正丁酸钠对人胃腺癌细胞染色体数目和DNA含量的影响

培养的人胃腺癌MGc 80-3细胞经过正丁酸钠处理7天后,生长抑制率达50.7%,约有90%的细胞形态发生分化,其超微结构亦有显著改变。而且,在染色体数目上,超二倍体细胞由对照组的78%增加到实验组的96%,超三倍体和超四倍体细胞则分别从6%和14%下降至2%。同时应用~3H-TdR放射自显影和福尔根细胞光度法测定未标记细胞(G_1期)DNA含量,结果显示实验组比对照组降低了。而且在实验组的同一制片中,未分化细胞DNA含量平均为超六倍体值(DI=3.67和3.56),其中90%的细胞超过6C;分化细胞DNA含量则平均为近四倍体值(DI=2.03和1.99),其中近60%的细胞少于4C。两者差异统计显著,表明形态分化的人胃腺癌细胞的遗传物质含量明显减少,但这些细胞并非就是正常二倍体细胞。     

Nitric oxide synthase is up-regulated in muscle fibers in muscular dystrophy

Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The neuronal NO synthase isoform (NOS1) was reported to be located exclusively in the sarcolemma. Its loss from the sarcolemma was associated with development of Duchenne muscular dystrophy (DMD). However, new studies evidence that all three NOS isoforms-NOS1, NOS2, and NOS3-are co-expressed in the sarcoplasm both in normal and in DMD skeletal muscles. To address this controversy, we assayed NOS expression in DMD myofibers in situ cytophotometrically and found NOS expression in DMD myofibers up-regulated. These results support the hypothesis that NO deficiency with consequent muscle degeneration in DMD results from NO scavenging by superoxides rather than from reduced NOS expression.     

Cytophotometric evidence for cell ‘transdifferentiation’ in planarian regeneration

Summary The role and fate of male germ cells in planarian regeneration was studied in a population ofDugesia lugubris s.1. which provided a suitable karyological marker to distinguish diploid male germ cells from triploid embryonic and somatic cells. The nuclear Feulgen-DNA content in non-replicating triploid muscle cells of the pharynx and in non-replicating male gonia of testes from intact animals were measured by the cytophotometric technique. The pharynx was then removed by transection and each anterior regenerant was allowed to completely regenerate this organ. Measurements of the Feulgen-DNA content in muscle cells of the regenerated pharynx showed that most of these cells (95%) have a DNA content typical of triploid cells; however, some muscle cells (5%) with a nuclear DNA content typical of male gonia alone were observed.These results were interpreted in the following way. After transection, young male germ cells move from the testes to the wound where they participate in blastema formation along with reserve and/or somatic dedifferentiated cells. During regeneration some of these cells of male origin differentiate into pharyngeal muscle cells. Our findings are discussed in relation to the occurrence of mataplasia in planarians.     

Vergleichende morphologische und zytophotometrisch-autoradiographische Untersuchung der menschlichen Erythropoiese

Zusammenfassung Um die Abhängigkeit der erythropoietischen Zellformen von ihrer Stellung im Generationszyklus (G₁, S und G₂) zu untersuchen, wurde das Knochenmark von vier Normalpersonen mit der Kombination von zytophotometrischer DNS-Bestimmung (Feulgen-Photometrie) und autoradiographischer Technik mit ³H-TdR (in vitro) untersucht. Die Zellen wurden vor dieser Untersuchung in den nach Pappenheim gefärbten Ausstrichen differenziert.Sowohl in der Gruppe der basophilen Erythroblasten (E₁-E₃) als auch bei den polychromatischen Normoblasten (E₄) wurde eine postmitotische (G₁) und eine prämitotische Ruhephase (G₂) nachgewiesen. Beide Zellgruppen waren zu ca. 65% mit³H-TdR markiert (S).Unter den basophilen Erythroblasten war bei E₁ eine G₂-Phase, jedoch keine G₁-Phase nachweisbar. Demgegenüber fand sich bei E₃ eine ausgeprägte G₁-Phase, hingegen keine G₂-Phase. Bei E₂ war sowohl eine G₁-Phase als auch eine G₂-Phase erkennbar. Nach diesen Befunden stellen die Erythroblasten E₁-E₃ keine Zellgruppen mit jeweils vollständigem Generationszyklus dar, sondern sind als ein Zellkompartment zu verstehen, in dem die G₁-Phase vorwiegend durch die kleineren Zellen, die zytologisch die Merkmale der basophilen Normoblasten besitzen, und die G₂-Phase vorwiegend durch die größeren Zellen vom Typ der Proerythroblasten und Makroblasten repräsentiert wird.

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Comparative morphological and cytophotometric-autoradiographical investigation of erythropoiesis in the human

Summary In the different types of normal human nucleated red cells the stages of the cell cycle (G₁, S and G₂) were investigated by combined application of the cytophotometric determination of the DNA content (Feulgen photometry) and of autoradiographic labelling using ³H-TdR in vitro. The individual cells were identified in Pappenheim stain.In the basophilic erythroblasts (E₁-E₃) as well as in the polychromatic erythroblasts (E₄) about 62–65% of the cells were labelled (S). The unlabelled cells partly were diploid and partly were tetraploid, representing G₁ and G₂. The oxyphilic erythroblasts (E₅) mostly were diploid and unlabelled (G₁).Within the basophilic erythroblasts G₁ was demonstrated mainly in E₃, and G₂ was demonstrated mainly in E₁. In E₂, G₁ as well as G₂ were present. The results indicate that all basophilic erythroblasts belong to one cell compartment, in which G₁ is represented by the smaller cells commonly subclassified as basophilic normoblasts and G₂ is represented by the large cells usually called proerythroblasts and macroblasts.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail,Planorbarius corneus

Summary The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with ³H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.     

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time -a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift -was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonie medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 μM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.     

Cytophotometrical measurement of nuclear DNA content in some coniferous and deciduous trees

Summary The DNA content of nuclei from meristematic root tip cells of five coniferous and one deciduous tree species and, for comparison, ofVicia faba was cytophotometrically determined. The DNA values of diploid nuclei fromGinkgo biloba are approximately a quarter lower than those fromVicia faba. The nuclear DNA values of the other tree species are merely a third to a ninth part of those ofVicia faba. In three tree species, as well as diploid, we have found nuclei of different polyploid level.The reliability of different cytochemical methods, which are used for determination of the nuclear DNA content, is critically analyzed. The DNA values of the investigated tree species are discussed in connection with the evolution of the DNA content in higher plants.Dedicated to Professor Dr. F. Mechelke in honour of his 60th birthday     

Myotomal myogenesis inBombina variegata L.

Summary InBombina variegata, striated myofibrils first appear in G₂ uninucleated primary myoblasts. Multinucleated muscle fibres form later as a result of the fusion of primary myobasts with secondary myoblasts of mesenchymal origin. The nuclei of the polykaryocytes vary in size and DNA content (nuclear dimorphism). The larger nuclei of the primary myoblasts retain tetraploid quantities of DNA, whereas the smaller nuclei of the secondary myoblasts are diploid. From this we conclude that fusion can take place between cells that are in different phases of the cell cycle (G₁–G₂). Our findings are compared with those on myogenesis in other chordate species and are confronted with the current commonly accepted model of vertebrate muscle differentiation.This work is dedicated to Professor Kazimierz Sembrat on his 55-th anniversary of research workThis research was supported in part by Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland     

Normalization of the DNA content of telophase cells from wheat calli by nutrient modifications

Summary Cytophotometric analyses were conducted to determine whether the DNA content of wheat callus varied by tissue culture medium or age of callus. Wheat,Triticum aestivum L. line PCYT-20, was cultured on three variations of the Murashige and Skoog (1962) growth medium. At the end of 2, 4, 6 and 8 weeks, samples were collected and prepared for Feulgen cytophotometry. Standards for the DNA measurements were readings from 100 telophase nuclei in wheat meristematic root tips. Amounts of DNA per nucleus present in telophase cells from callus grown on single-strength MS indicated that ploidy level increased 52%, 74% and 39%, respectively, over time from 2, 4, and 6 weeks as compared to the double-strength MS medium, and 29%, 60% and 32%, respectively, when coconut water was added to the single-strength MS culture medium. The shape of the mitotically-active cells in callus was more variable than in root tips cells. Callus grown on double-strength MS medium produced more shoots than callus grown on single-strength MS. Double-strength MS medium and, to a lesser extent, additional sucrose and organic nitrogen overcame the effects of 2,4-D on DNA amplification. Improved media may reduce the somaclonal variation induced by tissue culture.     

Cytophotometric studies on the reaction kinetics of acid phosphatase in motoneurons marked by injection of Evans Blue into the soleus muscle of the mouse

Summary Evans blue was injected into the soleus muscle of albino mice in order to mark retrogradely the corresponding motoneurons of the spinal cord. Subsequently, reaction kinetics of acid phosphatase were studied in the marked nerve cells. 41–51 motoneurons per animal were counted. They are located in the dorsolateral portion of the Rexed zone IX where they form motor cell columns approximately 1.5 mm in length. After identification of the motoneurons, the enzyme reaction for acid phosphatase was performed by covering the section with a gel film containing naphthol-AS-BI-phosphate-hexazonium-pararosanilin. The formation of the azo dye was measured cytophotometrically at 520 nm. During the first 10 min of registration, a linear decrease in transmission of 0.4 per cent/min was shown. Because of the unimodal distribution of changes in transmission, the motoneurons of the soleus muscle could not be characterized as fast or slow types on the basis of the reaction kinetics of acid phosphatase.Dedicated to Professor Berta Scharrer in honor of her 70th birthdayThe author wishes to thank G. Bigdeli Azari, M. Dietrich and D. Vaihinger for skillful technical assistance