Secretion of proteoglycans by chondrocytes. Influence of colchicine, cytochalasin B, and beta-D-xyloside.

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.     

Multiple transmitter neurons in Tritonia. I. Biochemical studies

The buccal ganglia of the marine mollusc Tritonia control a variety of movements associated with feeding, including gut motility. The buccal ganglia and gut contain a class of peptides termed small cardioactive peptides (SCPs). Cobalt backfilling of the nerve which innervates the gut stains several buccal neurons including two pairs of reidentifiable cells, B11 and B12. Both appear white under epiillumination, a characteristic of peptidergic neurons in gastropods. Enzymatic and biochemical analyses of extracts from microdissected B11 cell bodies demonstrate that this neuron contains two species of SCPs. Labeling in organ culture followed by dissection and extraction of cell bodies indicates that these peptides were synthesized in B11. One of these peptides appears to be identical to SCPB, one of two SCPs that have been sequenced. The other SCP present in these neurons is novel. Less extensive analyses of extracts of B12 somata suggest that it also contains the same SCPs. In addition to the peptides, B11 also contains large quantities of acetylcholine (ACh) as determined by a radioenzymatic assay of cell body extracts. B12 does not contain measureable ACh. The concentration of the two peptides and ACh in the B11 cytoplasm is approximately 1 mM. Neuron B11 appears to be an appropriate model system for studying the biochemical and physiological properties of multiple transmitter neurons.     

Biochemical studies on sporulation in blue-green algae. I. Glycogen accumulation.

Glycogen accumulation in vegetative cells of Anabaena sp. is demonstrated to be a light-dependent process. No glycogen accumulation is found in dark or in cultures supplemented with 10(-5) M DCMU in light. Large quantities of glycogen accumulate in cells undergoing sporulation and the amount increased with the onset of maturation of spores.     

Conformational studies of proteoglycans: theoretical studies on the conformation of heparin.

Various models proposed for heparin have been examined by a stereochemical approach involving contact distance criteria and potential energy calculations. The present study suggests that the model favored by Atkins and coworkers [Biochem. J. (1973) 135 , 729–733 and (1974) 143 , 251–252] is not stereochemically satisfactory. An alternative model has been proposed with N-acetyl-D -glucosamine and one of the uronides in the ⁴C₁ conformation and the other uronide (probably sulfated) in the ¹C₄ conformation. The observed variations in the tetrasaccharide periodicities (16.5–17.3 Å) in different crystalline modifications of heparin have been attributed to possible changes in the rotational angles about the interunit glycosidic bonds rather than a change in the pyranose ring conformation. The proposed model is also independent of the observed variation in the relative composition of uronic acid residues in heparin. These conclusions are in disagreement with those of earlier workers.     

Role of glomerular proteoglycans in IgA nephropathy

Mesangial matrix expansion is a prominent feature of the most common form of glomerulonephritis, IgA nephropathy (IgAN). To find molecular markers and improve the understanding of the disease, the gene and protein expression of proteoglycans were investigated in biopsies from IgAN patients and correlated to clinical and morphological data. We collected and microdissected renal biopsies from IgAN patients (n = 19) and from healthy kidney donors (n = 14). Patients were followed for an average time of 4 years and blood pressure was according to target guidelines. Distinct patterns of gene expression were seen in glomerular and tubulo-interstitial cells. Three of the proteoglycans investigated were found to be of special interest and upregulated in glomeruli: perlecan, decorin and biglycan. Perlecan gene expression negatively correlated to albumin excretion and progress of the disease. Abundant decorin protein expression was found in sclerotic glomeruli, but not in unaffected glomeruli from IgAN patients or in controls. Transforming growth factor beta (TGF-β), known to interact with perlecan, decorin and biglycan, were upregulated both on gene and protein level in the glomeruli. This study provides further insight into the molecular mechanisms involved in mesangial matrix expansion in IgAN. We conclude that perlecan is a possible prognostic marker for patients with IgAN. In addition, the up-regulation of biglycan and decorin, as well as TGF-β itself, indicate that regulation of TGF-β, and other profibrotic markers plays a role in IgAN pathology.     

Effect of benzyl beta-D-xyloside on the biosynthesis of chondroitin sulphate proteoglycan in cultured human monocytes.

Monocytes isolated from human blood differentiate into macrophage-like cells when maintained in vitro for 3-5 days on plastic or glass culture dishes. In the process the cells display characteristic morphological changes, and in addition, a transition in glycosaminoglycan biosynthesis, from the production of chondroitin 4-sulphate to the formation of a polysaccharide containing 20% 4,6-disulphated disaccharide units [Kolset, Kjellén, Seljelid & Lindahl (1983) Biochem. J. 210, 661-667]. Cells were incubated with inorganic [35S]sulphate on day 1 or day 6 in culture, in the presence or absence of benzyl beta-D-xyloside, and labelled polysaccharide was isolated from the culture medium. In the presence of xyloside, the secretion of proteoglycans (90% galactosaminoglycan) was inhibited in a dose-dependent fashion and replaced by release of single polysaccharide chains, the size of which decreased with increasing dose of xyloside. The single polysaccharide chains produced on day 6 in the presence of 0.5 mM-xyloside showed the same proportion of disulphated disaccharide units as did the corresponding control material. Day-1 polysaccharide contained negligible amounts of this component, irrespective of the presence or absence of xyloside. It is concluded that the regulatory mechanism that induces 'oversulphation' during the differentiation process operates independently of any association between the polysaccharide chains and the core protein. Moreover, cells maintained in the presence of 0.5 mM-xyloside throughout a 6-day culture period showed the same morphological change, indicative of differentiation into macrophage-like cells, as did untreated control cells. The xyloside did not significantly affect the cytotoxicity of the monocytes, or of the differentiated macrophage-like cells, toward tumour cells.     

Biochemical studies on bovine adenovirus type 3. I. Purification and properties.

Bovine adenovirus type 3 (BAV3) was purified and its properties were studied. On productive infection of CKT1 cells (a cell line derived from calf kidney) with BAV3, it was observed that viral DNA synthesis was initiated after about 24 h and its rate was maximal after about 40 h. Maturation of the virus occurred several hours after this. Purified BAV3 was separated into four discrete bands by CsCl density gradient centrifugation (complete, incomplete, empty, and degraded viruses). The complete BAV3 was similar in size and structure to human and avian adenoviruses. Polyacrylamide gel electrophoresis showed that the complete BAV3 virion contained at least 10 polypeptides. The total structural proteins of the virion had a similar amino acid composition to those of human adenoviruses. DNA of the complete virus was a linear duplex and its contour length was 12.3 +/- 0.9 mum. The So20,w value of the DNA was 32.9S and its buoyant density in CsCl was 1.717 g/ml. There was about 25% homology between the DNAs of BAV3 and human adenovirus type 5 by filter hybridization. It was also noted that BAV3 produced incomplete virus. The incomplete virus was similar in morphology to the complete virus and contained almost all the structural polypeptides of the latter, but lacked infectivity. However, its DNA had a deletion(s) (13%) which seemed to locate near a terminal.     

Biochemical studies on cell fusion. I. Lipid composition of fusion- resistant cells

A series of stable cell mutants of mouse fibroblasts were previously isolated (Roos, D. S. and R. L. Davidson, 1980, Somatic Cell Genet., 6:381-390) that exhibit varying degrees of resistance to the fusion-inducing effect of polyethylene glycol (PEG), but are morphologically similar to the parental cells from which they were derived. Biochemical analysis of these mutant cell lines has revealed differences in whole cell lipid composition which are directly correlated with their susceptibility to fusion. Fusion-resistant cells contain elevated levels of neutral lipids, particularly triglycerides and an unusual ether-linked lipid, O-alkyl, diacylglycerol. This ether lipid is increased approximately 35-fold over parental cells in the most highly PEG-resistant cell line. Fusion-resistant cells also contain more highly saturated fatty acyl chains (ratio of saturated to polyunsaturated fatty acids [S/P ratio] approximately 4:1) than the parental line (S/P ratio approximately 1:1). Cells which are intermediate in their resistance to PEG have ether lipid and fatty acid composition which is intermediate between the parental cells and the most fusion-resistant mutants. In a related communication (Roos, D. S. and P. W. Choppin, 1985, J. Cell. Biol., 100:1591-1598) evidence is presented that alteration of lipid content can predictably control the fusion response of these cells.     

Ultrastructural architecture of proteoglycans in the glomerular basement membrane. A cytochemical approach

Rat kidneys were perfused with fixative solutions containing either a) a polycationic dye (Alcian blue 8 GX, Astra blue 6 GLL, cuprolinic blue, ruthenium red), b) a monocationic dye (safranine 0), or c) Alcian blue in the presence of a 0.3 M MgCl2 concentration. Whereas solutions of a revealed the glomerular basement membrane proteoglycans as particles or threads 60 nm apart and arranged in a reticular pattern, solutions of b and c demonstrated new morphological aspects of these molecules. They appeared as tiny filamentous structures, about 100 to 160 nm long, ordered in a network-like pattern with a mesh of about 60-nm width. The filaments displayed lateral branches about 20 nm apart and about 25 nm long, projecting within the meshes. We suggest that the filamentous structures are the protein core, and the branches are the glycosaminoglycans of proteoglycan molecules. Because of this arrangement the negatively charged sites of the glomerular basement membrane would lie closer to each other than previously assumed.     

Biochemical and genetic studies on rabbit hemoglobin. I. Electrophoretic polymorphism of theβ chain

A genetic polymorphism of beta-chain rabbit (Oryctolagus cuniculus) hemoglobin is demonstrated by means of acid starch gel electrophoresis. The biochemical evidence presented suggests that a previously reported substitution of a neutral amino acid for a histidine is responsible for the detected genetic variation. Segregation analysis was performed in a sample of 15 matings with 49 offspring and confirmed the genetic hypothesis: two common alleles at an autosomal locus. The calculated gene frequencies in a random sample of 125 individuals are HBB*1 = 0.48 and HBB*2 = 0.52.     

Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. I. Biochemical characterization and electron transport of Ascaris microsomes.

Two subcellular fraction, P-1 and P-2, were isolated by differential centrifugation from 0.25 M sucrose muscle homogenates of the parasitic roundworm, Ascaris lumbricoides suum. Morphological studies indicated that P-1 fraction consisted of intact mitochondria, whereas P-2 fraction consisted almost exclusively of vesicular components. The difference spectrum of Ascaris microsomes showed a characteristic b-type cytochrome spectrum with three distinct absorption peaks at 560, 525, and 424 nm. However, the alpha-peak at 560 nm was asymmetric with a shoulder at 555 nm. This microsomal b-type cytochrome was reduced by NADH, which was inhibited by rotenone and HgCl2. The reduced b-type cytochrome was easily reoxidized by shaking. NADH-oxidase activity observed in Ascaris microsomes was inhibited by rotenone, but not by KCN, NaN3, and antimycin A. On the other hand, NADH-cytochrome c and NADH-neotetrazolium (NT) reductase activities in Ascaris microsomes were not inhibited by antimycin A and rotenone, but were inhibited by HgCl2. Further observations indicated that neither HgCl2 nor rotenone inhibited Ascaris microsomal NADH-ferricyanide (FC) reductase activity, but rabbit antibody prepared against the purified NADH-FC reductase inhibited the NADH-cytochrome c reductase activity, the reduction of b-type cytochrome and the NADH-oxidase activity, as well as microsomal NADH-FC reductase activity.     

Analysis of perinotochordal materials. I. Studies on proteoglycans synthesis

Perinotochordal proteoglycans have been shown to influence somite chondrogenic differentiation. However, information concerning the composition of the proteoglycan molecules synthesized by the notochord, or the exact type of molecule necessary for the induction of somite chondrogenesis is not known. The results of the present study indicate that the proteoglycan extracted from the 8 day old notochord culture consists of predominantly small proteoglycans, while the large aggregates form less than 30% of the total. The chondroitin sulfate composition also shows a cartilage type of proteoglycan molecules synthesized by the notochord.