Quantitative Bestimmung von Sulfhydrylgruppen mit “Mercurochrom”

Zusammenfassung Dibrommerkurifluoreszein (DBMF) reagiert stöchiometrisch und quantitativ mit der SH-Gruppe von Cystein, Glutathion und Thioglycolsäure. Polarographische und spektrometrische Titrationen ergeben, daß die Wellenlänge des ersten Absorptionsmaximums von DBMF (507 nm) bei den gebildeten Merkaptiden unverändert bleibt, während der molare Extinktionskoeffizient um rund 20% ansteigt. Serumalbumin, Ovalbumin, -Laktoglobulin und Glycerinaldehydphosphat-Dehydrogenase bilden nach Inkubation mit DBMF Addukte aus denen die reinen DBMF-Protein-Merkaptidkomplexe säulenchromatographisch isoliert wurden. Sie zeigen im Absorptionsspektrum eine bathochrome Verschiebung der Farbstoffbande (520 nm) mit um ca. 50% erniedrigtem Extinktionskoeffizienten (₅₂₀=32000–33850). Die mit diesem Wert aus den Maximumsextinktionen berechneten SH-Gehalte entsprechen den auf Grund von Literaturangaben zu erwartenden Daten. Eine selektive Reaktion, z.B. mit besonders zugänglichen oder hoch-reaktiven SH-Gruppen, konnte mit DBMF nicht festgestellt werden. Native tierische Tumorzellen zeigen nach 30 min Inkubation mit DBMF und Auswaschen mit isotonem Phosphat-Puffer in Kern und Plasma als Hauptbande das rotverschobene Maximum, an dem jedoch auch das unverschobene Maximum als mehr oder minder deutliche Inflexion beteiligt ist. Probeweise, mit ₅₂₀ ausgeführte Berechnungen des Protein-SH-Gehaltes zeigten 1,7–2,1·10^–14 Mole/Zelle an. Dieses vorläufige Ergebnis liegt zwischen den in früheren eingehenden Untersuchungen mit DDD-Echtblau mikrospektrometrisch (1,1–1,55·10^–14) und mit DTNB makroskopisch gefundenen SH-Gehalten von EATZ (3,1·10^–14). Ob und wie stark bei den mit DBMF gefundenen Werten auch unspezifische Adsorption beteiligt ist, läßt sich gegenwärtig noch nicht sicher beurteilen. Eine Reaktion mit Nukleinsäure konnte auf Grund von Modellversuchen jedoch mit Sicherheit ausgeschlossen werden.

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Quantitative determination of sulfhydryl groups with mercurochrome

Summary Dibrommercuryfluoresceine (DBMF) reacts stoichiometrically and quantitatively with the thiol group of cysteine, glutathione and thioglycolic acid respectively, at pH 7.0. Polarographical and spectrometrical titrations clearly show that in the spectra of the investigated mercaptides the wave length of the first absorption maximum of DBMF (507 nm) remains unchanged but the molar extinction coefficient increases by approximately 20%. Serum albumin, ovalbumin, -lactoglobulin and glyceraldehydephosphatedi-hydrogenase after incubation with DBMF, form adducts with the dye from which the pure mercaptide complexes were separated by means of column chromatography. These complexes show a bathochromic shift (520 nm) of the dye band which is decreased now by 50%. The molar extinction coefficient ₅₂₀ has been determined from 32,000 to 33,850. On the basis of these values SH-contents of the four proteins were obtained which are in good accordance with data previously published in the literature. No selective reaction, f.i. with more accessible or/and reactive SH-groups was observed. After 30 min incubation with DBMF and washing with isotonic phosphate buffer, native animal tumor cells show in the main absorption band the bathochromically shifted dye maximum. A first temptative estimation of the protein SH-groups yielded 1.7–2.1×10^–14 mole SH/single cell. This result lies between the SH-content determined microspectrometrically on cells stained with DDD-Fast Blue B (1.1–1.55×10^–14) and macroscopically on cell homogenates with DTNB (3.1×10^–14). Up to now, no certain information can be given whether or to what extent unspecific absorption effects possibly might be involved in the data obtained with DBMF treated cells, but interaction with nucleic acids can be excluded with certainty on the basis of relevant model experiments.

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Diese Arbeit wurde mit Unterstützung des Fonds zur Förderung der wissenschaftlichen Forschung, Wien, durchgeführt

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Herrn Prof. Dr. G. Zigeuner zum 60. Geburtstag gewidmet.     

The viscoelasticity of entangled actin networks: the influence of defects and modulation by talin and vinculin

Rheological measurements of the frequency-dependent complex elastic module G^*() of entangled F-actin solutions in the frequency range 10^–5 – 1 Hz were carried out in three dynamic regimes: 1.) A terminal relaxation from gel-like to liquid-like behaviour measured at frequencies < _d ^–1 2.) a rubber-type plateau and 3.) a regime determined by chain conformational transitions at frequencies > _i ^–1. A major point of interest was to clarify whether rheological, high precision measurements can yield quantitative information about the influence of talin and vinculin on the structure, chain dynamics, elasticity and viscoelasticity of actin filaments with time. We show that in the regime reflecting internal chain dynamics (10^–2 to 1 s time domain), F-actin behaves as a random coil of the Rouse type. This contrasts with dynamic light scattering and correlation spectroscopic studies of actin filament flickering, which indicate that filaments behave as semiflexible rods. The internal chain dynamics, which are determined by thermically excited bending undulations, exhibit a persistence length of 0.3–1 m Evidence is provided that this discrepancy is due to a cross-over of semiflexible rod behaviour at excitation wavelengths () below approximately 1 gm to random-coil behaviour at 1 µ (expected at a frequency 1 Hz). The random coil behaviour is largely determined by defects in actin filaments leading to sharp bends of the chain which act as semiflexible hinges. Talin produces drastic effects on the time course of viscoelasticity during actin polymerization. It promotes the rapid formation of short filament fragments ( 1 gmm, within time scales of min) which anneal slowly into long filaments (within several hours), most probably by fusion. The viscoelasticity depends on the coexistence of short and very long filaments indicated by the elongation of the rubber plateau. The most dramatic effect is a reduction of the ratio of the terminal ('Ed) to the Rouse relaxation time of _i by more than one order of magnitude (_d/_i = 100 compared to ratio _d/_i = 2000 for pure actin). From this it is concluded that talin causes a remarkable decrease in the effective segment length of the macromolecule and, thus induces an increase in chain stiffness. Vinculin on the other hand shows no such effect. Correspondence to: E. Sackmann     

Die Laut?u?erungen des Blutspechts,Dendrocopos syriacus

Zusammenfassung Blutspechtlaute sind den Lauten der Buntspechte ähnlich, aber doch deutlich anders.Paarkontakt: Auffallend ist der Unterschied zwischen - und -Wirbeln: -Wirbel 27,5 Schläge (37 Wirbel), -Wirbel 18,0 Schläge (6 Wirbel). Die Wirbel, vor allem die -Wirbel, sind deutlich länger als die Trommelwirbel vom Buntspecht. Zwischen dem 1. und 2. Schlag ist ein großer Abstand. Die Schlagfolge nimmt von Wirbelbeginn bis zu Wirbelende zu.Gurrende Laute sind bei der Ablösung und Kopula zu hören. Sie klingen ähnlich wie die entsprechenden Buntspechtlaute. Das gegenseitige Verstehen dieser Laute dürfte — falls es Bastard-Paare geben sollte — sehr wichtig sein (Paar-Synchronisation).Feindsituation: Die aggressiven Kreck-Laute tönen deutlich anders als die vom Buntspecht. Sie scheinen dem Kreck des Weißrückenspechts zu ähneln. Das gellende Schirken bei großer Gefahr besteht aus einzelnen Elementen, deren Lautgestalt einem umgekehrten Kix ähnlich ist.Unspezifische Erregung: Die Bedeutung des Kixens ist nicht festgelegt. Kixen kann man das ganze Jahr hindurch hören. Blutspechtkixen klingt weicher als das Kixen des Buntspechts. Die Tonhöhe hängt von der Erregung des Vogels ab.Güg-Reihen hörte ich von erregten Vögeln während der Ausflugphase der Jungen oder bei Störungen während des Brütens. Buntspechte haben keine entsprechende Lautreihe.

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Summary The vocalisation of Syrian Woodpecker is similar to that of the Great Spotted Woodpecker. But nevertheless the differences are striking.Pair-contact: In drumming there is an apparent difference between and : -drumming 27,5 strokes (37 drummings), -drumming 18,0 strokes (6 drummings). The drumming especially that of the differs significant from that ofmajor. Between the first and second stroke there is a wide distance. The frequence of strokes gradually increases. By changing at the nest (in relief ritual) and at copula gurr sounds are uttered (contact notes,Lawrence). They are similar to those ofmajor. Reciprocous understanding of this sounds would be very necessary, if there are hybrid-pairs (Pair-synchronisation).Aggressive notes: The aggressive Krek-sounds are apparently unlike those ofmajor. They seem to be similar to those ofD. leucotos. Shrilling sounds of alert consist in elements which in the spectrograms are similar to the figure of a Kix, upside down.Unspecific notes: The meaning of Kixen is not fixed. Kixen is to be heard throughout the year. The level of sound is in dependence to excitement.Series of Güg-notes are uttered by Syrian woodpeckers in great excitement, when the young leave the nest by the young or when the adults are disturbed in incubating.D. major has no corresponding vocalisation.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Heterocellular hereditary persistence of fetal hemoglobin (HPFH). Molecular mechanisms of abnormal γ-gene expression in association with β thalassemia and linkage relationship with the β-globin gene cluster

Summary We report a study of four families of Italian origin in which heterocellular HPFH is inherited linked to thalassemia over two or three generations. The HPFH + thalassemia carriers showed thalassemic blood pictures and elevated HbF and F-cell number without increase in the HbF/F-cell content. Association of this gene complex with a second thalassemia trait gives rise to a mild clinical picture characterized by 9–12 g/dl of mainly HbF in peripheral blood and no transfusion requirement. In two families, independent segregation of the HPFH or -thal trait was observed, and in one case the study of the DNA polymorphisms within the gene cluster indicated that the HPFH mutation lies outside that DNA region. In one family the coexistence of a polymorphic variant of the ^A chain (the ^AT chain) allowed us to demonstrate that the increased chain synthesis caused by the heterocellular HPFH determinant is directed by both chromosomes.     

Adrenergic regulation of chloride secretion across the opercular epithelium: The role of cyclic AMP

Summary Activation of the -adrenergic receptors of the opercular epithelium ofFundulus heteroclitus stimulates Cl^– secretion, while activation of the -adrenergic receptors inhibits Cl^– secretion (Degnan and Zadunaisky, 1979). The possible involvement of adenosine 3, 5-monophosphate (cAMP) in these adrenergic responses was investigated. Isolated opercular epithelia incubated in Ringer, containing 10 mM theophylline, had cAMP levels ranging between 5.3 and 19.3 pmoles·mg protein^–1 (mean=9.5±1.0 pmoles·mg protein^–1). Activation of the -receptors by 10^–5 M isoproterenol increased the mean cAMP level 430% (P<0.001). Blockage of the -receptors with propranolol greatly reduced the increase in cAMP in response to isoproterenol. Activation of the -receptors by 10^–5 M arterenol stimulated the mean cAMP level 270% (P<0.01). However, when the -receptors were blocked with propranolol, arterenol had no effect on the cAMP level. The possible involvement of Ca⁺⁺ in these adrenergic responses was investigated. Neither the stimulatory effect of isoproterenol, nor the inhibitory effect of arterenol on the Cl^– secretion were diminished in the absence of extracellular Ca⁺⁺. The Ca⁺⁺ ionophore, A23187, and the calmodulin inhibitor, trifluoperazine, had no effects on the Cl^– secretion. The Ca⁺⁺-channel blocker, D600, had a significant inhibitory effect (P<0.005). Guanosine 3,5-monophosphate (cGMP) had no effect on the Cl^– secretion.The results indicate that -adrenergic stimulation of Cl^– secretion across the opercular epithelium is accompanied by an elevation in tissue cAMP levels. -adrenergic inhibition of Cl^– secretion does not involve changes in the tissue cAMP. Neither of these responses appear to require Ca⁺⁺.     

Einfluß von Bromuracil auf die Mutationsauslösung und Inaktivierung durch UV und Röntgenstrahlen beim Phagen χ

Zusammenfassung Durch Anreicherung in BUdR- und FUdR-haltigem Medium wurde in die DNS des Serratiaphagen Bromuracil (BU) eingebaut. Im CsCl-Gradienten zeigte die Dichte der BU--Partikel einen Ersatz des Thymins durch BU von 25 bis 30% an. Die Häufigkeit zweier Plaquetypmutanten (c und b) war in der BU--Population signifikant (durchschnittlich 1,4 bzw. 2,4), die des dritten Typs (e) nur insignifikant über die in BU-freien Phagen erhöht. Im Vergleich zu letzterem betrug die absolute Häufigkeit der zusätzlichen c- und b-Mutanten in BU- 2,2 × 10^–4 bzw. 1,1 × 10^–4.Die UV-Inaktivierung von BU- war etwa um den Faktor 1,40 höher als die des normalen . Die Inaktivierung durch Röntgenstrahlen war dagegen nur um das 1,14fache erhöht. Die Häufigkeit der durch Röntgenstrahlen induzierten Plaquemutationen wurde durch den BU-Einbau nicht merklich beeinflußt. Dagegen war die UV-Induktion der Mutationen sehr viel mehr verstärkt als die UV-Inaktivierung. Die Sensibilisierungsfaktoren der drei Mutantentypen waren verschieden und am höchsten bei niederen UV-Dosen (3,4, 5,0 und 22 für c, b bzw. e). Das bedeutet eine Annäherung der Dosiskurven an linearen Verlauf. Die zur Auslösung von Mutationen erforderliche Trefferzahl wird somit durch BU-Einbau vermindert. Für -normal entsprachen die Kurven der UV-Mutationsinduktion dem 2-Treffertyp. Es wird geschlossen, daß die Hemmung der Dunkelreaktivierung (HCR) der Letalläsionen und Prämutationen in BU-DNA nicht die einzige Ursache für die Unterschiede in den UV-Sensibilitäten ist, sondern daß die Bildung anderer UV-Produkte als in der normalen DNA ebenfalls eine Rolle spielt.

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Summary Bromouracil (BU) was incorporated into the DNA of theSerratiaphage by means of culture in a medium with BUdR and FUdR. The density of BU-x particles in the CsCl-gradient indicated a replacement of about 24 to 32 % of the thymine by BU. The frequency of two types of plaquemutants (c and b) in the BU--population was increased significantly above the frequencies in BU-free phage by average factors of 1.4 and 2.4, resp. A third mutant type (e) was increased less. The absolut frequencies of additional c- and b-mutants in BU- compared with normal were about 2.10^–4 and 1.10^–4, resp.The UV-inactivation of BU- was higher by a factor of about 1.40 than that of normal , the inactivation by X-rays was only 1.14 times higher. The X-ray induced plaquemutation frequencies were not remarkably influenced by incorporated BU, but UV-mutation-induetion was increased much stronger than UV-inactivation. The sensitization factors were different for the three mutation types and highest at low UV-doses (3.4, 5.0 and 22 for c, b and e resp.). The UV-dose curves of mutation induction in normal are of the 2 hit type, but for BU- they were significantly less curved indicating the participation of 1-hit-processes in premutation production. It is concluded that inhibition of dark repair (HCR) of lethal lesions and premutations in BU-DNA is not the only reason for the differences in UV-sensitivities but that production of other species of UV-products in BU-DNA than in normal DNA plays also a role.

     

Morphological development and gibberellin production by different strains of Gibberella fujikuroi in shake flasks and bioreactor

Strain H-984 of G. fujikuroi grown for 38h in a shake flask with medium containing 20g glucose l^–1, 3g yeast extract l^–1, 2.5g NH4NO3 l^–1, 0.5g KH2PO4 l^–1, 0.1g MgSO4 l^–1, 1g CaCO₃ l^–1, and inoculated into a bioreactor with medium containing 60g glucose l^–1; 1g NH_4Cl l^–1; 3g KH2PO4 l^–1 and 1.5g MgSO₄ l^–1 produced 1100mg gibberellic acid l–1.     

Calculations of one-, two- and three-bond nuclear spin-spin couplings in a model peptide and correlations with experimental data

Summary We present ab initio calculations of the Fermi contact term and experimental correlations of six coupling constants, ³J_H ^N _H , ¹J_{C H} , ²J_CH , ¹J_{C N}, ²J_{C N} and ¹J_CN, in a peptide as functions of the backbone dihedral angles, and . Given estimates of experimental uncertainties, we find semiquantitative experimental correlations for ³J_H ^N _H , ¹J_{C N} and ²J_{C N}, qualitative correlations for ¹J_{C H} and ²J_CH , but no experimental correlations of practical utility for ¹J_CN, owing to its complex dependence on at least four dihedral angles. Errors in the estimation of dihedral angles from X-ray crystallographic data for proteins, which result from uncertainties in atom-to-atom distances, place substantial limitations on the quantitative reliability of coupling constant calculations fitted to such data. In the accompanying paper [Edison, A.S. et al., J. Biomol. NMR, 4, 543–551] we apply the results of the coupling constant calculations presented here to the estimation of and angles in staphylococcal nuclease from experimental coupling constants.Abbreviations AO atomic orbital - BPTI basic pancreatic trypsin inhibitor (bovine) - CI-2 chymotrypsin inhibitor 2 - E.COSY exclusive correlation spectroscopy (Griesinger et al., 1986) - ⁿJ_AB single bond (n=1), geminal (n=2), or vicinal (n=3) coupling constant between nuclei A and B - LCAO linear combination of atomic orbitals - NBO natural bond orbital - n lone pair orbitals - bonding orbitals - ^* antibonding orbitals - dihedral angle or molecular orbital wave function; r², correlation coefficient - RHF restricted Hartree-Fock; rmsd, root-mean-square deviation - 3-21G and 6-31G^* molecular orbital basis set designations (Hehre et al., 1986)     

Effects of insecticides on cultures of insect cells

Serial dilutions of 20 insecticides were examined for their effects on the growth of insect cells cultivated in vitro. No differences in susceptibility were found for cells derived from the moth Antheraea eucalypti and the mosquito Aedes aegypti.Rotenone was the most effective inhibitor investigated, decreasing the rate of cell division at 0.001 g/ml. Malathion and diazinon first showed effects at 12 g and 112/ml respectively. Toxicants first effective at 10 g/ml included pp-DDT, dieldrin, pyrethrins and sodium arsenate; at 100 g/ml they included lindane and carbaryl; at 1000 g/ml only nicotine sulphate.The majority of insecticides tested (principal exception rotenone) were very much more toxic to last instar A. aegypti larvae than to the insect cells, suggesting that the functions of highly organized tissues are more readily interfered with than those of individual cell types comprising them.

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Zusammenfassung Verdünnungsserien von 20 Insektiziden wurden auf ihren Effekt auf das Wachstum von in vitro kultivierten Insektenzellen untersucht. Die untersuchten Zellen stammten aus Kulturen von Ovariolen von Antheraea eucalypti-Puppen und von Gewebe von Aedes aegypti-Larven. Rotenon erwies sich als das wirksamste Insektizid: es verlangsamte die Zellteilung in einer Konzentration von 0.001 g/ml. Malathion wurde erst in einer Konzentration von 12 g/ml wirksam, Diazinon bei 112 g/ml. Mehrere Insektizide zeigten erste Wirksamkeit bei 10 g/ml; diese waren: pp-DDT, pp-DDD, pp-DDE, Methoxychlor, Aldrin, Dieldrin, Pyrethrine, Allethrin und Natriumarsenat. Insektizide mit einer ersten Wirksamkeit bei 100 g/ml waren Lindan, Isolan, Dimetilan, Carbaryl, DNOC und Piperonylbutoxid. Nikotinsulfat war erst bei 1 mg/ml oder bei höheren Konzentrationen wirksam. Zwischen den Antheraea- und Aedes-Zellen wurde kein Unterschied in der Empfindlichkeit gegen die verschiedenen Insektizide gefunden. Bei niedrigen Konzentrationen zeigten Malathion und Natriumarsenat erst nach dem 4. Tag bedeutendere Effekte. Ein schwacher synergistischer Effekt wurde beim Mischen von Pyrethrinen mit Piperonylbutoxid in niedrigen Konzentrationen beobachtet, nicht aber bei hohen Konzentrationen.Bei der Mehrzahl (17 von 19) der Insektizide waren die Zellen in 10 bis 10.000 mal höheren Insektizid-Konzentrationen zu überleben fähig als A. aegypti-Larven im letzten Stadium. Rotenon war das einzige Insektizid, dessen Toxizität auf Zellen stärker war (10.000 Mal) als auf intakte Larven.

     

Spectral position control of dye absorption band maximum in an orienting matrix

The continuous control of the maximum position of the dye absorption band (the zero of the derivative dD ()/d of the cell's optical density D ()) in a nematic matrix is demonstrated experimentally, as a result of changing the angle between the optical axis of a planar-oriented sample and the plane of polarization of absorbed light incident normal to the optical axis. The theory proposed describes quantitatively the experimental dependence (). The rotation of the polarizer with given frequency results in the spectral position modulation of the solute band maximum () within (=0°)–(90°)=700 cm^–1.     

Zum Brenzkatechinamin-Stoffwechsel der Nebenniere

Zusammenfassung Durch Injektion von ³H-Dopa wurden die Brenzkatechinamine der Maus in vivo markiert. Die Verteilung der radioaktiven Brenzkatechinamine in der Nebenniere wurde bis zu 22 d nach Markierung autoradiographisch verfolgt.Auf lichtmikroskopischen Autoradiogrammen war das Zytoplasma aller phäochromen Zellen relativ gleichförmig markiert. Unterschiede der Chromaffinität waren ohne Einfluß auf den ³H-Brenzkatechinamin-Gehalt. Elektronenmikroskopische Autoradiogramme (Foto-Emulsion NUC-307) zeigten aber, daß vor allem solche Bezirke des Zytoplasmas Radioaktivität enthielten, die reich an phäochromen Granula waren. Über Granula der Adrenalin-Zellen war im elektronenmikroskopischen Autoradiogramm die Silberkorndichte doppelt so groß wie über den Granula der Noradrenalin-Zellen.Die Radioaktivität aller chromaffinen Zellen nahm mit zunehmender Überlebenszeit annähernd exponentiell ab. Die durch Silberkornzählung auf Autoradiogrammen bestimmte Halbwertszeit der ³H-Brenzkatechinamine des Marks betrug 6–12 Tage.Die experimentelle Entspeicherung der ³H-Brenzkatechinamine durch Reserpin erniedrigte innerhalb von 1 d den ³H-Brenzkatechinamin-Gehalt aller chromaffinen Zellen des Marks in gleichem Maße, während Insulin zu einer von Zelle zu Zelle wechselnden Entspeicherung führte.Empfindlichkeit und Auflösung der elektronenmikroskopischen Autoradiographie mit NUC-307 (Monoschicht) wurden bestimmt. 38-Zerfallsprozesse (Tritium) werden zur Erzeugung eines entwicklungsfähigen Silberkorns benötigt. Das erreichte Auflösungsvermögen betrug 0,14 .

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Summary The catecholamines of mice were labeled in vivo by injecting ³H-Dopa. The distribution of the radioactive catecholamines in the adrenal gland was autoradiographically followed up to the 22nd day after labeling.The cytoplasm of all chromaffin cells was rather evenly labeled on light microscopic autoradiograms. Differences in the intensity of the chromaffin reaction were of no influence on the contents of radioactive catecholamines. However, electron microscopic autoradiograms (photographic emulsion NUC-307) showed that the radioactive material was located mainly in those areas of the cytoplasm which were rich in chromaffin granules. In these electron microscopic autoradiograms the silver grains were twice as dense over the adrenaline cells as over the noradrenaline cells.The radioactivity of all chromaffin cells decreased almost exponentially with increasing survival time. Determined by grain countings on autoradiograms, the half life of ³H-catecholamines in the medulla was between 6 to 12 days.The release of ³H-catecholamines, which was experimentally induced by reserpine, lowered the contents of ³H-catecholamines in all chromaffin cells of the medulla to the same extent within 1 day, whereas the degree of depletion after insulin varied from cell to cell.The sensitivity and photographic resolving power of electron microscope autoradiograms with NUC-307 (monolayer) was determined. On the average 38-particles were necessary per grain; the resolving power was 0.14 .

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Fräulein Helma Korff und Fräulein Ana Vöhringer danken wir für ihre Hilfe bei der Durchführung der Versuche und Auswertung der Autoradiogramme. Die Untersuchungen wurden durch Förderung seitens des Bundesministeriums für Wissenschaftliche Forschung und der Deutschen Forschungsgemeinschaft ermöglicht.     

Stable sulfur isotopic biogeochemistry of the Hubbard Brook Experimental Forest, New Hampshire

In natural ecosystems, differences often exist in the relative abundanceof stable S isotopes (°³⁴S) that can provide clues as tothe source, nature, and cycling of S. Values of °³⁴S inprecipitation, throughfall, soils, soil solution, and stream waters weremeasured at the Hubbard Brook Experimental Forest (HBEF), New Hampshire.Values of °³⁴S in precipitation and throughfall weresimilar to each other but differed seasonally. Precipitation°³⁴S values were higher in the dormant season[°³⁴S = 5.9±0.6 (17)][Mean + SE(N)]than in the growing season [°³⁴S = 5.0±0.6(40)] but throughfall growing-season values were higher[°³⁴S = 5.6±0.6(68)] than for the dormantseason [°³⁴S = 4.9±0.7 (9)]. Different treespecies did not affect throughfall °³⁴S values. In soilsolution, °³⁴S values were higher in the growing season(°³⁴S = 8.9±2.8; 8.8±1.7;and 4.0±0.6 for Oa, Bh, and Bs horizons, respectively) thanin the dormant season (°³⁴S = 5.6±1.5;3.7±2.4; and 3.4±1.2 for Oa, Bh, and Bshorizons, respectively). These seasonal differences in°³⁴S were probably caused by biological isotopicfractionation. The °³⁴S values in streams were generally2 lower and more variable than those in precipitation andthroughfall, suggesting fractionation and/or different isotopic sources inthe soil.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence against the operation of the Calvin cycle in growing cells

Chlorobium limicola has been proposed to assimilate CO₂ autotrophically via a reductive tricarboxylic acid cycle rather than via the Calvin cycle. This proposal has been a matter of considerable controversy. In order to determine which pathway is operative, the bacterium was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C-pyruvate, and the incorporation of ¹⁴C into alanine (intracellular pyruvate), aspartate (oxaloacetate), glutamate (-ketoglutarate), and glucose (hexosephosphate) was measured in exponentially growing cells in long term labeling experiments. During growth in presence of pyruvate, 20% of the cell carbon were derived from pyruvate in the medium, 80% from CO₂. Since pyruvate was not oxidized to CO₂, only those compounds should become labeled which were synthesized from CO₂ via pyruvate.The three amino acids and glucose were found to be labeled. Alanine had one fifth the specific radioactivity of the extracellular pyruvate, indicating that 20% of the intracellular pyruvate pool were derived from pyruvate in the medium, 80% were synthesized from CO₂. Glucose had twice the specific radioactivity of alanine, showing that hexosephosphate synthesis from CO₂ proceeded via the pyruvate pool. The latter finding is not consistent with the operation of the Calvin cycle, in which pyruvate is not an intermediate. The specific radioactivities of aspartate (oxaloacetate) and of glutamate (-ketoglutarate) were practically identical but considerably lower than that of alanine ( intracellular pyruvate). These findings are compatible with the operation of a reductive tricarboxylic acid cycle as mechanism of autotrophic CO₂ fixation. Degradation studies of the cell components support this interpretation. Offprint requests to: G. Fuchs     

Cyclosporin A-sensitive decrease in the transmembrane potential across the inner membrane of liver mitochondria induced by low concentrations of fatty acids and Ca2+

At low Ca²⁺ concentrations the pore of the inner mitochondrial membrane can open in substates with lower permeability (Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys., 195, 468-477). Recently, we showed that Ca²⁺ loading of mitochondria augments the cyclosporin A-dependent decrease in transmembrane potential () across the inner mitochondrial membrane caused by 10 M myristic acid but does not affect the stimulation of respiration by this fatty acid. We have proposed that in our experiments the pore opened in a substate with lower permeability rather than in the classic state (Bodrova, M. E., et al. (2000) IUBMB Life, 50, 189-194). Here we show that under conditions lowering the probability of classic pore opening in Ca²⁺-loaded mitochondria myristic acid induces the cyclosporin A-sensitive decrease and mitochondrial swelling more effectively than uncoupler SF6847 does, though their protonophoric activities are equal. In the absence of P_i and presence of succinate and rotenone (with or without glutamate) cyclosporin A either reversed or only stopped decrease induced by 5 M myristic acid and 5 M Ca²⁺. In the last case nigericin, when added after cyclosporin A, reversed the decrease, and the following addition of EGTA produced only a weak (if any) increase. In P_i-containing medium (in the presence of glutamate and malate) cyclosporin A reversed the decrease. These data show that the cyclosporin A-sensitive decrease in by low concentrations of fatty acids and Ca²⁺ cannot be explained by specific uncoupling effect of fatty acid. We propose that: 1) low concentrations of Ca²⁺ and fatty acid induce the pore opening in a substate with a selective cation permeability, and the cyclosporin A-sensitive decrease results from a conversion of to pH gradient due to the electrogenic cation transport in mitochondria; 2) the ADP/ATP-antiporter is involved in this process; 3) higher efficiency of fatty acid compared to SF6847 in the Ca²⁺-dependent pore opening seems to be due to its interaction with the nucleotide-binding site of the ADP/ATP-antiporter and higher affinity of fatty acids to cations.     

Synthesis of New 15-Ketosterol Analogues from Ergosterol

Ergosteryl acetate was converted through three stages into 3-acetoxy-24-methyl-5-cholesta-8(14),22-diene-15-one in 32% overall yield. The product was transformed to 3-hydroxy-24-methyl-5-cholesta-8(14),22-diene-15-one, 3-hydroxy-24-methyl-5-cholesta-8(14),22-diene-15-one, and 24-methyl-5-cholesta-8(14),22-diene-3,15-dione. The compounds were characterized by ¹H and ¹³C NMR spectra.     

Photoperiodism and thermoperiodism in the predatory mite Amblyseius potentillae are probably based on the same mechanism

Summary A comparative study was made of the photoperiodic and thermoperiodic induction of diapause in the phytoseiid mite Amblyseius potentillae. Sensitivity to thermoperiod was found to be highest during the protonymphal and deutonymphal stages, with some sensitivity still being present in the young adult. Summation of both photoperiodic and thermoperiodic cycles was shown to take place, which demonstrated the presence of a photoperiodic counter as well as a thermoperiodic counter in these mites. Vitamin A appeared to be necessary for some early step in the physiological mechanism of diapause induction and not just for the expression of the diapause response. The light sensitivity threshold for photoperiodic induction of diapause was found to be extremely low, viz. less than 0.02 W/cm². Moreover, the light sensitivity threshold appeared to be strongly temperature dependent in A. potentillae. Experiments in which the mites experienced various sequences of short-day photoperiods and short-day thermoperiods, applied either concurrently or in succession, showed that the information collected by the photoperiodic counter and the thermoperiodic counter is integrated into one induction sum. These results strongly suggest that photoperiodic and thermoperiodic induction of diapause in these mites is based on the same physiological mechanism.Abbreviations DD continuous darkness - LL continuous light - LD light-dark cycle (e.g. LD 16:8 is a cycle of 16 h of light and 8 h of darkness) - TC thermoperiodic cycle (e.g. TC 16:8 (27°: 15°) is a thermoperiod with a 16 h thermophase of 27 °C and an 18 h cryophase of 15°C)     

Mercury contamination in Lake Xolotlán (Managua)

Total mercury was measured in different compartments of Lake Xolotlán's (Managua) ecosystemviz., sediments, water, fish and men. Sediments from 18 localities at 5 depths inside the sediment (5, 10, 15, 20 and 25 cm) contained an average concentration of 0.62 g Hg.g^–1±0.46 at the surface, with extreme values of 0.16 and 1.8 g.g^–1. The highest concentration was observed at 25 cm depth in front of the chlor-alkaly factory (ELPESA). This maximum is associated with the period of highest production of this factory. The highest mercury concentrations in water were also measured close to the discharge of ELPESA,viz. 787 g.Hg^–1 in January and 506 g.g^–1 in April. The mean mercury concentrations measured in the muscles of the most consumed fish were 0.63 g.g^–1±0.22 (extreme values 0.22 and 1.45) inCichlasoma managuense, and 0.07 g.g^–1±0.14 (extreme values 0.004 and 0.63) inC. citrinellum. The concentration in the liver was 0.79 g.g^–1±1.29 inC. managuense and 0.62 g.g^–1±0.44 inC. citrinellum. Human hairs (n=98) of fishermen and their families contained 5.03 g.g^–1±6.2 (extreme values 0.02 and 38.22). The mean concentration measured in men was 6.22 g.g^–1±6.34 (n=58), and in women 3.39 g.g^–1±5.7 (n=40). The average mercury concentration of hairs of workers of ELPESA was 91.24 g.g^–1±156.9 (extreme values 0.46 and 724.53; n=32). We conclude that total mercury levels in the various ecosystem compartments are very high and mercury contamination in the lake may be considered as dangerous for human health.     

Der olfaktorische Saum der Katze

Zusammenfassung Das olfaktorische Epithel von erwachsenen Katzen wurde nach Perfusionsfixierung mit Glutaraldehyd und Nachfixierung mit Osmiumtetroxyd stereomikroskopisch im Totalpräparat und elektronenmikroskopisch in Schnittserien untersucht. Im Auflichtverfahren zeigt die Regio respiratoria eine blaugrüne und die Regio olfactoria eine seidighelle graublaue Interferenzfarbe. Bei stärkerer lichtmikroskopischer Vergrößerung sind die ausgerichteten Bündel der Sinnesgeißeln zu erkennen. Besondere Aufmerksamkeit wurde dem olfaktorischen Saum gewidmet, der sich in drei Schichten gliedert: 1. In eine Innenzone, die die olfaktorischen Vesikel und die Mikrozotten der Stützzellen beherbergt. 2. In eine Außenzone, in der die distalen Segmente der Sinnesgeißeln liegen und 3. in den strukturdichteren Terminalfilm. Die Geißelenden tauchen von unten in den Film ein und tragen dann auf ihrer Membranoberfläche entweder Filamenteverdichtungen oder sie sind von einem optisch leeren Hof umgeben. Die distalen Geißelsegmente können wahrscheinlich bis 80 lang sein, sie enthalten nur zwei mikrotubuläre Geißelfibrillen. Das proximale Geißelsegment trägt unmittelbar über dem Ursprung im olfaktorischen Vesikel eine Ringmanschette. Unterschiedliche Formen der Sinneszellendkolben sprechen dafür, daß die Perikarya ihr olfaktorisches Bläschen im Rahmen einer stetigen Mauserung ersetzen können. Der vonOkano et al. (1967) als jugendliche Stützzelle angesehene vierte Zelltyp des Riechepithels ist auch bei der Katze vorhanden. Ein weiterer fünfter Zelltyp hat basal im Epithel ein Perikaryon und wie die Sinneszellen einen dünnen peripheren Fortsatz, der mit steifen Mikrozotten in die Innenzone des olfaktorischen Saumes hineinreicht.Es wird diskutiert, wieweit Innenstrukturen des Geißelapparates an den Vorgängen der Aufnahme von Geruchsreizen und der Erregungsbildung beteiligt sein können.

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On the olfactory epithelium of the cat

Summary An examination of the olfactory epithelium of adult cats was carried out after perfusion with glutaraldehyde and postfixation with osmium tetroxide. Unsectioned preparations were studied with the stereomicroscope, and serial sections were examined under the electron microscope. With incident illumination light the regio respiratoria shows a blue-green interference colour while the regio olfactoria exhibits a distinctively silky bluegrey shade. A higher lightmicroscopic magnification reveals bundles of olfactory cilia arranged in parallel rows. Particular attention was paid to the fact that the olfactory border is composed of three layers, namely: 1. an inner layer, containing olfactory vesicles and the microvilli of supporting cells; 2. an outer layer with the distal segments of the olfactory cilia; and 3. a dense terminal film. The distal segments of the olfactory cilia are probably up to 80 microns long and usually contain two microtubules. They extend into the dense terminal film, having on their membrane a concentration of filamentous material or being surrounded by an electron opaque halo. The proximal segment of the cilium looks like a kinocilium bearing a circular cuff just above the olfactory vesicle. Different shapes of the olfactory vesicles indicate that the perikarya are capable of replacing their olfactory vesicles from time to time. The juvenile (fourth) cell type described byOkano et al. (1967) is also observed in the cat. A fifth cell type was found in the regio olfactoria of the cat; its cell body lies deep in the olfactory mucosa among the perikarya of the olfactory cells. A thin distal process reaches the inner layer of the olfactory border with straight stiff microvilli.It is also discussed to which extent the internal structure of the cilia is involved in olfactory perception, or in the initiation of a generator potential.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Absorption of circularly polarizedγ-radiation in L- and D-amino acids

No difference within the experimental error ( 10³) was observed in the absorption of right- and left-circularly polarized-radiation in L- and D-tryptophan and tyrosine. For the measurements the 14.4 keV transition of⁵⁷Fe was used and the analysis of the circularly polarized-radiation was performed by Mössbauer method.