LYTIC ACTIVITIES IN RENAL PROTEIN ABSORPTION DROPLETS : An Electron Microscopical Cytochemical Study

The digestive cycle following reabsorption of hemoglobin by cells of the proximal convoluted tubules in mouse kidney and the uptake of ferritin by glomerular mesangial cells in the kidney of normal and nephrotic rats were investigated by electron microscopical histochemical procedures. Mouse kidneys, sampled at closely spaced time points between 1 to 48 hours after intraperitoneal injection of hemoglobin, and rat (normal and nephrotic) kidneys, sampled at 30 minutes, 2 hours, and 48 hours after intravenous injection of ferritin, were fixed in glutaraldehyde, cut at 50 µ on a freezing microtome, incubated for acid phosphatase and thiolacetate-esterase, and postfixed in OsO₄. Satisfactory preservation of fine structure permitted the localization of the enzymatic reaction products on cell structures involved in uptake and digestion of exogenous proteins. The latter were identified either by their density (hemoglobin) or their molecular structure (ferritin). It was found that lysosomal enzymic activities and incorporated exogenous proteins occur together in the same membrane-bounded structures. In the cells of the proximal convolution, lytic activities become demonstrable within 1 hour after hemoglobin injection, appear first in apical vacuoles filled with hemoglobin, and persist in fully formed protein absorption droplets. At the end of the lytic cycle (~48 hours post injection), the cells have an increased population of polymorphic bodies which exhibit lytic activities. In smaller numbers, identical bodies occur in controls. It is concluded that they represent remnants of previous digestive events. The means by which the resorptive vacuoles acquire hydrolytic activities remain unknown. Fusion of newly formed vacuoles with residual bodies was not seen, and hemoglobin incorporation into such bodies was only occasionally encountered. Acid phosphatase activity was found sometimes in the Golgi complex, but enzyme transport from the complex to the resorbing vacuoles could not be established. Autolytic vacuoles containing mitochondria or mitochondrial remnants were frequently found during the early stages of hemoglobin resorption, but no definite conclusions about the mechanism involved in the segregation of endogenous material were obtained. In nephrotic rats ferritin was segregated in membrane-bounded bodies mainly in the mesangial cells and to a lesser extent in epithelial and endothelial cells. Most of these sites were marked by the reaction products of acid phosphatase and organophosphorus-resistant esterase and therefore identified as lysosomes connected with the digestion of incorporated exogenous proteins.     

Die Auslese tetraploider Formen von Lupinus angustifolius nach Röntgenbestrahlung und Ergebnisse der vergleichenden Untersuchungen mit den diploiden Ausgangsformen

Zusammenfassung Nach Röntgenbestrahlung wurden aus verschiedenen X₂-Generationen von Lupinus angustifolius mehrere breitblättrige Mutanten ausgelesen. Die stark vom Normalen abweichende Pollenform ließ vermuten, daß eine Erhöhung der Valenzstufe vorlag. Durch vergleichende Untersuchungen mit den Ausgangsformen und durch Chromosomenzählungen wurden sieben Mutantenstämme als Tetraploide bestätigt. Wuchshöhenmessungen und Auszählungen der Ansatzverhältnisse gestatten Schlußfolgerungen über die zu erwartende Leistungsfähigkeit der Tetraploiden.

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Selection of tetraploid forms of Lupinus angustifolius after X-radiation and results of comparative studies with the diploid parental strains

Summary After X-radiation some mutants with broader leaves were selected from different X₂-generations of Lupinus angustifolius. The pollen grains deviated much from their normal form and it was supposed that they were tetraploids. Comparative studies with the parental forms and counting of chromosomes proved seven mutant strains to be tetraploid. Measurements of plant height and counts of seed setting permit drawing a conclusion on the yield capacity of the tetraploids.

     

Die Photosynthese von Meerespflanzen in ihrer Beziehung Zum Salzgehalt

Zusammenfassung Werden Blattstücke vonPosidonia oceanica oder Thallusteile vonUlva lactuca unter sonst optimalen Assimilationsbedingungen aus Meerwasser in Süßwasser, überführt, so sinkt hier die apparente Assimilation beiPosidonia bis zu Null oder gar zu negativen Werten ab, beiUlva bis zu etwa 23% der Leistung im Meerwasser. Bei Rückführung in Meerwasser wird die Assimilationsrate ebenso schnell wieder erhöht, die Ausgangswerte werden allerdings innerhalb der Versuchszeit — einige 20-min-Perioden — meist nicht in voller Höhe wieder erreicht.Folgt der Wechsel zwischen Meer- und Süßwasser im Wechsel von 20 min, so reagieren die Versuchspflanzen mit der gleichen Präzision wie auf das Aus- und Einschalten einer Lichtquelle.Wird anPosidonia die photosynthetische Rate in verschiedenen Stufen verdünnten Meerwassers (Verdünnung nicht mit dest. Wasser, sondern mit Leitungswasser von bekanntem Bicarbonatgehalt und p_H) bestimmt und in Prozenten der Leistung in Meerwasser ausgedrückt, so ergibt sich die in Abb. 3 dargestellte Kurve. Innerhalb der Versuchszeit scheint jeder Meerwasserkonzentration ein bestimmter Photosynthesewert zugeordnet zu sein, ein Verhalten, das auf das Vorhandensein von Gleichgewichtszuständen hindeutet, die zwischen dem photosynthetischen System und dem Zustand des Außenmediums bestehen.Infolge der überaus raschen Reaktion der Pflanzen auf Änderungen im Außenmedium wird als Arbeitshypothese die Vermutung ausgesprochen, daß an der Änderung der photosynthetischen Rate bei Übertragung in und Rückführung aus Süßwasser in Meerwasser nicht Zustandsveränderungen imZellinneren, sondern Zustände der Membranen verantwortlich sind, welche das Ausmaß der CO₂-Permeation bestimmen und damit einen begrenzenden Faktor der Photosynthese darstellen.Mit 4 Textabbildungen     

Das Wachstum infiltrierter Keimlinge

Zusammenfassung Werden Keimlinge vonHelianthus annuus undVicia faba mittels einer Wasserstrahlpumpe mit Wasser infiltriert, so führt dies sofort in allen Organen der Pflanze zu einer sehr starken und mitunter völligen Hemmung des Wachstums. Wirkt der Unterdruck in Luft ein, so daß es hernach zu keiner Wasserfüllung der Interzellularen kommt, so unterbleibt jede Wachstumshemmung.Die Frage nach der Kausalbeziehung zwischen Infiltration und Wachstumshemmung konnte nicht geklärt werden, da die nächstiliegende Annahme, Infiltration führe zu einer Atmungshemmung, durch das Experiment nicht bestätigt werden konnte.Es wird darauf hingewiesen, daß die Zufuhr von Wirk- oder Nährstoffen durch Infiltration eine Methode ist, die in wachsenden Organen nur mit großem Vorbehalt angewendet werden darf, da eine im Wachstum weitgehend gehemmte Pflanze sich in einem anomalen Zustand befindet.Mit 8 Textabbildungen.     

Untersuchungen über das osmotische Verhalten der GrünalgeValonia ventricosa

Zusammenfassung 1.Valonia ventricosa ist eine pantropisch verbreitete marine Grünalge (einziger Fundort im Mittelmeer: Insel Ibiza). Die hier dargestellten Untersuchungen wurden an der Ostküste Venezuelas durchgeführt.2. Die osmotischen Werte (kryoskopisch bestimmt) liegen 1 bis 3 atm über dem Wert des Meerwassers.3. Wachstum und Alter der Zellen ändern den osmotischen Wert nicht.4. Aus Meerwasser in destilliertes Wasser überführt, tritt sehr rasche Abnahme des osmotischen Wertes des Zellsaftes ein (innerhalb von 160 Minuten von 26 auf 2 atm).5. Infolge der größeren relativen Oberfläche nimmt der osmotische Wert kleinerer Zellen viel schneller als der größerer.6. In konzentriertem Meerwasser (31 atm) ändern sich die Zellsaftwerte innerhalb von 3 Stunden nicht.7. In stärker konzentriertem Meerwasser sterben die Zellen rasch ab. Ihr Zellsaft wird damit zum Spielball der Außenbedingungen.8.Valonia ventricosa ist somit ein stenohaliner Organismus ohne erkennbare Fähigkeit zur Osmoregulation.9. Zugabe von CaCl₂ zum destillierten Wasser verlangsamt zunächst die Abnahme des osmotischen Wertes.10. Die chemische Untersuchung des Zellsaftes zeigt auch bei dieser Art das Vorherrschen des Kaliums, das gegenüber dem Meerwasser 66fach konzentriert ist.11. Die Zellwand färbt sich mit Methylenblau stark an, ist jedoch für diesen Farbstoff in keiner Richtung permeable. Angefärbte Zellwände scheinen eine geringe Ionenpermeabilität zu besitzen.12. Elektronenoptische Untersuchnugen der Zellwände zeigen, daß diese aus 40 bis 50 Lamellen bestehen, wobei benachbarte Lamellen eine überkreuzte Paralleltextur (Fischgrätenmuster) besitzen. Da die Zellen — im Gegensatz zu anderenValonia-Arten — nicht in destilliertem Wasser platzen, muß die Zellwand entweder hohe Drucke auszuhalten vermögen oder eine relativ geringe Wasserpermeabilität besitzen.

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Investigations on the osmotic behaviour of the green algaValonia ventricosa

Among the marine Chlorophyta,Valonia ventricosa represents a pantropic species; one of its few extratropical localities is the island Ibiza in the Mediterranean. Our physiological investigations were carried out during several months between 1960 and 1963 in the coastal waters of Venezuela (Mochima Bay near Cumana). The following results were obtained: 1. The osmotic values (measured with the kryoskopic method) are 1 to 3 atm higher than those of the seawater (salinity 36–37 ). 2. Neither size nor age of the cells influence the osmotic characteristics of the cell sap. 3. A transfer from marine to distilled water causes a rapid decrease of the osmotic values (within 160 minutes from 26 to 2 atm). Due to the bigger relative surface, this decrease is more rapid in small cells than in the bigger ones. 4. In concentrated seawater with 31 atm the osmotic values of the cells did not change within 3 hours. 5. In more concentrated or in diluted seawater, the cells are irreversibly damaged within a short time.Valonia ventricosa can therefore be considered as a stenohaline aliga without any recognizable osmoregulation. 6. Addition of CaCl₂ delays the decrease of the osmotic value. 7. Chemical analysis of the cell sap demonstrates the well-known prevalence of potassium, which is 66 times more concentrated than in seawater. 8. The cell wall can be easily stained with methyleneblue and in this case the permeability for anorganic ions is probably reduced. 9. Photographs taken with the electron-microscope show in cross section the multilammellate nature of the cell wall and the change of the fibrillar-direction from one lamella to the other, giving the picture of a cross-fibrillar structure. Since the cells — in contradiction to those of otherValonia species — do not burst in distilled water, it must be assumed that the cell wall structure is able to resist high pressures (about 26 atm) or is characterized by a relatively low water-permeability.

     

Properties and cellular localization of a luciferin binding protein in the bioluminescence reaction of Gonyaulax polyedra

A luciferin binding protein LBP involved in the bioluminescence reaction of Gonyaulax polyedra was purified and used for antibody production. Luciferin bound to LBP is fluorescent and can be used as a marker in living cells, allowing the localization of LBP in cortical organelles to be visualized. In cell sections, the same peripheral localization was observed using anti-LBP and immunofluorescence microscopy. The amount of LBP is ten-fold greater from cells from in night phase compared to those from in day phase, as determined both by immunoblots of cell extracts, and in vivo fluorescence. These changes correlate with the circadian changes in bioluminescence of living cells.     

Interspecific incompatibility due to stylar barriers in tuber-bearing and closely related non-tuber-bearing Solanums

Summary The phenomenon of interspecific incompatibility between various wild tuber-bearing and closely related non-tuber-bearing Solanum species was studied. One area of investigation included an examination of possible protein interactions in the incompatibility reaction using SDS electrophoresis. Pollen tube inhibition and morphology were examined in conjunction with biochemical analysis. Two sets of crosses were examined: interspecific tuber-bearing species crosses and interspecific tuber-bearing × non-tuber-bearing species crosses. These crosses had consistent pollen tube inhibition in the upper one-third of the style. The upper third of the styles of incompatibly pollinated, compatibly pollinated, and unpollinated styles was studied under fluorescence microscopy to observe pollen tube growth and morphology. Interspecific tuber-bearing × non-tuber-bearing species crosses demonstrated consistent pollen tube inhibition just below the stigma with frequent pollen tube swelling and bursting and extensive callose deposition along the pollen tube wall. Interspecific tuber-bearing species crosses had pollen tube inhibition further down the style with pollen tube tip tapering and extensive callose deposition. Stylar proteins of the lower two-thirds of the styles were analyzed with SDS electrophoresis. No unique protein differences were found to be specifically associated with the interspecific incompatibility reaction in this portion of the style.Cooperative investigation of the U.S. Department of Agriculture, Agricultural Research Service, and the Wisconsin Experiment Station. Supported in part by the USDA, Cooperative States Research Service Competitive grant no. 83-CRCR-1-1253     

Failure of (SJL/J x PL/J)F1 hybrid mice to develop immunologic tolerance to V beta 17a+ T cells results in F1 anti-SJL mixed lymphocyte reaction.

It has been reported previously that spleen cells from (SJL x PL) F1 hybrid mice are not tolerant to SJL parental cells as assessed by a one-way MLR. The possibility that the F1 anti-SJL reaction was due to the effect of lymphokines produced by the irradiated SJL T cells in response to I-Eu expressed on the F1 hybrid cells was eliminated since inclusion of anti-I-E mAb was without effect. Cell separations showed the responder cells to be plastic and nylon wool nonadherent Ia- T cells. Separation of the SJL spleen cells showed that the stimulator cells were nonadherent, passed through a nylon wool column, and were Ia-. the F1-anti-SJL MLR was blocked 70 to 90% by inclusion of mAb KJ23a in the culture medium that indicated that the stimulatory cell population was V beta 17a+ T cells. This was confirmed by the use of V beta 17a+ and V beta 17a-T cell clones as stimulators. To determine whether failure to develop tolerance to this T cell subset in F1 hybrid mice might be responsible for the F1-anti-parent MLR, (SJL x PL)F1 mice were treated at birth and 48 h thereafter with anti-I-E mAb for 7 wk. Spleen cells from antibody-treated F1 mice were nonreactive with irradiated SJL parental cells in contrast to spleen cells from control mice which indicated that V beta 17a+ T cells were eliminated by negative selection before the development of tolerance.     

Intra- and interspecies analyses of the carcinoembryonic antigen (CEA) gene family reveal independent evolution in primates and rodents

Summary Various rodent and primate DNAs exhibit a stronger intra- than interspecies cross-hybridization with probes derived from the N-terminal domain exons of human and rat carcinoembryonic antigen (CEA)-like genes. Southern analyses also reveal that the human and rat CEA gene families are of similar complexity. We counted at least 10 different genes per human haploid genome. In the rat, approximately seven to nine different N-terminal domain exons that presumably represent different genes appear to be present. We were able to assign the corresponding genomic restriction endonuclease fragments to already isolated CEA gene family members of both human and rat. Highly similar subgroups, as found within the human CEA gene family, seem to be absent from the rat genome. Hybridization with an intron probe from the human nonspecific cross-reacting antigen (NCA) gene and analysis of DNA sequence data indicate the conservation of noncoding regions among CEA-like genes within primates, implicating that whole gene units may have been duplicated. With the help of a computer program and by calculating the rate of synonymous substitutions, evolutionary trees have been derived. From this, we propose that an independent parallel evolution, leading to different CEA gene families, must have taken place in, at least, the primate and rodent orders.