Influence of systematic errors on the evaluation of the S phase portions from DNA distributions of solid tumors as shown for 328 breast carcinomas

The portion of cells in S phase has proved to be a valuable prognostic indicator of early relapse and life expectancy, particularly in breast carcinoma. Comparisons of published data on samples of primary breast carcinoma biopsies showed that the values obtained by analyses of flow cytometric DNA distributions were generally higher than those of determinations based on the tritiated thymidine (3H-ThdR) labeling index (LI). Flow cytometric DNA analyses of 328 biopsy samples of primary breast carcinomas revealed that these differences could be explained by varying contributions of debris background. Since this influence is inversely proportional to the cell counts in each channel, it may cause considerable errors, particularly in the S phase channels, which normally contain the lowest counts of the DNA distributions. Two different mathematical rationales were tested in order to separate DNA distributions from the debris superimposition. No appreciable differences were found with respect to the essential results. After appropriate subtraction of the background levels, the previously reported discrepancies between cytometrically determined S phase portions and 3H-ThdR LI values disappeared, and good agreement was achieved for the comparable tumor samples of the present study. In conclusion, debris background subtractions should generally precede the DNA histogram analyses, particularly of solid tumors, in order to obtain reliable S phase values.     

Identification of novel Y chromosome encoded transcripts by testis transcriptome analysis of mice with deletions of the Y chromosome long arm

Background  

The male-specific region of the mouse Y chromosome long arm (MSYq) is comprised largely of repeated DNA, including multiple copies of the spermatid-expressed Ssty gene family. Large deletions of MSYq are associated with sperm head defects for which Ssty deficiency has been presumed to be responsible.     

Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation

ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD to arginine, asparagine, or cysteine residues in target proteins. This post-translational protein modification is the mechanism by which cholera-toxin and other bacterial toxins cause pathology in human host cells. Molecular cloning has identified five toxin-related GPI-anchored cell surface ARTs in the mouse (ART1, ART2.1, ART2.2, ART3, and ART4) and three in the human (ART1, ART3, and ART4). ART2-which has sparked interest because of its ability to activate the cytolytic P2X7 purinergic receptor by ADP-ribosylation-is encoded by two functional gene copies in the mouse genome while the human genome carries two inactivated ART2 pseudogenes. We generated stable transfectants for FLAG-tagged versions of each of the functional human and mouse ARTs. Using genetic immunization we raised monoclonal antibodies that recognize the native human ARTs on the surface of living cells. Some of these mAbs recognize an epitope shared with the mouse ART orthologue but not with more distant ART paralogues. Screening of primary cells and established cell lines by FACS revealed expression of ART1 by monocytes, neutrophils and myeloid leukemia cell lines but not by cell lines derived from solid tumors. ART1 and ART4 have been assigned the designations: CD296, and CD297, respectively.     

Probing the expression and function of the P2X7 purinoceptor with antibodies raised by genetic immunization

The cytolytic P2X7 purinoceptor is widely expressed on leucocytes and has sparked interest because of its peculiar ability to induce a large nonselective membrane pore following treatment of cells with ecto-ATP. Antibodies raised against synthetic P2X7 peptides generally work well in Western-Blot analyses but fail to recognize the native protein on the cell surface. Genetic immunization is a useful technique to raise antibodies directed against proteins in native conformation. Using this technique we have generated highly specific polyclonal (rabbit) and monoclonal (rat) anti-P2X7 antibodies that readily detect mouse P2X7 on the surface of living cells by immunofluorescence analyses and flow cytometry. Binding of these antibodies to P2X7 is reduced within seconds after treatment of cells with ATP, suggesting that ligand binding induces a conformational shift and/or the shedding of P2X7. By site directed mutagenesis we have mutated three conserved arginine residues (R294A, R307A, R316A) in the extracellular loop of P2X7 near the second transmembrane region. Each of these mutations results in loss of ATP response. FACS and immunoblot analyses reveal that the R294A mutant is expressed at higher levels than wild-type P2X7 in transfected cells, whereas the R307A and R316A mutants are barely detectable because there is no or very little protein synthesis of these constructs. In accord with its resistance to ATP-induced activation the R294A mutant is not down-modulated from the cell surface by ATP-treatment.     

Dynamic modeling of complex biological systems: a link between metabolic and macroscopic description

In this study, a class of dynamic models based on metabolic reaction pathways is analyzed, showing that systems with complex intracellular reaction networks can be represented by macroscopic reactions relating extracellular components only. Based on rigorous assumptions, the model reduction procedure is systematic and allows an equivalent 'input-output' representation of the system to be derived. The procedure is illustrated with a few examples.     

Mantle displays of freshwater mussels elicit attacks from fish

1. Gravid females of some North American freshwater mussel species (Bivalvia: Unionidae) display highly modified mantle margins and other reproductive structures which mimic small fish, terrestrial insects, or aquatic macro-invertebrates. We report the responses of fish to these lures, based on the results of laboratory encounters between the following pairs of displaying mussels and fishes: Lampsilis cardium and Micropterus coosae; L . perovalis and M . coosae; and Villosa nebulosa and Percina nigrofasciata . In all three encounters, the lures elicited attacks from fish.
2. Encounters between Lampsilis spp. and M. coosae resulted in gill infestations of the fish by larval mussels, which are obligate parasites on fish. An encounter between V . nebulosa and P . nigrofasciata did not result in infestation.
3. The use of these lures to attract fish may greatly increase the chances of parasite/host encounters and may also reduce the chances of infestation of unsuitable hosts.     

Weitere Untersuchungen über die formative Wirkung des Lichtes und mechanischer Reize auf Pflanzen

Zusammenfassung der Ergebnisse Auf das Längenwachstum der Hypokotyle vonSinapis alba wirkt intermittierendes Licht stärker wachstumshemmend (etiolementsverhindernd) als (bei gleicher Reizsumme) kontinuierliches; die Hemmung wird weiterhin mit zunehmendem Zeitabstand zwischen den Teilreizen stärker. Hieraus kann geschlossen werden, daß jeder Teilreiz ein Refraktärstadium von mehreren Minuten Dauer induziert, das erst abgeklungen sein muß, wenn ein zweiter Reiz voll wirksam werden soll.Die formative Wirkung des Lichtes auf das Blatt ist anderer Natur; das zeigen auch ältere Beobachtungen über die unterschiedliche Abhängigkeit von der Lichtqualität.Auch durch mechanische Reize wird das Längenwachstum der Hypokotyle vonSinapis um so mehr gehemmt, je größer der Zeitabstand zwischen den Teilreizen der intermittierenden Reizung ist; auch hier machen sich also Refraktärstadien geltend.Diese Beobachtungen stützen die schon früher gezogene Schlußfolgerung, daß eine Komponente der formativen Wirkung des Lichtes in der Auslösung von Erregungsvorgängen im engeren Sinne besteht, wie sie ähnlich auch durch mechanische Reizung bedingt werden können.Die Beeinflussung der Gewebedifferenzierung in den Internodien vonVicia Faba durch mechanische Reize ist teilweise übereinstimmend mit der durch photische Reizung bedingten, so daß man für einen Teil dieser Beeinflussungen, namentlich für die Ausbildung von Wandverdickungen, vermuten darf, daß sie bei beiden Reizarten gemeinsam durch die Vermittlung jener Erregungsvorgänge entsteht.Mit 6 Textabbildungen.     

Immunochemical demonstration of keratin and vimentin in cytologic aspirates

Antibodies to intermediate filament proteins were used to characterize tumor cells present in peritoneal and pleural effusions and in thin needle aspirates from palpable lymph nodes. Metastatic adenocarcinoma cells (breast, ovary, endometrium, cervix, colon and stomach) as well as squamous-cell carcinomas and mesotheliomas stained specifically with antibodies to keratin while mesenchymally derived tumor cells (lymphomas, melanoma, fibrosarcoma and neurofibrosarcoma) were positive only for vimentin. Especially in cases of lymph node aspirates, keratin staining in cells was a direct indication of metastatic carcinoma. Antibodies to these different components of the cytoskeleton can thus be used in cytopathologic diagnosis when a definitive diagnosis cannot be made on the basis of conventional cytologic features.     

Clonal sublines of rat neurotumor RT4 and cell differentiation. VI. Chromosome analysis

The RT4 neurotumor cell system consists of clonally derived cell lines where a stem cell type segregates in vitro into three biochemically and morphologically different cell types, one glial and two neuronal types. This process has been termed cell-type conversion (M. Imada and N. Sueoka, 1978, Dev. Biol. 66, 97-108). Detailed cytogenetic analysis of the RT4 cell lines are described. Giemsa-banding analysis of 12 independent clonal isolates of the four different RT4 cell types showed a relatively stable karyotype. The stem cell line, RT4-AC, is diploid and most stable, and it has one 4q+ marker chromosome in place of a normal No. 4. This 4q+ marker was identified in all cell types of the RT4 system and was not observed in other cell lines of BDIX origin. The 4q+, therefore, is a chromosomal marker of the RT4 system. Consistent chromosome rearrangement was not found in any one of the cell-type conversions of the RT4-AC cells into the three derivative cell types. The relative stability of the karyotype of the different clonal isolates gives the RT4 system an advantage in studies of genetic regulation and expression of cell-type conversion in vitro. Also the 4q+ marker can be used to identify RT4 cells in coculture experiments or to distinguish RT4 cells in cases of suspected cell-line contamination.