Claude Palévody

Noninvasive fluorine-19 NMR study of fluoropyrimidine metabolism in cell cultures of human pancreatic and colon adenocarcinoma.

SummaryFluorine-19 NMR spectrometry was used to monitor the metabolism of two antineoplastic fluoropyrimidines, 5-fluorouracil (5FU) and 5′-deoxy-5-fluorouridine (5′dFUrd), in cell cultures of human pancreatic (Capan-1) and colon (HT-29) adenocarcinoma. The preliminary results showed, for the two tumor cell lines treated with 5FU, the presence in nonperfused cells of three signals corresponding to intracellular metabolites: 5FU, F-nucleotides and F-nucleosides. When the cells were perfused only the signals of F-nucleotides and 5FU were present. The F-nucleosides observed during the analysis of the nonperfused cells came from the conversion of F-nucleotides. During the NMR recording of Capan-1 cells at 37 °C the first metabolite of the catabolic pathway of 5FU, 5,6-dihydro-5-fluorouracil, occurred. At the beginning of the NMR recording of Capan-1 cells treated with 5′dFUrd, two signals corresponding to F-nucleotides and F-nucleosides (consistent with 5′dFUrd) were observed; during the analysis, a supplementary signal corresponding to 5FU appeared. Even after pretreatment with methotrexate the signal of 5FU incorporated into RNA was not detected. Our experiments, performed in attempts to observe the signal of the ternary complex between thymidylate synthetase (TS), 5-fluoro-2′-deoxyuridine-5′-monophosphate (FdUMP) and 5,10-methylene-tetrahydrofolate (5,10-CH2FH4), allowed detection in some cases of a broad signal, whose chemical shift was similar to that reported in the literature following incubation of TS with FdUMP and 5,10-CH2FH4, but our results were not always reproducible.

Modifications ultrastructurales des glandes vésiculaires des Machilidae (Insecta, Thysanura) au cours des cycles de mue

SummaryDuring the period between apolysis and ecdysis, the vesicular glands show many important transformations which affect not only the cuticular ductules, but all the cells. The cytoplasm of the glandular cells undergoes a partial autolysis, whereas other parts of the cells present a high secretory activity. Immediately after the apolysis the cellular reservoir empties and disappears almost completely; soon after, refills with secretion. The most interesting transformations concern each ciliary cell, always associated with a glandular cell. In the first phase of the moulting cycle, the dendrite of the ciliary cell grows a ciliumlike extension (= distal region of the dendrite), which penetrates into the corresponding ductule; the new intima of this ductule is laid around the cilium. At the same time, the proximal region of the dendrite forms a circular fold around the base of the cilium and begins to secrete a material which will form the end apparatus. This latter is finished during the second phase of the cycle. The third phase is characterized by the degeneration of the distal region of the dendrite and the circular fold. Thus, the end apparatus is not a secretion of the ductule-carrying cell, but of the ciliary cell. At the end of the moulting period, just before ecdysis, the vesicular gland again takes the structure characteristic of the intermoult: the reservoir of the glandular cell is very large; the cuticular apparatus is almost formed; the dendrite of the ciliary cells shows, at its apex, a short “cilium” (= ciliary region s. str. + short distal region) surrounded by microvilli, free in the secretion of the reservoir.

Expression and characterization of alkaline phosphatases during differentiation of human pancreatic cancer (Capan‐1) cells in culture

Summary— Human pancreatic cells of the Capan‐1 cell line differentiate in culture. During the exponential growth phase, the cells are undifferentiated, only becoming differentiated during the stationary phase. The formation of domes in this phase is related to the exchange of water and electrolytes. The present study was designed to characterize the localization and expression of alkaline phosphatases (AP) in Capan‐1 cells during growth in culture. Biochemical, cytoenzymatic and immunocytochemical methods were employed combined with light and electron microscopic examination. AP essentially of the placental type were expressed progressively during the exponential growth phase, and were seen to be distributed over the surface of the Capan‐1 cells. In the stationary phase, the AP became localized on the surface of microvilli. The precipitates of the enzyme reaction highlighted regular four‐boided structures. Biochemical assays showed a progressive increase in activity of this enzyme in cells during both the exponential and stationary growth phases. However, in the stationary phase between days 7 and 8, there was a fall in enzyme activity, with a corresponding increase in this activity in the culture medium. Cytological examination indicated that this fall could be accounted for by loss of AP‐positive membranes by vesiculization of apical microvilli and release of microvesicles into the culture medium. Immunoblots showed that Capan‐1 cells expressed two types of AP, a placental type (70 kDa) and to a lesser extent a liver type (80 kDa). Expression of the placental type was attributed to a neoplastic derepression of the coding gene, while the liver type was assumed to be a normal gene expression of human duct cells. The placental type AP might thus serve as a marker of transformation, and the liver type as a marker of differentiation.

Calcium phosphate deposits in domes of human pancreatic adenocarcinoma (Capan-1) cell cultures

Summary— Human pancreatic cells of the Capan‐1 line form domes in culture during the stationary growth stage. The domes are thought to be a result of the transport of water and electrolytes by the Capan‐1 cells. In older Capan‐1 cultures, the epithelial sheets formed thickenings from several layers of cells of which the outermost ones were joined by tight type junctions. In the intracellular space, deposits of insoluble calcium salts were observed. Culture of Capan‐1 cells in the presence of fibroblasts prolonged survival of the cultures with intact domes for more than 80 days. The Capan‐1 cells proliferated forming multilayers and closed cavities which we called super‐domes. X‐ray spectrometry and electron diffraction analysis showed that the abundant deposits inside these cavities consisted of calcium phosphate in an apatite structure. The number of these deposits increased with time in culture, and they appeared to be formed at the sites of contact with an extracellular matrix consisting of cell debris. Deposits were not observed within the culture medium. Cells from domes were stained cytochemically for ATPases and alkaline phosphatases and examined by light and electron microscopy. The Capan‐1 cells surrounding the domes were differentiated, polarized cells containing placental type alkaline phosphatases on their apical membranes and Ca2+‐ATPases on their basolateral membranes. These enzymes were thought to play a role in the accumulation of phosphate and Ca2+ ions in the dome cavities, which then formed crystals in the presence of organic compounds produced by lysis of cells of the deepest layers of the super‐domes. The crystals of hydroxyapatite observed in standard Capan‐1 cell cultures and those cocultured with fibroblasts were assumed to be a result of transepithelial transport of Ca2+ and phosphate ions by these cells.

Internalization of indium-labeled LDL through a lipid chelating anchor in human pancreatic-cancer cells as a potential radiopharmaceutical for tumor localization

Low‐density lipoproteins (LDL) labeled with indium via a lipid‐chelating agent, the bis(stearylamide) of diethylenetriaminepentaacetic acid (L), were evaluated as a potential radiopharmaceutical (111In‐L‐LDL) for tumor localization by studying their internalization in human pancreatic cancer cells (Capan‐1). Using Dil‐LDL (l,l′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine perchlorate‐LDL), this cell line was shown to bind human LDL with a high‐affinity saturable component and a low‐affinity non‐saturable (40%) component. The single saturable high‐affinity binding site had a KD of 27.5 ± 2.1 μg/ml and a maximal binding of 610 ± 7.5 ng/ml protein. Electron‐microscopic examination of the In‐L‐LDL particles revealed the peripheral distribution of the electron‐dense indium atoms at the outer surface of LDL. The modified LDL were then shown to be internalized by the cells. After conjugation of In‐L‐LDL to colloidal gold to follow the different stages of internalization, electron‐microscopic examination showed that the In‐L‐LDL gold conjugates were stuck to the external sheet of the plasma apical and microvilli membrane, into earlier and later endosomes and into multi‐vesicular bodies, suggesting the penetration of the In‐L‐LDL particles into lysosomal vacuoles. The observation of In‐L‐LDL‐gold conjugates in deep‐seated cytoplasm suggests that LDL could be employed as a drug‐transport vehicle for targeting cytotoxics or radionuclides close to the cell nucleus. Int. J. Cancer, 70:315–322, 1997.

Variations cytologiques des corps allates au cours du cycle reproducteur du Collembole Folsomia candida

Abstract In the parthenogenetic females of the Collembola, Folsomia candida , study of the nucleoplasmic ratio and fine structure of the corpora allata cells reveals an activity cycle synchronized with the ovarian cycle. The maximum cytoplasmic volume, connected with a relatively great development of the endoplasmic reticulum, is reached during the vitellogenic phase. The simultaneous control of the ovary by moulting hormones and by a corpus allatum hormone is discussed.

Organogenèse et ultrastructure des sensilles placoïdes des antennes de Diadromus pulchellus wesmael (Hymenoptera : Ichneumonidae)

Abstract The placoid sensilla (PS) of the antenna of D. pulchellus (Hymenoptera : Ichneumonidae) are interpreted as composed of 2 superposed cuticular chambers. Organogenesis of a PS during imaginal moulting reveals the existence of 30–40 neurons accompanied by glial cells (a periaxonal and a peridendritic), and 5 enveloping cells, which secrete the epicuticle of the sensillum. These cells are distributed in a trichogen type (2 cells), a tormogen type (2 cells), and an odd cell enveloping the whole. In the differentiated sensillum, the distal dendritic processes are branched and elongated into the upper chamber. The 4 internal enveloping cells form microvilli that remain in the lower chamber; the external enveloping cell has vesicular cytoplasm, and it separates the internal cells from the cuticle.

Progressive development of gap junctions during growth of human pancreatic duct cells (Capan‐1) in vitro and in vivo

Summary— Among their numerous functions, gap junctions play a crucial role in proliferation, differentiation and secretion processes, although their existence and potential role in ion secretion in human pancreatic ducts have yet to be established. To investigate the morphogenesis and the role of gap junctions in human pancreatic duct cells, the Capan‐1 cell line maintained in culture or heterotransplanted into nude mice was employed as model system. Capan‐1 cells polarize during their growth in vivo and in vitro forming duct‐like structures. Furthermore in culture, after confluence, these cells form domes, which is indicative of ion exchange processes. After treatment with tannic acid and freeze‐fracture, gap junctions were observed along the basolateral membranes of Capan‐1 cells on electron microscopic examination. The presence of alkaline phosphatases on gap junctions was demonstrated cytoenzymatically. In addition, cell‐to‐cell communication was visualized by microinjection of Lucifer yellow. During differentiation of Capan‐1 cells in culture, the frequency of intercellular communications increased markedly over the period (days 11–13) when the cells form duct‐like structures. The increase in gap junctions was demonstrated by analysis of the polarized cells organized in duct‐like structures that are commonly observed in the tumors formed by heterotransplantation of Capan‐1 cells into nude mice. Furthermore, gap junctions associated with tight junctions were also observed in the cells forming such structures. The role of gap junctions in ion exchange was evaluated by counting the number of domes in cultures treated with heptanol. Heptanol (an uncoupling agent of gap junction communication) completely inhibited dome formation in a reversible way, and reduced the frequency of intercellular communications by 44%. These results suggest that the gap junctions expressed by Capan‐1 cells are involved in ion secretion by the human cancerous pancreatic duct cell line, Capan‐1. In the present study, we show that: i) the expression of gap junctions is linked to development of the spatial conformation of the cells; and ii) gap junctions may be involved in ion secretion.

Structure et fonctionnement de la spermatheque chez l'endoparasite solitaire Diadromus pulchellus wesmael (Hymenoptera : Ichneumonidae)

Abstract Studies of the fine anatomy and ultrastructure of the spermathecae of endoparasitic females of Diadromus pulchellus Wesmael (Hymenoptera : Ichneumonidae) were made in order to understand the role of this organ in sex-ratio regulation. The spermatheca, approximately 50 μm in diameter, includes a spherical capsule and a relatively voluminous gland, each connected by a duct to the spermathecal duct that opens into the vagina. The spermatheca is enveloped by a complex muscular system. Circular muscle fibres surround the capsule and the canal. The capsule wall is made up of thick cuticle resting upon well-developed epithelial cells, containing numerous vesticles derived from the endoplasmic reticulum. The gland has the typical structure of epidermal glands but lamella-shaped lipid secretions are present. Lastly, the vaginal wall includes a zone of differentiated cells surrounding the opening of the spermathecal canal. Emphasis is placed on the role of the muscular system and the importance of glandular secretions.