REUTILIZATION OF LYMPHOCYTE DNA BY CELLS OF INTESTINAL CRYPTS AND REGENERATING LIVER

Lymphoid cells from mice injected 54 hours and 30 hours earlier with ³H-thymidine were washed and transfused into isogenic recipients at 29 to 30 hours after partial hepatectomy. The recipients were killed 28 to 30 hours later, and liver, intestine, Peyer''s patch, spleen, and the transfused cells were examined in autoradiographs exposed 6 months. Approximately 80 per cent of the labeled transfused cells were classed as lymphocytes. The labeled DNA contained in the transfused cells was partitioned to about 14 times as many recipient liver and intestinal cells, appearing in 72 to 78 per cent of hepatocyte nuclei, in 30 to 35 per cent of liver reticuloendothelial nuclei, and in 90 to 95 per cent of intestinal crypt nuclei. The label was not comparably widespread in the lymphoid organs, but was limited to a few intensely labeled lymphocytes and a somewhat larger number of very weakly labeled cells. When heat-killed cells rather than living cells were transfused, intensely labeled lymphocytes were absent from the lymphoid organs, but the labeling of cells in the recipients was otherwise identical. The results suggest that (a) reutilized DNA is derived from dead cells, (b) reutilized DNA is mainly degraded to nucleosides and nucleotides, the usual immediate de novo DNA precursors, before reincorporation into DNA, and (c) DNA reutilization may occur in the lymphoid organs, but on a less active scale than in intestine or regenerating liver.     

UPTAKE AND TRANSFER OF PARTICULATE MATTER FROM THE PERITONEAL CAVITY OF THE RAT

1. Colloidal mercuric sulfide or thorium dioxide injected intraperitoneally passes into the cytoplasm of the mesothelium of the mesentery and of the diaphragm as early as 15 to 30 minutes after the injection. 2. Between 15 minutes and 12½ hours the number of particles within the mesothelial cells increases as the time between injection and termination of the experiment is lengthened. 3. The particulate matter is usually localized in the cytoplasm within clear vacuoles or bodies having a relatively dense matrix. 4. A greater quantity of the absorbed material is commonly observed within the cytoplasm of the diaphragmatic than of the mesenteric mesothelium.     

THE METABOLISM OF LABELLED ETHANOLAMINE IN NEURONAL AND GLIAL CELLS OF THE RABBIT IN VIVO1

Abstract— Adult rabbits were injected intraventricularly with [¹⁴C]ethanolamine and the incorporation of the base into the phosphatidylethanolamine and ethanolamine plasmalogen (and their water-soluble precursors) of isolated neuronal and glial cells was investigated. All the radioactivity was incorporated into the base moiety of the ethanolamine lipids for the time intervals examined in both types of cells. In neurons, maximum labelling of the two ethanolamine lipids occurred at 7 h after administration, whereas the highest specific radioactivity for glial phosphatidylethanolamine and ethanolamine plasmalogen was reached at 20 and 36 h, respectively. The two lipids had a faster turnover in neurons than in glia, and in both populations incorporated the base at a faster rate than did whole brain tissue. The maximum incorporation rates for phosphorylethanolamine and CDP-ethanolamine were reached in both types of cell at about 6 h after administration but the content of radioactivity per unit protein for phosphorylethanolamine was much higher in glial than in neuronal cells. It is concluded that the site of most active synthesis of ethanolamine phospholipids in vivo is the neuronal cell, with a possible transfer of intact lipid molecule to the glial compartment.     

棉酚对大鼠前列腺细胞增殖的影响

我们从体内及体外二个方面研究了棉酚对大鼠前列腺细胞的影响。在体内研究中,给成年SD大鼠口服棉酚对其前列腺作组织学观察;在体外研究中,将棉酚溶液直接加入培养系统中,对包括组织学结构、细胞生长速度、DNA合成状态及细胞分裂周期等多项指标进行分析。实验结果指出,在形态学上,经棉酚处理的大鼠前列腺的体积及重量均下降,与对照组有明显差异;二组前列腺的腺泡在组织学结构上也有明显不同:对照组前列腺的腺泡中充满突出的褶襞,由饱满的立柱形的上皮细胞构成这些褶襞及腺泡壁;而在实验组的腺泡中褶襞较少,构成褶襞及腺泡壁的为方形或扁方形的细胞。而腔内具有褶襞的腺泡总量实验组低于对照组约14%。根据体外实验的结果,可见随着棉酚浓度的增加,细胞增殖水平和DNA合成水平也相应下降,而呈剂量及时间的相关效应,其中10μg/ml的剂量能引起最大的抑制作用。根据对细胞分裂周期的分析,在棉酚组细胞进入S期的比例仅为全部细胞的31%,而对照组则为41%,这进一步说明了由于棉酚阻碍细胞进入S期从而抑制了细胞的增殖。     

毛喉萜对小鼠骨髓细胞和腹腔巨噬细胞表面胰岛素受体和GM－CSF受体的下调作用

放射性标记受体分析表明毛喉萜（ＦＳＫ）可以降低小鼠骨央细胞和腹腔巨噬细胞表面的胰岛素（ＩＮＳ）和粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）受体数目,而且对ＧＭ－ＣＳＦ的作用有剂量和时间依赖性,经ＦＳＫ处理的巨噬细胞酸性磷酸酶活性增加,微丝更加舒展。     

THE FINE STRUCTURE OF THE CELLS IN MOUSE SARCOMA 37 ASCITIC FLUIDS

The tumour cells and the reaction cells in Sarcoma 37 ascitic fluids have been studied in thin sections with the electron microscope. The reaction cells were either leucocytes or much larger acidophilic peritoneal cells of the same dimensions as the tumour cells; the peritoneal cells formed as much as 20 per cent of the large cell population. The fine structure of the cells is described and some new observations recorded. It has been found that the cell membrane of eosinophil granulocytes has a laminated composition and the characteristic granules of these cells a double limiting membrane. The pores in the double nuclear membrane of the peritoneal cells have been observed to have a fine line running across them. In the tumour cells, a rounded granular body with a central dense area has been found in the region of the centrosome; these cells were also seen to contain rows of parallel smooth surfaced cisternae lying 150 mµ apart similar to those hitherto only observed in spermatids. There was a feltwork of fine filaments in the cytoplasm of the centrosome region of the tumour cells. The cytoplasmic fine structure underlying the basophilia of the tumour cells and the acidophilia of the peritoneal cells is compared and discussed.     

COLONY-FORMING ABILITY OF BONE MARROW CELLS OF W ANAEMIC MICE ON MACROPHAGE LAYER FORMED IN PERITONEAL CAVITY OF MICE

The colony-forming ability of haematopoietic cells of W anaemic mice was examined on the macrophage layer formed in the peritoneal cavity of mice. Bone marrow cells of W anaemic mice formed a considerable number of colonies on the macrophage layer, notwithstanding they did not form any colonies in the spleen of the same recipients. As the colony-forming ability of the bone marrow cells was not reduced by the incubation with ³H-thymidine, most of the cells which formed colonies on the macrophage layer seemed to stay in G₀ state. The interrelationship between the spleen colony-forming cells, the macrophage-layer colony-forming cells, and in vitro colony-forming cells was discussed.     

DISTRIBUTION AND TURNOVER OF PHOSPHATE COMPOUNDS IN GROWING CHLORELLA CELLS

1. Using the Chlorella cells which had been uniformly labeled with³²P, the distribution of phosphorus in various fractions ofcell material was investigated. Uniformly ³²P-labeled Chlorellawas further grown in a P-free medium or in a standard "cold"medium, and the change of distribution of ³²P (as well as theuptake of exogenous P) in various cell fractions was followed.
2. Analysis of the ³²P-labeled algal cells showed that the highestin P-content was the fraction of RNA followed by those of polyphosphates,lipid, nucleotidic labile phosphate compounds, DNA and protein(in decreasing order). ATP and ADP were found to be only minorfractions of the total labile phosphates.
3. On incubating the³P-labeled alga in a P-free medium, the P.contentsin the fractionsof DNA, protein, lipid and ATP increased, thosein polyphosphatesand ADP decreased, and that in RNA remainedalmost unchanged.When the ³²P-labeled alga was further grownin the normal "cold"medium, DNA and protein increased withthe expenditure of endogenous³²P, but with practically no incorporationof external P. Inthe meantime the P in polyphosphates decreasedconsiderably,and the RNA fraction incorporated a large amountof externalP but only a little of endogenous³²P.
4. It was inferred that,under the experimental conditions of thepresent study, thephosphorus used in the syntheses of DNA andprotein was primarilytaken from polyphosphates, while thatused in the synthesesof RNA, phospholipid and polyphosphateswas, for the most part,taken from the extracellular P-source.

¹A part of this paper was read at the Vth International Congressof Biochemistry, Moscow, August 10–16, 1961. (Received June 4, 1961; )     

PERITONEAL EXUDATE MONONUCLEAR PHAGOCYTE COLONY-FORMING CELLS: THEIR PROLIFERATIVE STATE IN VIVO AND SENSITIVITY TO CYTOTOXIC AGENTS

We investigated the proliferative state of peritoneal exudate macrophage colony-forming cells (PE-CFC) by exposing them to [³H]thymidine with a high specific activity in vitro and by administering cytosine arabinoside and methotrexate in vivo. Since [³H]TdR had no appreciable killing effect on PE-CFC and since the drug treatment caused no reduction in the appearance of PE-CFC, we concluded that the majority of PE-CFC, if not all, are not in active cell cycle. The production of PE-CFC was, however, suppressed by the administration of nitrogen mustard, cyclophosphamide, BCNU and vinblastine.     

AUTORADIOGRAPHIC DETECTION OF DNA POLYMERASE CONTAINING NUCLEI IN SARCOMA 180 ASCITES CELLS

A technique has been developed allowing the autoradiographic detection of incorporation of ³H-thymidine-5'-triphosphate (³H-TTP) into nuclear DNA of smears of Sarcoma-180 (S-180) mouse ascites tumors under the direction of the cell's own nuclear DNA polymerase. Dried smears are dipped into an agar solution, which strips cytoplasm from the nuclei, and are then air dried and incubated with a buffered mixture containing four nucleotide triphosphates (one labeled), Mg⁺⁺, and Ficoll, with the cell's own DNA acting as primer. The incorporation of ³H-TTP into the nuclei, like the cell free DNA polymerase assay, is largely dependent on the presence of all four nucleotide triphosphates and Mg⁺⁺ and produces a product which is DNase sensitive and RNase resistant.
DNA polymerase activity, as studied in a cell free assay, decreases with tumor age. This correlates well with a decreasing ³H-TTP labeling index in autoradiographs of aging tumors. The ³H-TTP labeling index has also been shown to exceed but parallel the in vivo ³H-thymidine (³H-TdR) pulse labeling index for all tumor ages examined.
In at least some cell systems DNA polymerase seems characteristic of cells in cycle. The autoradiographic detection of nuclei containing the enzyme offers a new tool for the study of tumor cytokinetics.     

A SELECTIVE LETHAL EFFECT OF PENICILLIN ON SARCOMA CELLS GROWING WITH NORMAL TISSUE IN ROLLER TUBE CULTURES

An agent present in pharmaceutical Squibb and in Reichel penicillin preparations was found to exert a selective lethal effect upon rat and mouse sarcoma cells growing with normal cells in tissue cultures.     

卵巢癌细胞与腹膜间皮细胞相互作用对VEGF和bFGF表达的影响

目的探讨卵巢癌细胞与腹膜间皮细胞(HPMC)相互作用对血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)表达的影响。方法用Millicell将卵巢癌细胞SKOV3与HPMC进行非接触性共培养,用RT-PCR检测细胞VEGF及bFGF mRNA表达,ELISA检测细胞条件培养液中VEGF及bFGF蛋白水平。结果共培养后,SK-OV3 VEGF及bFGF mRNA表达分别为3.62±0.23及3.41±0.25,条件培养液中VEGF及bFGF蛋白水平分别为(523.5±24.9)pg/ml及(156.4±17.3)pg/ml,与SKOV3单独培养时相比,差别均有显著性(P<0.01)。HPMC共培养后VEGF及bFGF mRNA表达分别为3.96±0.09及3.54±0.21,条件培养液中VEGF及bFGF蛋白水平分别为(1567.62±45.42)pg/ml及(682.9±33.7)pg/ml,均明显高于HPMC单独培养时的水平(P<0.01)。结论卵巢癌细胞与HPMC均可合成VEGF及bFGF;二者共培养时,相互刺激表达更高的VEGF及bFGF。     

CYTOCHEMICAL LOCALIZATION OF ADENOSINE TRIPHOSPHATASE IN THE MITOTIC APPARATUS OF HELA AND SARCOMA 180 TISSUE CULTURE CELLS

ATPase was localized in distinct regions of the mitotic apparatus of HeLa and Sarcoma 180 tissue culture cells. ATPase was demonstrated in the metaphase spindle of HeLa and Sarcoma 180 cells fixed in cold buffered 2 per cent formalin (pH 6.5 to 6.8) containing 2 x 10^-3 M CaCl₂. A high concentration of ATPase was frequently observed at the poles of the spindle. ATPase was also demonstrated in the interzonal region of both cell types during anaphase. The narrowing of the band of ATPase activity localized in the interzonal region during telophase indicates that ATPase activity is associated with the central spindle. In polar views of Sarcoma 180 cells fixed in cold, unbuffered, 2 per cent formalin, ATPase was frequently localized in granules in the region of the inner circumference of the ring of chromosomes formed at metaphase. ATPase in the mitotic apparatus of HeLa and Sarcoma 180 cells was shown not to be due to non-specific alkaline phosphatase. Mitotic apparatus ATPase in Sarcoma 180 cells was suppressed by an —SH inhibitor.     

成年猴雪旺细胞的在体增殖和体外迁移的研究

为了探讨成年猴雪旺氏细胞的在体增殖和体外迁移的能力,我们对用神经结扎术结扎的A组6只3-13岁雄性恒河猴的腓肠神经进行植块培养,部分细胞培养在聚酯纤维上,2-4周后作抗S-100抗体免疫组化染色和电镜观察;B组2只未做结扎的新生猴腓肠神经培养作为对照.结果显示A组雪旺氏细胞平均在培养的第5天从神经段中迁出,年幼者早于成年猴;细胞在纤维上以螺旋状向前迁移;雪旺氏细胞抗S-100蛋白抗体染色阳性;电镜显示,雪旺氏细胞包卷纤维,但是,未见髓鞘形成.B组神经段培养2周仍无雪旺氏细胞迁出.研究表明,结扎神经使其发生瓦勒氏变性,经植块培养、纯化,能够获得可用于移植的成年猴的雪旺氏细胞.     

RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS

The incorporation of fucose-³H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-³H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-³H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-³H. At 3–5 min following fucose-³H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.