Effect of insulin on sulfated proteoglycan synthesis in cultured smooth muscle cells from pig aorta

The effect of insulin upon proteoglycan synthesis was studied in cultured smooth muscle cells from pig aorta blocked in the G0 phase by serum deprivation. Insulin enhanced [35S]sulfate incorporation into cell layer and medium-secreted proteoglycans. The increase in incorporation of the precursor was not due to a mitogenic response by smooth muscle cells to the hormone and the specific radioactivity of proteoglycans showed that the stimulation reflected a real increase in sulfated proteoglycan synthesis. Maximal stimulation was observed, for the cell layer as well as for the medium, 40 h after the addition of 1.7 x 10(-7) M insulin and reached respectively 65 and 53%. This stimulation was about 80 and 60% of the level achieved with 10% fetal calf serum for cell layer and medium-secreted proteoglycans, respectively. The half-maximal effect was attained, for both the cell layer and the medium, in the presence of 2.1 x 10(-9) M insulin. Proteoglycans secreted into the medium, in the presence of 1.7 x 10(-8) M insulin for 40 h, showed a higher proportion of complexes (24%) than those synthesized in control medium (11%) and at least 95% of the monomers from culture treated with insulin were characterized by a smaller hydrodynamic size than those synthesized by cells maintained in control medium. This decrease in the size of proteoglycans was partly due to a decrease in the size of their glycanic chains.     

The Use of Proton Chemical Shifts to Define the Solution Structure of a Dimeric Peptide

For flexible peptides, nuclear Overhauser Effects (NOE) experiments do not provide enough information to ensure a correct definition of their solution structure. The use of distance constraints, derived from the knowledge of proton chemical shifts, is developed to restrict the number of possible conformations. In the case of flexible molecules, randomization appears as an important factor of the correct estimation of the chemical shifts from the 3D structure. The refinement of the solution structure of the highly flexible AVP-like parallel dimer is described to illustrate this process.     

Effect of dibutyryl cyclic AMP and theophylline on lipoprotein lipase secretion by human monocyte-derived macrophages

The effect of dibutyryl cyclic AMP and theophylline on lipoprotein lipase secretion was investigated after a 24 h pretreatment of human monocyte-derived macrophages. Both the effectors decreased in a dose-dependent manner the enzyme activity recovered in the culture medium. The decrease in lipoprotein lipase activity appeared to be related to reduced enzyme synthesis without apparent modification of its stability and half-life and was conversely associated with an increase of lysosomal acid hydrolase activities. This effect was reversible on removal of the nucleotide. The present findings suggest that cyclic AMP may play a role in lipoprotein lipase expression in human macrophages and therefore may participate in the regulation of lipoprotein uptake by these cells, which are strongly implicated in the atherogenic process.     

Mapping of the gene for anti-müllerian hormone to the short arm of human chromosome 19

The gene coding for human anti-Müllerian hormone (AMH) was localized to subbands p13.2----p13.3 on chromosome 19, using in situ hybridization and Southern blot analysis of a panel of man-mouse and man-hamster somatic cell hybrids.     

Extrachromosomal circular DNAs in Drosophila melanogaster: Comparison between embryos and Kc0% cells

We established the size distribution of extrachromosomal covalently closed circular DNA molecules from embryos of various Drosophila melanogaster strains and from Kc0% tissue culture cells. In embryos, more than 80% of the circular DNA molecules are smaller than 2.5 kb and all the distributions show a peak of molecules of between 200 and 400 bp. The Kc0% cell distribution differs mainly from that of embryos in that 48% of the molecules have a size between 4 and 8 kb. Correlating with this, circular molecules homologous to copia, 412 and 297 were detected only in Kc0% cells. The three tandemly repeated families containing the 5S genes, the histone genes and the 240 bp repeat of the ribosomal DNA intergenic spacer, which had previously been identified in circular DNAs from embryos, were also found in cultured cells. A fourth tandemly repeated family corresponding to the 1.688 g/cm³ satellite DNA was detected, both in embryos and Kc0% cells. It consists of circular multimeric molecules containing multiple copies of the 359 bp repeated unit. No circular DNA molecules homologous to the actin genes, the type I ribosomal DNA insertion, or the F and I transposable elements were found in embryos or Kc0% cells. Thus it appears that the extrachromosomal circular DNA molecules from embryos and from tissue culture cells differ mainly in the presence of circular copies of the copia-like transposable elements.     

Mutation of tyrosine residues 1162 and 1163 of the insulin receptor affects hormone and receptor internalization

Insulin internalization and degradation, insulin receptor internalization and recycling, as well as long term receptor down-regulation were comparatively studied in Chinese hamster ovary (CHO) cell lines, either parental or expressing the wild-type human insulin receptor (CHO.R) or a mutated receptor in which the tyrosine residues in positions 1162 and 1163 were replaced by phenylalanines (CHO.Y2). The two transfected cell lines presented very similar binding characteristics, and their pulse labeling with [35S]methionine revealed that the receptors were processed normally. As expected, the mutation of these twin tyrosines resulted in a defective insulin stimulation of both receptor kinase activity and glycogen synthesis. We now present evidence that compared to CHO.R cells, which efficiently internalized and degraded insulin, CHO.Y2 cells exhibited a marked defect in hormone internalization, leading to impaired insulin degradation. Moreover, the mutated receptors were found to be less effective than the wild-type receptors in transducing the hormone signal for receptor internalization, whereas the process of receptor recycling after internalization seemed not to be altered. In parental CHO cells, insulin induced long term receptor down-regulation, but was totally ineffective in both transfected cell lines. These results reveal that the tyrosines 1162 and 1163 in the kinase regulatory domain of the receptor beta-subunit play a pivotal role in insulin and receptor internalization.     

Evidence for a modulatory role of protein kinase C on glycosaminoglycan biosynthesis during the spontaneous differentiation of Caco-2 cells

The role of protein kinase C (PKC) in the regulation of glycosaminoglycan (GAG) sulfation was investigated during the spontaneous differentiation of Caco-2 cells. The total cellular activity of PKC as well as its subcellular distribution was examined from d 5 (non-differentiated cells) to d 15 (enterocytic differentiated cells): during this period, PKC was redistributed from the membrane to the cytosol, but the amount of PKC activity was not modified. This redistribution of PKC was concomitant with an increase in 35S-sulfate incorporation in GAG. 4-beta phorbol 12 beta-myristate, 13-alpha acetate (PMA) and 1-2 dioctanoyl-glycerol (DIC8), 2 PKC activators, decreased 35S-sulfate incorporation in GAG; by contrast, 4 alpha-phorbol 12,13 didecanoate (4 alpha-PDD), an inactive phorbol ester, proved to be ineffective. These results suggest that membrane-bound PKC which is the active form of the enzyme, may exert on GAG sulfation a modulatory role, which is gradually attenuated as Caco-2 cell differentiation progresses.     

Biosynthesis of glycosaminoglycans in the human colonic tumor cell line Caco-2: structural changes occurring with the morphological differentiation of the cells

The human colon cancer cell line Caco-2 cultured in vitro displayed morphological differentiation which was shown to be a growth-related event. We have investigated this phenomenon further in relation to the cell surface glycosaminoglycans produced by growing (5-day, i.e., prior to differentiation) and confluent (9-day, i.e., after morphological and functional differentiation) cultures. Neosynthesized [35S]glycosaminoglycans were purified on DEAE-cellulose; at confluency, they were bound more strongly to the column than the corresponding fractions from the growing cells. Analysis of Kav values of heparan sulfate and chondroitin sulfates from growing and confluent cells indicated an increase in chain length of both glycosaminoglycans in morphologically differentiated cells. Heparan sulfate was the main 35S-labeled glycosaminoglycan of the cell surface of both 5-day and 9-day cultures. Paper chromatography of the unsaturated disaccharides obtained by chondroitinase digestion showed that chondroitin sulfate chains were primarily 6-sulfated in the 2 studied extracts. Heparan sulfate chains were isolated as chondroitinase-resistant material and treated with nitrous acid. Analysis of N- and O-sulfate group-related radioactivity showed an increase in the amount of 35S-label in the form of N-sulfate groups and an increase in the O-35S-sulfation pattern in heparan sulfate from morphologically differentiated cells. Thus, the structural features of both chondroitin sulfates and heparan sulfate were significantly different when the growing cells became morphologically differentiated.     

Effect of endothelial-cell-conditioned medium on proteoglycan synthesis in cultured smooth muscle cells from pig aorta

The effect of porcine endothelial-cell-conditioned medium on proteoglycan synthesis by pig aorta smooth muscle cells was studied under serum-free conditions. Maximal stimulation of [35S]-sulfate incorporation (50%) into medium-secreted and cell layer proteoglycans was observed after 20 min and 4 h incubation, respectively. This stimulation can be explained neither by increased secretion nor by oversulfation of medium-secreted [35S]-labeled proteoglycans. Those [35S]-proteoglycans secreted (for 24 h) in the presence of endothelial cell-conditioned medium were characterized by a higher hydrodynamic size than those secreted in the presence of control medium, without modification of glycosaminoglycan chain length. Agreement between the stimulation of incorporation of [35S]-sulfate into glycanic chains (50.1%) and [14C]-serine residues associated with glycosaminoglycans (49.9%) involved an increase in the number of glycanic chains linked to protein cores. The lesser stimulation of [14C]-serine incorporation into secreted proteins (18%) suggested that stimulation of glycosaminoglycan synthesis was not the direct consequence of enhanced protein synthesis. Proteoglycan synthesis was studied in the presence of para-nitrophenyl-beta-D-xyloside. Fractionation of medium-secreted [35S]-proteoglycans and xyloside-initiated glycosaminoglycans revealed that stimulation of [35S]-glycosaminoglycan protein core acceptor for glycanic chain initiation. Our results suggest that the factor(s) secreted by endothelial cells are able to modify smooth muscle cell proteoglycan synthesis by stimulating the first step of protein core glycosylation. This stimulation was accompanied by an increase in proteoglycan hydrodynamic size.