Ein Beitrag zum histochemischen Verhalten der Kern-DNS in Interphase und Mitose,dargestellt durch kombinierte Säurehydrolyse und Akridinorangefluorochromierung

Zusammenfassung Aufbauend auf vorangegangenen Arbeiten über die unterschiedliche Darstellung von DNS durch Kombination einer Säurehydrolyse mit einer Akridinorange-fluorochromierung, sollte untersucht werden, ob sich, damit unter standardisierten Bedingungen Unterschiede der DNS an alkoholfixierten Kernen bei Tumoren, im Laufe der Mitose sowie bei Kernen nekrotischer Zellen darstellen lassen.Ein rascherer Fluoreszenzumschlag zum Langwelligen bereits nach kurzer Hydrolyse (1 min, n/HCl, 37° oder 60°) war zu beobachten: bei allen nekrotischen Zellkernen, bei den Kernen eines Teiles der untersuchten Tumoren (in 45 von 85 Fällen), innerhalb eines Interphasekernes im Heterochromatin und in allen Fällen an den Mitosechromosomen. Unter Berücksichtigung unserer heutigen Vorstellungen über den Bindungsmechanismus des Akridinorange an die Nukleinsäuren des Zellkernes und über die Veränderungen der DNS-Molekülstruktur nach Säurehydrolyse kommen als morphologisches Substrat für den Fluoreszenzumschlag in Betracht: eine geringere sekundärstrukturelle Festigkeit des DNS-Moleküls, ein geringerer Polymerisationsgrad und ein erhöhter Gehalt an ribonukleaseresistentem DNS-RNS-Komplex.Unabhängig von der noch problematischen Deutung der Befunde ist die angewandte Methode in der Lage, Unterschiede in der Struktur der Nukleinsäuren dieser Zellkerne zur Darstellung zu bringen, die sich bisher einem Nachweis entzogen haben.

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Summary Based on previous papers on the variable demonstration of DNA by means of a combination of acid hydrolysis and conjugation with acridine orange, a study was conducted to investigate if under standardized conditions alcohol fixed nuclei of tumours show differences in their DNA during mitosis and necrosis. A rather sudden change of the fluorescence to the long wave region after short hydrolysis (1 min, n/HCl, 37° or 60°) was observed: in all necrotic cell nuclei; in the nuclei of some of the tumours (45 out of 85); in the heterochromatine of an interphase nucleus, and in all cases of chromosomes in mitosis. Taking into consideration the current knowledge on the linking mechanism of acridine orange to nucleic acids of the cell nucleus, and on changes of the molecular structure of DNA following acid hydrolysis the morphologic substrate responsible for the change of the fluorescence could be: a lesser secondary structural stability of the DNA molecule; a lower degree of polymerisation and a higher level of a ribonuclease resistant DNA-RNA complex.Although it is rather difficult to interprete the findings, it can be said that the method described reveals differences in the structure of certain cell nuclei, which so far have not been demonstrable.

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Einige Ergebnisse dieser Arbeit wurden auf der 4. Arbeitstagung der Arbeitsgemeinschaft Morphologie in der DDR am 2./3. Oktober 1964 in Leipzig vorgetragen und sind Teil einer Habilitationsschrift, die dem Senat der Medizinischen Akademie Carl Gustav Carus, Dresden, vorgelegen hat.     

A comparison of sperm morphology and silver nitrate staining characteristics in the domestic ferret and the black-footed ferret

Ejaculated sperm from the domestic ferret (Mustela putorius furo) and the black-footed ferret (Mustela nigripes) were compared for differences in morphological abnormalities and argentophilic protein distribution. Thawed domestic ferret sperm was also compared to fresh sperm to determine whether there were any effects on cell morphology due to cryopreservation. There were statistically significant differences between the two species of ferret in two of the categories scored. The domestic ferret had a higher frequency of cells that were bent in the midpiece and in the principal piece, and a higher frequency of headless and tailless cells when compared to the black-footed ferret. There were no statistically significant differences in cell morphology between the fresh and cryopreserved ejaculates of the domestic ferret employing a standard egg yolk cryoextender. Silver nitrate staining distribution was different between the two species in both the head and tail region.     

Explanation and prediction in vegetation science

A definition of vegetation science is given, spanning 6 levels of integration and stressing the interrelations among them. The problems of realism are discussed. The selection of levels is related to the adequate correspondence between conceptualization and research aims. Pattern and process are introduced as the central concepts of vegetation science. The perception of reality is dependent on the spatial and temporal scale chosen. The concept of noise is discussed in relation to stochasticity and randomness of events. Traces of essentialism are found both in classification of communities and habitat ecology. Classification is important, particularly the coexistence of alternative classification approaches. Organicism as a basis of vegetation research is rejected because the organismic view is inadequate on higher integration levels. The concept of function is redefined in a non-teleologic way.Present vegetation ecological research is inductivistic. One possible alternative to inductivism is falsificationism. The major domain of this approach is hypothesis testing, which will become more important. Progress can only be reached by a maximum degree of communication among scientific individuals.Predictive ecology is partly based on historic explanation, partly on complementary approaches. Characters of vegetation worthwhile to be predicted are listed and the necessary requirements for vegetation science to become predictive are discussed. A major requirement is the development of succession and life-history theory. A further elaboration of the individualistic concept will be a main task of vegetation science in the near future.     

The polymorphic marker DXS304 is within 5 centimorgans of the fragile X locus

The fragile X syndrome, which is the most common cause of inherited mental retardation, poses important diagnostic problems for genetic counseling. The development of diagnostic strategies based on DNA analysis has been impaired by the lack of polymorphic markers very close to the disease locus. Here we report that the polymorphic probe U6.2 (locus DXS304) is much closer to the fragile X locus than all the previously reported markers. A recombination fraction of 0.02 between DXS304 and the fragile X locus was estimated by multipoint linkage analysis (confidence interval 0.002 to 0.05). Our data suggest that DXS304 is distal to the fragile X locus. This marker thus represents a major improvement for carrier detection and prenatal diagnosis in fragile X families.     

X-chromosome localization of the functional gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex

The functional gene locus for the E1 alpha subunit of the human pyruvate dehydrogenase complex has been localized to the p22.1-22.2 region of the X chromosome by in situ hybridization and analysis of somatic cell hybrids with various human X-chromosome rearrangements. Another locus showing significant cross-hybridization with an E1 alpha cDNA probe was detected on chromosome 4, in the region q22. The X-chromosome localization of the pyruvate dehydrogenase E1 alpha subunit gene provides a number of possible explanations for the clinical and biochemical variability which is a major feature of human pyruvate dehydrogenase deficiency.     

Single chloride channels in endosomal vesicle preparations from rat kidney cortex

Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H⁺ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl^– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl^–=Br^–=I^–>SO ₄ ^2– F^–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl^–-flux measurements and studies on the H⁺ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H⁺ uptake into the endosomes.     

Fusion of cultured dog kidney (MDCK) cells: I. Technique,fate of plasma membranes and of cell nuclei

Summary The evaluation of the intracellular signal train and its regulatory function in controlling transepithelial transport with electrophysiological methods often requires intracellular measurements with microelectrodes. However, multiple impalements in epithelial cells are hampered by the small size of the cells. In an attempt to avoid these problems we fused cells of an established cell line, Madin Darby canine kidney cells, originally derived from dog kidney, to giant cells by applying a modified polyethylene glycol method. During trypsin-induced detachment from the ground of the petri dish, individual cells grown in a monolayer incorporate volume and mainly lose basolateral plasma membrane by extrusion. By isovolumetric cell-to-cell fusion, spherical giant cells are formed within 2 hr. During this process a major part of the individual cell plasma membranes is internalized. Over three weeks following cell plasma membrane fusion degradation of single cell nuclei and cell nuclear fusion occurs. We conclude that this experimental approach opens the possibility to investigate ion transport of epithelia in culture by somatic cell genetic techniques.     

Evolution of chemotactic-signal transducers in enteric bacteria.

The methyl-accepting chemotactic-signal transducers of the enteric bacteria are transmembrane proteins that consist of a periplasmic receptor domain and a cytoplasmic signaling domain. To study their evolution, transducer genes from Enterobacter aerogenes and Klebsiella pneumoniae were compared with transducer genes from Escherichia coli and Salmonella typhimurium. There are at least two functional transducer genes in the nonmotile species K. pneumoniae, one of which complements the defect in serine taxis of an E. coli tsr mutant. The tse (taxis to serine) gene of E. aerogenes also complements an E. coli tsr mutant; the tas (taxis to aspartate) gene of E. aerogenes complements the defect in aspartate taxis, but not the defect in maltose taxis, of an E. coli tar mutant. The sequence was determined for 5 kilobases of E. aerogenes DNA containing a 3' fragment of the cheA gene, cheW, tse, tas, and a 5' fragment of the cheR gene. The tse and tas genes are in one operon, unlike tsr and tar. The cytoplasmic domains of Tse and Tas are very similar to those of E. coli and S. typhimurium transducers. The periplasmic domain of Tse is homologous to that of Tsr, but Tas and Tar are much less similar in this region. However, several short sequences are conserved in the periplasmic domains of Tsr, Tar, Tse, and Tas but not of Tap and Trg, transducers that do not bind amino acids. These conserved regions include residues implicated in amino-acid binding.     

Integrin phosphorylation is modulated during the differentiation of F-9 teratocarcinoma stem cells

The retinoic acid-induced differentiation of F-9 teratocarcinoma cells in monolayer culture is accompanied by the accumulation of fibrillar fibronectin deposits, the appearance of a highly structured actin cytoskeleton, and the redistribution of integrin to apparent sites of substrate contact. We have studied the 140-kD fibronectin receptor during this process and report that although the integrin molecule is present in equivalent amounts before and after differentiation, the level of integrin phosphorylation decreases dramatically as the cells differentiate. This loss of phosphorylation coincides temporally with the observed changes in actin, fibronectin, and integrin organization. The phosphorylation state of integrin thus may mediate developmentally regulated cell-matrix interactions.     

Localization of the photoreceptor gene ROM1 to human chromosome 11 and mouse chromosome 19: sublocalization to human 11q13 between PGA and PYGM.

Rom-1 is a retinal integral membrane protein that, together with the product of the human retinal degeneration slow gene (RDS), defines a photoreceptor-specific protein family. The gene for rom-1 (HGM symbol: ROM1) has been assigned to human chromosome 11 and mouse chromosome 19 by Southern blot analysis of somatic cell hybrid DNAs. ROM1 was regionally sublocalized to human 11p13-11q13 by using three mouse-human somatic cell hybrids; in situ hybridization refined the sublocalization to human 11q13. Analysis of somatic cell hybrids suggested that the most likely localization of ROM1 is in the approximately 2-cM interval between human PGA (human pepsinogen A) and PYGM (muscle glycogen phosphorylase). ROM1 appears to be a new member of a conserved syntenic group whose members include such genes as CD5, CD20, and OSBP (oxysterol-binding protein), on human chromosome 11 and mouse chromosome 19. Localization of the ROM1 gene will permit the examination of its linkage to hereditary retinopathies in man and mouse.