Interferenzmikroskopische und cytophotometrische Untersuchungen zur Analyse von Zellkernen

Zusammenfassung In vergleichenden interferenzmikroskopischen und cytophotometrischen Untersuchungen wurde versucht, die Globulinfraktion von Zellkernen quantitativ zu erfassen. Dazu wurden Thymuslymphozyten und nach Behrens isolierte Leberzellkerne der Ratte und Nierenzellkerne vom Schwein im Ausstrichpräparat bis zu 30 Std in physiologischer Kochsalz- und Tyrodelösung extrahiert. Die Abnahme der Kerntrockenmasse wurde interferenzmikroskopisch gemessen und der DNS-, Histon- und Gesamtproteingehalt cytophotometrisch bestimmt (Feulgen-Reaktion, Färbung mit Gallocyanin-Chromalaun, Fastgreen bei pH 8 und pH 2, Naphtholgelb S).Bei Verwendung von Glycerin als Eindeckmittel fand sich bei Leberzellkernen nach Extraktion mit 0,14 m NaCl-Lösung von 3–5°C eine Trockengewichtsabnahme von 18% nach 2 Std, von 29% nach 10 Std und von 34% nach 30 Std. Bis zu 2 Std wurden Proteine, nach 10 Std Nukleohistone extrahiert. Da der nach 2 Std gemessene Proteinverlust gut mit dem Globulingehalt von Leberzellkernen übereinstimmt, wurde offenbar die Globulinfraktion erfaßt. Nach Extraktion mit Tyrodelösung bei Zimmertemperatur lag der Substanzverlust in der gleichen Größenordnung (2 Std: 12%, 10 Std: 20%, 30 Std: 29%).Bei Verwendung von Tyrodelösung als Eindeckmittel blieb nach Extraktion isolierter Nierenzellkerne mit 0,14 mNaCl-Lösung die Kerntrockenmasse bis zu 2 Std unverändert, da die Globulinfraktion schon während der interferenzmikroskopischen Messung in Lösung geht. Auch bei Thymuslymphozyten fand sich erst nach 24 Std ein Verlust von 18% der Kerntrockenmasse durch Extraktion von DNS und Histonen.Während der Suspension von Thymusgewebe in 0,14 mNaCl-Lösung nahm innerhalb von 4 Std das Kerntrockengewicht um 40% zu, wahrscheinlich infolge Proteinaufnahme.Die gewonnenen Ergebnisse werden hinsichtlich der Fehlerquellen und im Vergleich zu cytophotometrischen Messungen und biochemischen Globulinbestimmungen diskutiert.

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Analysis of cell nuclei with the interference-microscope and cytophotometryI. Determination of globulins

Summary The globulinfraction of cell nuclei was determined by dry mass determinations in comparison with cytophotometric measurements.Lymphocytes from the thymus gland, and liver nuclei from the rat and kidney nuclei from the pig, isolated with Behrens' technique, were extracted in smears, up to 30 hrs, in physiological saline and tyrode solution. The decrease of nuclear dry mass was measured with the interference microscope, and the content of DNA, histones and total proteins was determined by cytophotometry (Feulgen-reaction, staining with gallocyanin chrome alum, fast green at pH 8 and pH 2, naphtol yellow S).Using glycerol as embedding medium, after extraction with 0,14 M saline at 3—5° C the dry weight of liver nuclei decreased by 18% after 2 hrs, by 29% after 10 hrs and by 34% after 30 hrs. Nuclear proteins were extracted up to 2 hrs, nucleo-histones were removed after 10 hrs. Since the loss of proteins is in good agreement with the globulin content of liver nuclei, obviously the nuclear globulins have been extracted until 2 hrs. After extraction with tyrode solution at room temperatue, the loss of dry matter was in the same range (2 hrs: 12%, 10 hrs: 20%, 30 hrs: 29%).Using tyrode solution as embedding medium, the dry weight of kidney nuclei remained unchanged for 2 hrs when extracted with 0,14 M saline, since the globulin-fraction is lost while nuclei are measured by interferometry. With thymocytes only a loss of 18% of nuclear dry weight was found after 24 hrs due to removal of DNA and histones.Suspending the thymus gland in 0,14 M saline, the nuclear dry weight increased by 40% in 4 hrs, probably due to adsorption of proteins.The results were discussed in the light of the errors of the measurements and compared to biochemical and histochemical datas.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Direct DNA sequencing of PCR amplified genomic DNA by the Maxam-Gilbert method

We present a method to directly sequence PCR amplification products utilizing the chemical method of Maxam and Gilbert. This procedure yields clearly readable DNA sequences of 100-400 base pairs in length derived from human genomic DNA in four days' time.     

Dose-dependent improvement of myoclonic hyperkinesia due to Valproic acid in eight Huntington's Disease patients: a case series

Background  

Chorea in Huntington's Disease (HD) is usually treated with antidopaminergic neuroleptics like haloperidol, olanzapine and tiaprid or dopamine depleting drugs like tetrabenazine. Some patients with hyperkinesia, however, react to treatment with antidopaminergic drugs by developing extrapyramidal side effects. In earlier studies valproic acid showed no beneficial effect on involuntary choreatic movements. Myoclonus is rare in HD and is often overseen or misdiagnosed as chorea.     

Hemoglobins of the Lucina pectinata/bacteria symbiosis. I. Molecular properties, kinetics and equilibria of reactions with ligands

Three hemoglobins have been isolated from the symbiont-harboring gill of the bivalve mollusc Lucina pectinata. Oxyhemoglobin I (Hb I), which may be called sulfide-reactive hemoglobin, reacts with hydrogen sulfide to form ferric hemoglobin sulfide in a reaction that may proceed by nucleophilic displacement of bound superoxide anion by hydrosulfide anion. Hemoglobins II and II, called oxygen-reactive hemoglobins, remain oxygenated in the presence of hydrogen sulfide. Hemoglobin I is monomeric; Hb II and Hb III self-associate in a concentration-dependent manner and form a tetramer when mixed. Oxygen binding is not cooperative. Oxygen affinities are all nearly the same, P50 = 0.1 to 0.2 Torr, and are independent of pH. Combination of Hb I with oxygen is fast; k'on = (estimated) 100-200 x 10(6) M-1 s-1. Combination of Hb II and Hb III with oxygen is slow: k'on = 0.4 and 0.3 x 10(6) M-1 s-1, respectively. Dissociation of oxygen from Hb I is fast relative to myoglobin: koff = 61 s-1. Dissociation from Hb II and Hb III is slow: koff = 0.11 and 0.08 s-1, respectively. These large differences in rates of reaction together with differences in the reactions of carbon monoxide suggest differences in configuration of the distal heme pocket. The fast reactions of Hb I are comparable to those of hemoglobins that lack distal histidine residues. Slow dissociation of oxygen from Hb II and Hb III suggest that a distal residue may interact strongly with the bound ligand. We infer that Hb I may facilitate delivery of hydrogen sulfide to the chemoautotrophic bacterial symbiont and Hb II and Hb III may facilitate delivery of oxygen. The midpoint oxidation-reduction potential of the ferrous/ferric couple of Hb I, 103 +/- 8 mV, was independent of pH. Potentials of Hb II and Hb III were pH-dependent. At neutral pH all three hemoglobins have similar midpoint potentials. The rate constant for combination of ferric Hb I with hydrogen sulfide increases 3000-fold from pH 10.5 to 5.5, with apparent pK 7.0, suggesting that undissociated hydrogen sulfide is the attacking ligand. At the acid limit combination of ferric Hb I with hydrogen sulfide, k'on = 2.3 x 10(5) M-1 s-1, is 40-fold faster than combination with ferric Hb II or myoglobin.(ABSTRACT TRUNCATED AT 400 WORDS)     

REDUCING INCIDENTAL MORTALITY OF FRANCISCANA DOLPHIN PONTOPORIA BLAINVILLEI WITH ACOUSTIC WARNING DEVICES ATTACHED TO FISHING NETS

We conducted a double blind experiment in an artisanal gillnet fishery in Argentina to determine the effectiveness of acoustic deterrents (pingers) at reducing bycatch of the Franciscana dolphin ( Pontoporia blainvillei ). The fishery was conducted by small inflatable and fiberglass vessels operating between 0.5 and 7 krn from the coast. Each vessel carried an independent observer who was rotated from vessel to vessel throughout the course of the experiment. Information on the number of dolphins captured, geographic position, depth, configuration of fishing gear, soak time, biomass of fish caught, and sea lion predation in a string/net producing any damage was recorded. Equivalent numbers of active and silent pingers were used during the experiment. A total of 45 dolphins were caught in the silent nets, and seven were caught in the active pinger nets, demonstrating a highly significant reduction in bycatch for this species. However, sea-lions ( Otaria flavescens ) damaged the fish in active pinger nets significantly more than silent nets, and the damage increased over the course of the experiment. Although pingers show promise as a management tool for this species, pinniped depredation suggests that higher pinger frequencies will be needed to avoid a "dinner bell" effect.