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.     

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.     

EDU and ozone protection: Foliar glycerolipids and steryl lipids in snapbean exposed to O3

Effects of the antiozonant EDU, N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N'-phenylurea, on the content and composition of foliar lipids in snapbean ( Phaseolus vulgaris L. cv. Bush Blue Lake 290) before and after a single, acute ozone (O₃) exposure were assessed. Pretreatment with EDU conferred protection against O₃-induced necrosis and losses of glycerolipids and chlorophyll. Systemic treatment of snapbean plants with EDU did not significantly alter membrane lipids in the first trifoliate leaf. Leaves of untreated controls had lost ca 50% of both galacto- (GL) and phospholipids (PL) by the end of a 3 h exposure to 0.4 μl l⁻¹ O₃. A decline in the ratio of mono- to di-galactosyldiacylglycerol (MGDG/DGDG) was associated with the loss of GL, and a decline in the ratio of linoleic to linolenic acid (18:2/18:3) was associated with the loss of PL in untreated controls. EDU-treated plants showed no significant loss of foliar GL and PL. The MGDG/DGDG ratio declined only slightly, and the 18:2/18:3 ratio in PL increased during O₃ exposure of EDU-treated seedlings. The level of total membrane sterols, including free sterols (FS), acylated steryl glycosides (ASG) and steryl glycosides (SG), did not change during O₃ exposure of either treated or untreated plants. However, in the controls the proportions of ASG and SG increased at the expense of FS, and the ratio of stigmasterol/sitolsterol increased in FS and SG. In EDU-treated plants, a relatively small increase in SG was offset by a decrease in FS, and there was no change in the stigmasterol/sitosterol ratio in ASG, SG or FS. The results indicate that EDU may confer tolerance to O₃ through induction of enzyme systems involved in the elimination of activated oxygen species and free radicals.     

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.