Endocytosis of a small dermatan sulphate proteoglycan. Identification of binding proteins.

Endosomal preparations from human osteosarcoma cells and from fibroblasts contain 51,000- and 26,000-Mr proteins which bind a small dermatan sulphate proteoglycan after SDS/polyacrylamide-gel electrophoresis and Western blotting. Binding can be inhibited by unlabelled proteoglycan core protein. The proteins co-precipitate with a proteoglycan core protein-antibody complex. Scatchard analysis of immobilized endosomal proteins yielded a KD of about 37 nM for the proteoglycan. In intact cells proteins of the same size can be found. They are sensitive to trypsinization. A 51,000-Mr protein is the predominant membrane protein with strong binding to immobilized dermatan sulphate proteoglycan. There are additional proteoglycan-binding proteins with Mr values of around 30,000 and 14,000 which are insensitive to trypsin treatment. In contrast with the 51,000- and 26,000-Mr proteins, they resist deoxycholate/Triton X-100 extraction several days after subcultivation.     

Characterization of Cyclic AMP-Dependent Phosphorylation of Neuronal Membrane Proteins

We examined the patterns of cyclic AMP-dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 microM) for the reaction. We also found that the detergents Triton X-100 and gramicidin S enhanced cyclic AMP-dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP-dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non-neural (bovine skeletal muscle) cyclic AMP-dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non-neural regulatory subunits behaved similarly. In addition to cyclic AMP-dependent phosphorylation, the alpha-subunit of pyruvate dehydrogenase (Mr = 41,000) is phosphorylated in a cyclic AMP-independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system.     

Comparison of small proteoglycans from skin fibroblasts and vascular smooth-muscle cells.

Physicochemical and chemical properties of small proteoglycans containing galactosaminoglycan chains from cultured human skin fibroblasts and human smooth-muscle cells were compared to determine the extent of structural similarity. The proteoglycan secreted by smooth-muscle cells was of larger molecular size and of higher buoyant density, due to longer glycosaminoglycan chains, than the secretion product of skin fibroblasts. Additionally, both proteoglycans differed in the ratio of iduronic acid and glucuronic acid residues. On the other hand, degradation of secreted [3H]leucine-labelled proteoglycans with chondroitin ABC lyase followed by SDS/polyacrylamide-gel electrophoresis resulted in the appearance of core protein bands of identical size (Mr 48,000 and 45,000, depending on the number of asparagine-bound oligosaccharides). An Mr value of 40,000 was determined for the core protein of cells pretreated with tunicamycin. An antibody against the core protein from fibroblast secretions was cross-reactive with the core protein from smooth-muscle cells. Core protein accumulating intracellularly after treatment with carbonyl cyanide m-chlorophenylhydrazone exhibited, on reduction and alkylation, an isoelectric point of 7.8 in both cell types. Limited proteolysis by staphylococcal V8 serine proteinase or endoproteinase Lys-C led in both instances to the formation of peptides of identical size. Peptides bearing asparagine-bound oligosaccharides were free of glycosaminoglycan chains. Similar peptide patterns were obtained when 125I-labelled core proteins were digested with either trypsin or chymotrypsin. Thus small proteoglycans from fibroblasts and smooth-muscle cells can be differentiated by their glycosaminoglycan moieties but not by the nature of their core proteins.     

Sister chromatid exchange analysis of the 15q11 region in Prader-Willi syndrome patients

Summary Prader-Willi syndrome (PWS) is a sporadic disorder in which about half of cases have a 15q12 deletion. Although a small number of cases have other rearrangements involving 15q12, the rest of the cases appear to have normal chromosomes. Clinical similarities among all these patients regardless of the karyotype strongly suggests a common etiology. To investigate the nature of this common etiology, we analyzed sister chromatid exchange (SCE) at the 15q11-13 region in 10 PWS patients with the chromosome deletion, 12 PWS patients with normal chromosomes, and 11 normal control individuals. While SCE at the q11-13 region was absent on the 15q12 deleted chromosome, the percentage of SCE on chromosome 15 at q11 was statistically higher for PWS with normal chromosomes (10.1%) compared to that for normal controls (1.9%) and the normal homologue (2.2%) in deleted patients (²=7.7982, df=2, P<0.025). The data suggest relative instability of DNA at the 15q11 region in PWS patients.     

Identification and characterization of genes involved in excision of the Lactococcus lactis conjugative transposon Tn5276.

The 70-kb transposon Tn5276, originally detected in Lactococcus lactis NIZO R5 and carrying the genes for nisin production and sucrose fermentation, can be conjugally transferred to other L. lactis strains. Sequence analysis and complementation studies showed that the right end of Tn5276 contains two genes, designated xis and int, which are involved in excision. The 379-amino-acid int gene product shows high (up to 50%) similarity with various integrases, including that of the Tn916-related conjugative transposons. The xis gene product, like almost all known excisionase (Xis) proteins, is a small (68-residue), basic protein. Expression of both the Tn5276 int and xis genes is required for efficient excision of the ends of Tn5276 in Escherichia coli that appeared to be circularized in the excision process. Mutational analysis of the xis and int genes showed that excision efficiency is dependent on the integrity of the int gene but that an intact xis gene is also required for efficient excision.     

Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

Distribution of negatively charged liposomes on rat liver hepatocytes and non-hepatocytes in cell suspension

The in vitro interactions between negatively charged multilamellar liposomes and purified rat liver parenchymal and non-parenchymal cells were studied. The liposomes were labelled with [¹⁴C]cholesterol and contained [³H]methotrexate. For both cell types the time course of liposomal attachment to the cells slowed down gradually after a rapid initial phase lasting ca 90 min. The rate of attachment at 4 °C was 3–7 times lower than that at 37 °C, and the metabolic inhibitors dinitrophenol and iodoacetic acid caused reduction of 20–30%. Up to 45% of the cell-associated liposomal radioactivity could be detached within 1 h incubation with unlabelled liposomes. Whereas liver parenchymal cell suspension seemed to exhibit similar characteristics in vitro as in vivo, the non-parenchymal cells in vitro showed a 20–50-fold reduction in the rate of liposomal attachment compared to in vivo.