Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids

Changes in membrane properties during the differentiation process in K562 cells have been investigated. A decrease of lectin-induced agglutination has been detected. The agglutination assay revealed to be an early and sensitive test to monitor the induced differentiation of the K562 cells. Naturally occurring fluorescent fatty acids (cis- and trans-parinaric acids) and the recently developed multifrequency phase and modulation technique were used to study cell membrane properties. Changes in fluorescence lifetime and polarization are clearly associated with cell differentiation, suggesting the involvement of the cellular plasma membrane in the differentiation process.     

The effect of glyceraldehyde on red cells: Haemoglobin status,oxidative metabolism and glycolysis

Glyceraldehyde induces changes in the flux of glucose oxidised through the hexose monophosphate pathway, the concentrations of intermediates in the Embden-Meyerhoff pathway, the oxidative status of haemoglobin and levels of reduced and oxidised pyridine nucleotides and glutathione in red cells. Glyceraldehyde autoxidises in the cellular incubations, consuming oxygen and producing glyoxalase I- and II-reactive materials. Major fates of glyceraldehyde in red cells appear to be: (i) adduct formation with reduced glutathione and cellular protein; (ii) autoxidation and reaction with oxyhaemoglobin and pyridine nucleotides, and (iii) phosphorylation of d-glyceraldehyde and entry into the glycolytic pathway as glyceraldehyde 3-phosphate. The production of glycerol from glyceraldehyde by red cell l-hexonate dehydrogenase appears not to be a major reaction of glyceraldehyde in red cells. These results indicate that high concentrations of glyceraldehyde (1–50 mM) may induce oxidative stress in red cells by virtue of the spontaneous autoxidation of glyceraldehyde, forming hydrogen peroxide and α-ketoaldehydes (glyoxalase substrates). The implications of glyceraldehyde-induced oxidative stress for the in vitro anti-sickling effect of dl-glyceraldehyde and for the polyol pathway metabolism of glyceraldehyde are discussed.     

Ethylene and growth control in the fringed waterlily (Nymphoides peltata): Stimulation of cell division and interaction with buoyant tension in petioles

The effect of ethylene on petiole growth of the Fringed Waterlily (Nymphoides peltata (S.G. Gmelin) O. Kuntze) changes during leaf ontogeny. During early development (before expansion of laminae), ethylene causes an increase in both cell number and cell size; later in development, promotion of rapid cell expansion is the dominant effect. The early effects may contribute to the accommodation of new leaves to water columns of different depth. The later effects on cell expansion only are shown to contribute to the rapid accommodation of floating leaves when changes in water level submerge the laminae. This kind of accommodation results from an interaction between accumulated ethylene, which increases wall extensibility, and the tension in petioles due to natural buoyancy which, it is suggested, supplements the driving force for cell expansion. Cell age (position) within a petiole and age of the whole petiole influence the growth response to ethylene alone and the amount of extra growth produced by applying tension when ethylene is present. In young petioles, apical cells are highly sensitive to ethylene and tension causes little further growth; older cells in both immature and mature petioles show little response to ethylene unless the petiole is under tension. Young (but not mature) petioles respond slowly to applied tension even in the absence of ethylene. It is concluded that as cells age the driving force for expansion limits increasingly their capacity to respond to the wall-loosening effects of ethylene. Dual sensitivity to ethylene and buoyant tension facilitates rapid accommodation responses but sensitivity of young petioles to tension alone may exclude Nymphoides from habitats where current velocity is appreciable.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO₂: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm. This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute.     

Characterization of a stable,anchorage-dependent clone obtained from a spontaneously transformed mouse cell line

Summary A variant nontransformed clone, I21, was selected from the spontaneously transformed mouse fibroblast line, IT22. Selection was done by plating IT22 in methylcellulose and picking single cells after 2 d. Cultures derived from these single cells were selected again and one clone, I21, derived from the second round of selection was characterized extensively. I21 and IT22 have the same plating efficiency (PE) on plastic, but in agarose they differ by 1000-fold. In comparison to IT22, I21 has a normal morphological appearance, a lower saturation density, a higher viability in stationary phase, an increased doubling time, an increased chromosome content, and is unable to form tumors in nude mice. I21 has remained remarkably stable in culture and has not reverted to the transformed phenotype for at least 300 generations in culture. Over 100 clones of I21, expanded to 10⁶ cells, failed to show an increased PE in agarose. Even expansion of the rare colonies of I21 that grow in agarose failed to produce clones similar to IT22. The research was supported by the Medical Research Council and the National Cancer Institute of Canada. R. Godbout was supported by a 1967 Science Scholarship and by an MRC Studentship. B. L. Gallie is a Research Associate of the Ontario Cancer Treatment and Research Foundation.     

Comparative use of fructose and glucose in human liver and fibroblastic cell cultures

Summary The effect of fructose as a substitute for glucose in cell culture media was investigated in human skin fibroblast and liver cell cultures. Cells were grown for between 2 and 10 days in identical flasks in four different media, containing 5.5, mmol·1⁻¹ and 27.5 mmol·I⁻¹ glucose and fructose, respectively. In the presence of fructose, cell growth was stimulated, but less in liver cells than fibroblasts. At Day 6, increases were observed in [³H]thymidine incorporation, protein levels, and amino acid consumption, and a reduction was noted in ATP levels. In media containing 5.5, mmol·1⁻¹ glucose or fructose, consumption of fructose was four times lower than that of glucose at Day 3 and did not rise until Day 6. In fructose media, the lactate production was very low (four to five times less than that of glucose) and the pH values were always higher. Some findings were different for the fibroblasts and liver cells, owing to the specific characteristics of these two cell types in culture; this applied especially to the effects of glucose and fructose concentrations of 27.5 mmol·1⁻¹. Several possible explanation for the stimulation of cell growth in fructose medium were discussed. This work was supported by grants for the Institut National de la Santé et de la Recherche Médicale (ATP 82-79-114) and the Unité d'Enseignement et de Recherche, Le Kremlin-Bicêtre, Université Paris-Sud (C. R. 848).     

Inactivation of the (+) gamete agglutinin during the mating-type reaction in chlamydomonas

Sex cell contact in Chlamydomonas is due to complementary sex-specific glycoproteins (mating-type substances, MTSs). Their interaction causes an instantaneous but labile flagella agglutination between sexually different gametes. The dynamic nature of this contact permits partner exchange between agglutinated gametes and accounts for the transitoriness of the contact, flagella adhesion being terminated upon ensuing pairing. This paper describes molecular events that underlie the adhesion potential of differentiated (+) gametes. In the contact-establishing interaction with its receptors on the (?) flagella, the agglutinin of differentiated (+) gametes is inactivated. Compensating for this inactivation, the adhesion potential of gametes in agglutination is sustained by continuous replenishment of the inactivated MTS by newly synthesized units. If this glycoprotein neosynthesis is blocked by tunicamycin (TUM), the adhesiveness of differentiated (+) gametes ceases. It is postulated that this complex interaction with incapacitation and neosynthesis forms the basis of the dynamic nature of the flagella contact and eventually accounts for its termination at pairing.     

Establishment and characterization of a new human hepatocellular carcinoma cell line

Summary A human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α₁-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.     

Interrelationships between water and cellular metabolism inArtemia cysts

Cysts of the crustaceanArtemia are a useful model for studies on intracellular water because they are capable of essentially complete and reversible desiccation. We have used a variety of techniques on this system, the present work being an attempt to estimate the density of intracellular water (ρ_w). The density of individual cysts was evaluated from sedimentation velocity. Heptane displacement methods were used to determine the volume of a known mass of cysts, from which the density was calculated. The two methods produce comparable results. It was shown that the densities and water contents of large masses of cysts accurately reflect those of individual cysts. Cyst densities (ρ_c) were determined over the entire range of water content from 0 to 0.63 weight fraction of water (W _f), and temperature dependence was measured for 0.61W _f over 2–41°C. The following refer to 25°C. No marked change was detected in ρ_c until the water content exceeded 0.15W _f, at which ρ_c decreased as a linear function of Wf to maximum water content. However, the cyst does not behave ideally in the sense that the densities of the nonaqueous components and added water are not additive as a function ofW _f. The partial specific volume of water in cysts at maximum hydration was estimated to be 3% larger than that of pure water. These observations are compared with density measurements on other systems, and with previous findings on the physical properties of water in this system.     

小鼠内细胞团标志蛋白的性质、分离和纯化

我们用二相电泳方法在小鼠畸胎瘤系统中发现小鼠胚胎内细胞团标志蛋白双联体(分子量39,000,p14.4—4.5)也存在于未分化的胚胎癌细胞株(PSAI-NG2、F9、PCC3)中,但在已分化细胞株滋养层母细胞癌TDM1中用二相电泳方法不能检查到这蛋白。把EC细胞分为细胞质、细胞核、染色体三部分再进行检查,可以清楚地看到这蛋白主要存在于染色体部分。把染色体再分成0.35M NaCl可溶性,2M NaCl可溶性及2M NaCl不溶性三个组份时,则这蛋白出现在0.35M NaCl可溶性组分中,表明它是与染色体疏松结合的非组蛋白染色体蛋白。这蛋白是一种耐热性蛋白,在80℃受热10分钟不能使它变性。用制备型SDS聚丙烯酰胺凝胶电泳对经热处理的0.36M NaCl可溶性染色体蛋白进行分离,可得到电泳纯的小鼠内细胞团标志蛋白。由于这蛋白约占细胞总蛋白0.6%,与染色体疏松结合,又与早期胚胎细胞的多能性相关,提示它在维持多能细胞染色体构象方面有重要作用。