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.     

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.     

Variants of the anti-Müllerian hormone gene in a compound heterozygote with the persistent Müllerian duct syndrome and his family

Summary The human genome contains a large number of interspersed simple repeat sequences that are variable in length and can therefore serve as highly informative, polymorphic markers. Typing procedures include conventional multilocus and single locus probing, and polymerase chain reaction aided analysis. We have identified simple sequences in a cosmid clone stemming from the human Y chromosome and consisting of (gata)_n repeats. We have compared these with two equivalent simple repeat loci from chromosome 12. After amplifying the tandemly repeated motifs, we detected between four and eight different alleles at each of the three loci. Codominant inheritance of the alleles was established in family studies and the informativity of the simple repeat loci was determined by typing unrelated individuals. The polymorphisms are suitable for application in linkage studies, practical forensic case work, deficiency cases in paternity determination, and for studying ethnological questions. The mutational mechanisms that bring about changes in simple repeats located both on the autosomes and on the sex chromosomes, are discussed.Professor Dr. Otto Prokop (Humboldt-Universität Berlin) on the occasion of his 70th birthday     

19-Hydroxyprostaglandin B2 as an internal standard for on-line extraction-high-performance liquid chromatography analysis of lipoxygenase products

Variable recovery of polar compounds is a potential pitfall in the on-line extraction-reversed-phase HPLC analysis of lipoxygenase products. Therefore, the addition of a polar internal standard to biological samples, which permits the assessment of the recovery of the polar components in individual samples, appears to be essential. 19-Hydroxyprostaglandin (PG) B2 was found to be a suitable compound for this application. It was easily prepared from deproteinized human semen by base-catalyzed dehydration of 19-hydroxy-PGE2, and purified in a single step by reversed-phase HPLC. Similarly to PGB2, already used as internal in HPLC, 19-hydroxy-PGB2 has excellent chemical stability and carries a strong uv chromophore that enables detection at 280 nm. Most importantly, it is eluted just prior to the polar arachidonic acid metabolites 20-hydroxy- and 20-carboxy-leukotriene B4, 12-oxo-dodecatrienoic acid and the lipoxines A and B. The measurement of the ratio of PGB2 and 19-hydroxy-PGB2 used in combination as internal standards in HPLC analysis of lipoxygenase products, provides a simple and reliable way to assess sample to sample recovery of the polar components when using on-line extraction procedures.     

Phosphorylation of ribosomal proteins during meiotic maturation and following activation in starfish oocytes: its relationship with changes of intracellular pH

An increased phosphorylation of ribosomal protein S6 has been shown to be correlated with an increase of intracellular pH (pHi) and with stimulation of protein synthesis in many systems. In this research changes in ribosome phosphorylation following hormone-induced meiotic maturation and fertilization or activation by ionophore A23187 were investigated in starfish oocytes. The hormone was found to stimulate, even in the absence of external Na+, the phosphorylation on serine residues of an Mr 31,000 protein identified as S6, as well as that of an acidic Mr 47,000 protein, presumably S1, on threonine residues. Phosphorylation of ribosomes was an early consequence of hormonal stimulation and did not decrease after completion of meiotic maturation. Fertilization or activation by ionophore of prophase-arrested oocytes also stimulated ribosome phosphorylation. Only S6 was labeled in this case, but to a lesser extent than upon hormone-induced meiotic maturation. Changes in pHi were monitored with ion-specific microelectrodes throughout meiotic maturation and following either fertilization or activation. The pHi did not change before germinal vesicle breakdown (GVBD) following hormone addition, but it increased before first polar body emission. It also increased following fertilization or activation by ionophore or the microinjection of Ca-EGTA. In all cases, alkalinization did not depend on activation of an amiloride-sensitive Na+/H+ exchanger. Microinjection of an alkaline Hepes buffer or external application of ammonia, both of which increased pHi, prevented unfertilized oocytes from arresting after formation of the female pronucleus and induced chromosome cycling. Phosphorylation of S6 was still observed following fertilization or induction of maturation when pHi was decreased by external application of acetate, a treatment which suppressed the emission of polar bodies. Protein synthesis increased in prophase-arrested oocytes after fertilization or activation. It also increased after ammonia addition, although this treatment did not stimulate S6 phosphorylation.