Subcellular localization, oligomerization, and ATP-binding of Caenorhabditis elegans CED-4.

Caspase family cell death proteases are activated during apoptosis through the oligomerization of caspase-binding "adapter" proteins. In the nematode Caenorhabditis elegans one adapter protein, CED-4, exists. Here we report an analysis of CED-4 protein expressed in insect Sf9 cells by infection with recombinant baculovirus. During expression, CED-4 assumed a perinuclear spherical or reticular localization where it was partly resistant to extraction with nonionic detergents. Both purified FLAG-CED-4 and GST-FLAG-CED-4 proteins were present in solution as large complexes. FLAG-CED-4 complexes were estimated by gel filtration to have a molecular weight of approximately 500 kDa to >1.2 MDa, while GST-FLAG-CED-4 complexes appeared somewhat smaller. Unlike its mammalian homologue Apaf-1, CED-4 exhibited a marked preference for ATP over dATP in filter binding studies and in competition experiments. ATP hydrolysis was required neither for complex stability nor for binding of CED-3. These features are likely to be relevant for CED-4's function as a caspase adapter.     

Über eine Bertalanffy-analoge Fluorochromierung mit 3-Dimethylamino-6-methoxyacridin

Zusammenfassung Drei neue Acridinfarbstoffe, 3-Dimethylamino-6-methoxyacridin 1, 3-Amino-6-methoxyacridin 2 und 3-Amino-7-methoxyacridin 3 wurden synthetisiert und ihre Eigenschaften als Fluorochrome für LM- und HeLa-Zellen untersucht. Die Substanzen sind Basen (pK_A: 1 8,76; 2 8,01; 3 7,65), die in neutralem und in saurem wäßrigem Medium Kationen bilden durch Protonierung des Aza-Stickstoffatoms vom Heterocyclus. Die Fluorochrome färben fixierte LM- und HeLa-Zellen bei pH=6. Die Fluoreszenz zeigt Metachromasie in Analogie zur Färbung nach Bertalanffy mit Acridinorange AO. Die Färbung hat höhere Photostabilität als bei AO. Die besten Eigenschaften als Fluorochrom hat 1, das im Detail untersucht wurde.Die Vorschrift der Färbung wurde optimiert. Kerne gefärbter Zellen fluoreszieren gelb-grün, Cytoplasma und Nucleoli orange bis braun-rot. Enzymatische Abbauversuche zeigen, daß der Farbstoff in den Kernen an DNA, im Cytoplasma bzw. den Nucleoli an RNA gebunden ist.Die Absorptions- und Emissionsspektren der fluorochromierten Zellen wurden mikrospektralphotometrisch untersucht. Die Absorptionsspektren von Kern und Cytoplasma sind sehr ähnlich. Das Maximum der längstwelligen Absorptionsbande beobachtet man bei beiden bei 21400 cm^–1 (467 nm) mit einer Schulter bei ca. 20100 cm^–1 (498 nm).Die Fluoreszenzspektren von Kern und Cytoplasma metachromatisch gefärbter Zellen sind verschieden. Das Maximum der Emission des Cytoplasmas bzw. der Nucleoli, 16200 cm^–1 (617 nm), ist langwellig gegenüber dem Fluoreszenzmaximum des Kerns, 18200 cm^–1 (549 nm), verschoben. Diese Verschiebung verursacht den metachromatischen Fluoreszenzeffekt.In Ergänzung wurde die Konzentrationsabhängigkeit der Absorptions- und Fluoreszenzspektren des Kations 1 in wäßriger Lösung, pH=6, im Konzentrationsintervall 6×10^–6–6×10^–4 M untersucht. Lage und Form der Banden hängen nur weinig von der Konzentration ab: Mittelwert des längstwelligen Absorptionsmaximums ca. 21500 cm^–1 (465 nm), der langwelligen Schulter ca. 20300 cm^–1 (493 nm) und des Fluoreszenzmaximums ca. 18300 cm^–1 (547 nm). Mit steigender Konzentration nimmt der molare Extinktionskoeffizient ab. Diese Abnahme und isosbestische Punkte in den Absorptionsspektren weisen auf die Bildung von Dimeren mit steigender Konzentration hin.Die Absorptionsspektren der metachromatisch gefärbten Zellen und des Farbstoffs in Lösung sind sehr ähnlich. Ein sorgfältiger Vergleich der Spektren macht es wahrscheinlich, daß die DNA-gebundenen Farbstoffkationen im Kern monomer, die RNA-gebundenen im Cytoplasma bzw. den Nucleoli dimer sind. Ähnliches wurde bei AO gefunden. Die spektralen Effekte sind jedoch bei 1 viel kleiner.Die Fluoreszenz der RNA-gebundenen Farbstoffkationen des Cytoplasmas ist stark langwellig gegenüber der Kernfluoreszenz verschoben. Absorptions- und Emissionsspektren lassen sich unter der Annahme deuten, daß der RNA-gebundene Farbstoff des metachromatisch gefärbten Cytoplasmas bzw. der Nucleoli im Grundzustand Dimere D, im ersten angeregten Zustand Excimere E bildet. Die Excimeren sind gegenüber den Dimeren stabilisiert; der Bindungsabstand R zwischen den Molekülen ist verkürzt. Das Potentialschema V(R) für Grund- und ersten angeregten Zustand wird im Detail diskutiert. Danach kann D nur in Absorption, E nur in Emission beobachtet werden. Unsere experimentellen Befunde stehen mit einer Excimerenhypothese in Übereinstimmung.Absorptions-, Fluoreszenz- und Absorptionspolarisationsspektren von 1 (Kation und freie Base) wurden in glasartig erstarrtem Ethanol bei 77 K gemessen. Die Spektren wurden mit quantenmechanischen Modellrechnungen in SCF-CI-PPP-Approximation verglichen. Danach sind die beiden Absorptionsbanden des freien Kations in wäßriger Lösung bei ca. 20300 und 21500 cm^–1 und des gebundenen Kations bei 20100 und 21400 cm^–1 dem 0-0- und 0-1-Übergang der längstwelligen Elektronenbande von 1 zuzuordnen.

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Bertalanffy-analogical fluorescence staining with 3-dimethylamine-6-methoxyacridine

Summary Three new acridine dyes, 3-dimethylamino-6-methyoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pK_A: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH=6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail.Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli.The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm^–1 (467 nm) with a shoulder at ca 20100 cm^–1 (498 nm).The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm^–1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm^–1 (549 nm). This shift causes the metachromatic fluorescence effect.In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH=6, in the concentration range 6×10^–6–6×10^–4 M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm^–1 (465 nm), shoulder at ca 20300 cm^–1 (493 nm), fluorescence maximum ca 18300 cm^–1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration.The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar. A careful comparison of the spectra make it probable that the dye cations bound to DNA in the nucleus are monomer and to RNA in the cytoplasm or nucleoli are dimer. The same has been observed with AO. But our spectral changes are smaller.The fluorescence of the dye cations bound to RNA in the cytoplasm is strongly red-shifted compared to the fluorescence of the nucleus. Absorption and emission spectra of metachromatically stained cytoplasm can be explained on the assumption that the RNA bound dye forms dimers D in the ground state and excimers E in the first excited state. Compared with D the excimers are stabilized and the bond distance R between the molecules is shortened. The potential energy curves V(R) of the ground state and the first excited state are discussed in detail. Accordingly D can only be observed in absorption and E in fluorescence. Our experimental results agree with the excimer hypothesis.Absorption, fluorescence and absorption polarisation spectra of 1 (cation and free base) have been measured in rigid ethanol at 77 K. The spectra are compared with quantum mechanical calculations in SCF-CI-PPP-approximation. According to that the two absorption bands of the free cation in aqueous solution at ca 20300 and 21500 cm^–1 and of the bound cations at ca 20100 and 21400 cm^–1 are classified as 0-0- and 0-1-transitions of the long wave length absorption of 1.

     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)