilage samples with³⁵S-sulfate. Cartilage was obtained from different areas of the femoral condyles and samples from these areas were further divided into three layers, where the superficial layer contains articular cartilage and the basal layers consist of growth cartilage. There was no significant difference in the overall amount of³⁵S-proteoglycans synthesized in different areas of the condyles. However, the total production of³⁵S-proteoglycans per mg tissue was highest in the basal layer in all areas. This was not due to a larger number of cells; the superficial layer contained more DNA per mg tissue than the basal layer. Gel chromatography on Sepharose CL-2B of the cartilage extracts, which resulted in the separation of large proteoglycans (K _av 0.4) from proteoglycans of small hydrodynamic size (K _av 0.8), showed that the relative amount of large proteoglycans increased with the distance from the articular surface. Again, no difference in the relative amounts of large and small proteoglycans were found when cartilage from different areas were compared. Osteochondrotic cartilage was detected in the pigs aged 12–18 weeks. In areas where osteochondrotic cartilage were present, the total production of³⁵S-proteoglycans was lowered and the relative amount of large proteoglycans was less than that found in the adjoining areas devoid of osteochondrotic lesions. The data available indicate that the higher relative amount of small proteoglycans in the osteochondrotic cartilage was partly caused by degradation of the large proteoglycans (aggrecan).     

cDNA cloning of carrot (Daucus carota) soluble acid beta-fructofuranosidases and comparison with the cell wall isoenzyme.

Carrot (Daucus carota), like most other plants, contains various isoenzymes of acid beta-fructofuranosidase (beta F) (invertase), which either accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular beta F). Using antibodies against isoenzyme I of carrot soluble beta F, we isolated several cDNA clones encoding polypeptides with sequences characteristic of beta Fs, from bacteria, yeast, and plants. The cDNA-derived polypeptide of one of the clones contains all partial peptide sequences of the purified isoenzyme I and thus codes for soluble acid beta F isoenzyme I. A second clone codes for a related polypeptide (63% identity and 77% similarity) with characteristics of isoenzyme II. These two soluble beta Fs, have acidic isoelectric points (3.8 and 5.7, respectively) clearly different from the extracellular enzyme, which has a basic isoelectric point of 9.9. Marked differences among the three nucleotide sequences as well as different hybridization patterns on genomic DNA gel blots prove that these three isoenzymes of carrot acid beta F are encoded by different genes and do not originate from differential splicing of a common gene, as is the case in the yeast Saccharomyces cerevisiae. All three carrot acid beta Fs, are preproenzymes with signal peptides and N-terminal propeptides. A comparison of the sequences of the soluble enzymes with the sequence of the extracellular protein identified C-terminal extensions with short hydrophobic amino acid stretches that may contain the information for vacuolar targeting.