摘要:

Summary—Anglogenesis is a key step in organ development and remodeling during embryogenesis or tissue regeneration. Some pathological events such as tumor growth or diabetic retinopathy also lead to angiogenesis formation. Several molecules have already been identified as promoting anglogenesisin vivo. Whether their bioactivity is mediated by other angiogenic growth factors or not is still unclear. We identified and purified recently a new anglogenic growth factor. Its unique specificity for vascular endothelial cells led us to provisionally name it vasculotropin (VAS). We describe the biochemical properties of VAS and its biological functions. Structural data showed that VAS is related to the SIS family.In vivoVAS was recognized as an inducer of angiogenesis and vascular permeability.In vitro, despite a moderate action on proliferation, VAS strongly stimulates the cell migration. The screening of the presence of cellular receptors and VAS production showed that the cells which bind VAS do not synthesize it, whereas the cells which synthesize VAS do not bind it. Thus, VAS seems to act through a paracrine pathway. We also present data suggesting that VAS has a lymphokine activit

ISSN:0248-4900    

DOI:10.1016/0248-4900(91)90002-5

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Summary—The aim of this study was to investigate a new method to enhance the efficiency to create mammalian cell lines. Cell immortalization was achieved by intranuclear microinjection of a recombinant DNA construct composed of a constitutive promoter controlling the genes encoding immortalizing proteins; the sequences coding for the large T and small t antigens were fused downstream of regulatory elements from the vimentin gene, the activation of which characterizes the vast majority of cells growingin vitro. Data show that the efficiency of the immortalizing procedures using the SV40 early genes could be enhanced by the control elements derived from the human vimentin (HuVim) 5′ sequences that contained nucleotides −878 to +93 from the CAP site. This HuVim 830−T/t recombinant was used to create cell lines from numerous primary cultures of different origins: rabbit, porcine and human endothelial cells, rabbit and bovine epithelial cells. A set of large T‐expressing cells was derived, and these cells retained characteristics of differentiated cells: binding ofUlex europaeuslectin and synthesis of Factor VIII for human endothelial cells; network of cytokeratin for bovine oviductal cells and rabbit mamm

ISSN:0248-4900    

DOI:10.1016/0248-4900(91)90003-6

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

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 di

ISSN:0248-4900    

DOI:10.1016/0248-4900(91)90004-7

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Summary—Expression of the c‐fosproto‐oncogene by ovine conceptuses was analyzed by Northern and slot blots and indirect immunohistofluorescence in relation to the expression of the embryonic interferon‐α (oTP) during implantation. c‐foswas expressed initially in the trophoblast, and then in the allantois, when this tissue began to develop (day 17). In the embryonic tissues, the c‐fosproto‐oncogene was weakly expressed up to day 22 and increased thereafter. In the trophoblast, the expression of c‐fosproto‐oncogene was transient, occurring when the oTP gene was transcribed at a maximal level at the beginning of implantation (days 14–15), and decreased thereafter, following the pattern of oTP gene expression. This decline is due essentially to the arrest of c‐fosand oTP gene expression by the trophoblastic cells which established cellular contacts with the uterine epithelium during th

ISSN:0248-4900    

DOI:10.1016/0248-4900(91)90005-8

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Summary—In an attempt to identify cell surface molecules involved in recognition phenomena between cells such as keratinocytes and melanocytes and putatively target biological responses modifiers to keratinocytes, we undertook the detection of cell surface sugar specific receptors: membrane lectins. Keratinocyte membrane lectins were found to bind synthetic glycoproteins (neoglycoproteins) carrying either α‐l‐fucosyl or α‐l‐rhamnosyl residues. Fluorescence microscopy observations indicate that cultured keratinocytes are able to bind these two neoglycoproteins while frozen sections of human skin labelled with neoglycoprotein‐coated covaspheres show that the selectivity of the binding to keratinocytes is restricted to α‐l‐rhamnosyl‐BSA. Keratinocytes were adapted to grow on collagen; harvesting conditions allowing the analysis of keratinocytes by flow cytometry are described. This technique allows the quantification of the binding at 4°C, and the estimation of the endocytosis of F‐, neoglycoproteins: F‐, α‐l‐Rha‐BSA and F‐, α‐l‐Fuc‐BSA were efficiently internalized. Thereafter, α‐l‐rhamnose‐substituted liposomes containing 5‐(6)carboxyfluorescein were prepared in order to follow the delivery of the fluorescent dye into cells. This was measured both by flow cytometry and by spectrofluorimetry. The expression of surface lectins was checked upon action of cytokines (IL1α, IL1β, IL2and TNF) which are kno

ISSN:0248-4900    

DOI:10.1016/0248-4900(91)90006-9

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Summary—The ability of second messengers to modulate receptor‐mediated endocytosis was studied on isolated rat hepatocytes. A 20‐min preincubation with vasopressin was used as a modulation. We observed a 20% inactivation of both surface and intracellular receptors, with no change in the affinity of those remaining active. The internalization and dissociation of a synchronous wave of ligand was not affected, but its degradation was partially inhibited. Our observations suggest that second messengers such as intracellular calcium and diacylglycerol play a complex role in the intracellular trafficking associated with endocy

ISSN:0248-4900    

DOI:10.1016/0248-4900(91)90007-A

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Summary—Bovine tracheal gland (BTG) cells in culture show an epithelial‐fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum‐free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number. The maintenance of the epithelial serous phenotype of BTG cells during the first passages (1 to 4) suggests that the study of the regulation of these gland cells should be limited to these early pas

ISSN:0248-4900    

DOI:10.1016/0248-4900(91)90008-B

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Summary—In rat parotid gland,3H‐protein secretion is stimulated by β‐adrenergic receptor activation (via cAMP) and also by cholinergic receptor activation (via IP3, calcium and diacylglycerol). The disorganization of microfilament system by cytochalasin D induced an inhibition of β‐adrenergic induced3H‐protein secretion whereas it did not modify the cholinergic muscarinic one. Cytochalasin D induced the formation of vacuoles in the parotid cell. In this work we show that the activation of muscarinic receptors (with carbachol) partially abolished the inhibitory effect of cytochalasin D on β‐adrenergic induced secretion. Since carbachol induced both intracellular calcium increase and protein kinase C activation, we decided to test separately the effect of calcium (using the calcium ionophore A23187) and protein kinase C activation (using phorbol ester) on the inhibitory effect of cytochalasin D on β‐adrenergic induced secretion. A23187, in the presence of calcium in the external medium was able to partially abolish cytochalasin D effect (iere‐establishing protein secretion) whereas activation of protein kinase C by phorbol 12–13 di‐butyrate had no effect. These results suggest that protein kinase C is not involved in re‐establishing a ‘normal’ secretion phenomenom whereas calcium does interfere. Furthermore, our fluorescence study shows that, when cytochalasin D is present in the incubation medium, the actin network is disturbed even in the presence of arbachol. This indicates that a calcium entry in the cell is not sufficient to restore a ‘normal’ actin network. Taken altogether, these results show that the total reorganization of the previously disorganized actin network is not necessary to restore β‐adrenergic secretion since carbachol is able to partially abolish the inhibitory effect of cytochalasin D on protein secretion whereas it has no effect on the effect of cytochalasin D on the actin network. However, this study confirms that, in rat parotid gland, the actin network plays an important role in

ISSN:0248-4900    

DOI:10.1016/0248-4900(91)90009-C

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Summary—A two‐fold increase in the amount of phospholipids was observed inBabesia divergensinfected human red blood cells.In vitroincubation with [32P]‐phosphorus and [3H]‐glycerol demonstrated thatB divergenshas the ability to synthesize the phospholipid backbone. On the other hand, the low incorporation of [14C]acetate indicated the absence of ade novofatty acid synthesis and suggested the necessity of an exogenous lipid source for the parasite. Several intra‐erythrocytic growth cycles ofB divergenscould be achievedin vitro, using a serum‐free medium supplemented only with fractions of human high density lipoproteins (HDL). At an HDL concentration of 0.5 mg/ml (protein concentration) and with a 1% starting parasitaemia, parasite growth was similar to that observed under standard culture conditions with 10% human serum, at least for the first 24 h, a time equivalent to three parasite erythrocytic life‐cycles. Lipid transfer from HDL to the intra‐erythrocytic parasites was demonstrated by uptake and exchange of fluorescent NBD‐phosphatidylcholine (NBD‐PC) loaded HDL at different temperatures. Kinetic experiments with [3H]‐oleyl‐PC‐loaded HDL demonstrated a unidirectional transfer of lipids from radiolabelled HDL to the parasite; partial conversion of PC to phosphatidylethanolamine (PE) was also observed. In the semi‐defined medium, the HDL fraction appeared to be the major source of lipids for the growth ofB div

ISSN:0248-4900    

DOI:10.1016/0248-4900(91)90010-K

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Summary—Autoradiographic and immunofluorescent techniques have been used to analyse the relationship between puffing, transcription and occurrence of DNA/RNA hybrids inD melanogastersalivary gland chromosome 2L. Experiments of3H‐uridine incorporation have indicated that similar rates of RNA synthesis are observable in well developed puffs as well as in some diffuse bands and interbands. On the other hand, puffs of similar size incorporate3H‐uridine at quite different rates. The presence of RNA polymerase II seems to follow a coincident pattern with that of3H‐uridine incorporation. Our results indicate that the rate of transcription does not determine either the formation of a puff or its potential size. Instead, we have found a positive correlation between the amount of DNA/RNA hybrids and puff size, independently of the transcription rates. Transient accumulation of transcribed RNAs in their chromosomal compartment could therefore play a relevant role in the determination of pu

ISSN:0248-4900    

DOI:10.1016/0248-4900(91)90011-B

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY