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.     

Fluorescent antibody method Conjugation Of Fluoresceinisothiocyanate With Immune ?-Globulin

ИЗyчaл0C044A; Bлияниe НeкOTOpыX ФaкTopoB (_pH, OTHoшeниe КpacиTeля к бeлкy, КOнпeнTpaция КOMпoнeнTOB, пpиcyTcTBиe opraничecкиX pacTBopиTeлeй B peaгиpyюшeй cмecи, TeMпepaTypa, Bpeмя peaкции) Ha эффeкTиBн0CTъ кOнъюгaции γ-глoбyлинa c флyopecцeинизOTиoциaнaTOM. КoличecTBO КpacиTeля, CBязaннoгo c γ-глoбyлинOM, знaч иTeлънO пoBышaлocъ, кoгдa пoBышaлcя B пpeдeлax 7,0–10,0_pH peaгиpyющeй cмeCи, Toгдa кaк ocTaлъныe фaкTopы нe oкaзыBaли нa мeчeныe бeлки B иccлeдyeмыx пpeдeлax cyщecTBeннoгo Bлияния. БылO ycTaнOBлeнo, чTO cпeцифичecкaя флyopecцeнпия cBязaннoгo кpacиTeля зaмeTнo пoнижaлacъ c пOBышeниeм мeчeннOcTи бeлкOB, a пoэTOмy нaибoлee цeлecooбpaзным пpeдcTaBляeTCя иcпoлъзOBaниe для мeTOдa флyopecцeнTныx aнTиTeл-кOнъюгaTOB c BecoBым COOTнOшeниeм бeлкOB и кpacиTeля, paBным пpиблизиTeльнO 60.     

Membrane Ig-cytoskeletal interactions. III. Receptor cross-linking results in the formation of extensive filamentous arrays of vimentin

The proposed function of intermediate filaments is to provide a cell type-specific structural framework that maintains cell shape and organelle distribution and mediates signal transduction through its connections with the plasma membrane and the nucleus. Vimentin is the intermediate filament protein expressed in B lymphocytes. Immunocytochemical analysis of the high salt-stable cytoskeletons from B cells stimulated with anti-Ig revealed an increased accumulation of vimentin in the cytoskeleton compared to nontreated controls. This increased accumulation of vimentin in the cytoskeleton was manifested by the organization of vimentin into extensive filamentous arrays (EFA) as viewed in the fluorescent microscope. In contrast to the effects of anti-Ig, activation of B cells with LPS did not induce the organization of vimentin into EFA. This suggested that signals unique to anti-Ig directed EFA formation. Immunocytochemical results were verified by biochemical analysis showing that vimentin was more abundant in isolated cytoskeletons from anti-Ig activated B cells, than cytoskeletons isolated from LPS-activated B cells. These observations established a relationship between increased content of vimentin in the cytoskeleton and the formation of EFA. By testing a wide variety of activating agents, we were able to correlate increased vimentin expression in the cytoskeleton to activating agents that cross-link membrane Ig. It appeared that treatment of B cells with LPS prohibited the induction of EFA by anti-Ig because cotreatment with both anti-Ig and LPS resulted in decreased vimentin accumulation in the cytoskeleton to a level less than that in resting cells. The significance of these results with regard to B cell biology is discussed.     

Physiological Events in Clostridium acetobutylicum during the Shift from Acidogenesis to Solventogenesis in Continuous Culture and Presentation of a Model for Shift Induction

The pH of continuous cultures of Clostridium acetobutylicum growing at pH 5.6 was allowed to decrease to 4.3 after acid production and thereby to shift the cultures from acetate and butyrate to acetone and butanol formation. Several parameters were determined during the shift. An increase in the intracellular acid concentration to 440 mM was recorded. An excess of undissociated butyric acid but not of acetic acid just before the shift to solventogenesis was followed by a decline in acid production and subsequently by the uptake of acids. The intracellular ATP concentration reached a minimum before the onset of solventogenesis; this presumably reflects the ATP-consuming proton extrusion connected with the increase in the ΔpH from 0.7 to 1.4 units. The pool of NADH plus NADPH exhibited a drastic increase until solventogenesis was induced. The changes in the ATP and ADP and NADH plus NADPH pools during these pH shift experiments were the beginning of a stable metabolic oscillation which could also be recorded as an oscillation of the culture redox potential under steady-state solventogenic conditions. Similar changes were observed when the shift was induced by the addition of butyrate and acetate (50 mM each) to the continuous culture. However, when methyl viologen was added, important differences were found: ATP levels did not reach a minimum, acetoacetate decarboxylase activity could not be measured, and butanol but not acetone was produced. A model for the shift is proposed; it assumes the generation of two signals, one by the changed ATP and ADP levels and the other by the increased NAD(P)H level.     

Compensation of respiratory alkalosis induced after acclimation to simulated altitude

Conscious intact rats previously acclimated for 3 wk to barometric pressure of 370-380 Torr (3WHx) were made alkalotic for 3 h by a decrease in inspired O2 fraction from 0.10 to 0.075 at ambient barometric pressure (730-740 Torr). Controls were normoxic littermates (Nx) in which inspired O2 fraction was lowered from approximately 0.21 to 0.10 for 3 h. Arterial PCO2 decreased progressively and similarly in both groups (65-70% of control at 15 min). Initially, arterial pH increased less in 3WHx (0.09 +/- 0.004 vs. 0.15 +/- 0.008). As hypocapnia continued, delta[HCO3-]/delta pH (mmol.l-1.pH) became more negative in Nx, from -15.2 +/- 2.5 at 15 min to -37.0 +/- 2.9 at 3 h, indicating nonrespiratory compensation of alkalosis. In 3WHx, delta[HCO3-]/delta pH did not change during alkalosis. Cumulative renal excretion of base (mueq/100 g) during alkalosis increased by 73.2 +/- 11.1 in Nx and 25.4 +/- 7.3 in 3WHx. This difference was mainly due to a larger increase in HCO3- excretion in Nx. The data suggest that the smaller compensation of hypocapnic alkalosis in 3WHx is partly due to the smaller increase in renal base excretion. Because base availability limits renal base excretion, the smaller renal response of 3WHx may be secondary to the low plasma HCO3- concentration that accompanies altitude acclimation.     

Initial steps of sophoroselipid biosynthesis by Candida bombicola ATCC 22214 grown on glucose

 Candida bombicola ATCC 22214 produces the glycolipid sophoroselipid when cultivated on a medium with glucose as the sole carbon source. Under phosphate-limiting conditions the product yield rises from 0.033 to 0.143 and the specific product formation rate rises from 0.004 h^-1 to 0.007 h^-1. Enhanced sophoroselipid synthesis is initiated by the decline of the specific activities of NAD- and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42) to 2% and 0% of the initial activities respectively. Constantly high specific activity of citrate synthase (EC 4.1.3.7) causes an accumulation of isocitrate and citrate in the mitochondria. Both acids are transported into the cytosol where citrate is cleaved by ATP: citrate lyase (EC 4.1.3.8) giving rise to acetyl-CoA, the precursor of fatty acid synthesis. The ATP: citrate lyase is unaffected by different energy charges; the apparent K _m values for coenzyme A, ATP and citrate are 23 μM, 250 μM and 256 μM respectively. NADPH for fatty acid synthesis might be generated by further metabolism of oxaloacetate, the other product of the citrate-cleaving reaction, by oxidation of the isocitrate by the cytosolic NADP-dependent isocitrate dehydrogenase or via the hexose monophosphate shunt. A possible explanation for sophoroselipid formation during exponential growth is given. Received: 7 November 1995/Received revision: 19 March 1996/Accepted: 25 March 1996     

A patient with an interstitial deletion in Xp22.3 locates the gene for X-linked recessive chondrodysplasia punctata to within a one megabase interval

A male patient carrying an interstitial deletion in Xp22.3 and affected by Kallmann syndrome, X-linked ichthyosis and mental retardation, but without chondrodysplasia punctata or short stature, was investigated with molecular probes from the distal Xp22.3 region. By means of a novel probe, M115, from the relevant region, the distal deletion breakpoint was shown to be between 3.18 and 3.57 Mb from Xptel. As the patient is not affected by X-linked recessive chondrodysplasia punctata, the gene for this disease can therefore be located to within an interval of less than one megabase proximal to the pseudoautosomal boundary. If the chondrodysplasia punctata gene is associated with a CpG island, this leaves only two islands at 2760 and 3180 kb from the Xp telomere as the most promising candidate sites for this gene.     

20-OH-ecdysone swells nuclear volume by alkalinization in salivary glands of Drosophila melanogaster

Ecdysteroids play an important role in the larval moulting process of insects. Ecdysone-induced stimulation causes specific puffs in polytene chromosomes of salivary gland cells resulting in nuclear swelling. During this process, changes of intracellular ion composition are thought to act as an early regulatory mechanism of gene activation. By use of video-imaging analysis and electrophysiological techniques, we examined ecdysone-induced nuclear swelling in Drosophila salivary glands in situ and its dependence on pH and calcium. Isolated glands of the third larval stage were superfused with a solution mimicking the haemolymph. Addition of 5×10^–6 mol/l 20-OH-ecdysone led, after a lag period of 50 min, to a sustained Ca²⁺-dependent increase of nuclear volume by 23.0±2.3%. Amiloride, a blocker of plasma membrane Na⁺/H⁺ exchange, prevented 20-OH-ecdysone-induced nuclear swelling. Decreasing pH in the superfusate from 7.15 to 6.8 led to nuclear shrinkage by 16.9±3.9%. Measurments of pH in salivary gland cells with ion-sensitive microelectrodes disclosed an alkalinization of 0.23±0.05 pH units after stimulation with 20-OH-ecdysone. We postulate that 20-OH-ecdysone activates the amilorde-sensitive plasma membrane Na⁺/H⁺ exchanger. This leads to intracellular alkalinization and concomitant decondensation of the nuclear chromatin visible as nuclear swelling. Thus, cell alkalinization could be a potentially important stimulatory mechanism in mediating ecdysteroid-induced activation of the cell nucleus.