Extracellular matrix metabolism by chondrocytes 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells

THe incorporation of [³H]glycine into acid-insoluble protein and of [³H]acetate into glysoaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gardients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of poly-ribosomes that was reversed by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence on L-glutamine was also demonstrated for other avain connective tissues.     

Cleavage of proteoglycan aggregate by leucocyte elastase.

The partial degradation of proteoglycan aggregate by human leucocyte elastase yielded products that banded with Mr 190,000, 140,000, 88,000, and 71,000 when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. Analysis of these bands revealed that the 190,000- and 140,000-Da bands contained chondroitin and keratan sulfate stubs and had N-terminal amino acid sequences corresponding to a sequence starting at residue 398 of the core protein of rat or human aggrecan. With increased time of digestion, the staining intensities of the 190,000-, 140,000-, and 88,000-Da bands decreased relative to the 71,000-Da band. Analysis of the 88,000- and 71,000-Da bands showed that they contained peptides substituted only with keratan sulfate stubs and that each band contained two peptides with different N-terminal sequences. One of these corresponded to a sequence that started at residue 398 of rat or human aggrecan and the other to the N-terminal sequence of bovine aggrecan. Under conditions of complete digestion, bands of 71,000 and 56,000 Da which contained only keratan sulfate stubs were observed on SDS-polyacrylamide electrophoresis. The 71,000-Da band was shown to have a single sequence similar to that starting at residue 398 of human and rat aggrecan and thus represents the globular domain 2 (G2) of the core protein of aggrecan. The 56,000-Da band was shown to have a sequence similar to that of the N-terminal sequence of bovine aggrecan indicating that this peptide corresponds to the globular domain 1 (G1) of the molecule. These results suggest that leucocyte elastase cleaves the core protein of aggrecan between valine 397 and isoleucine 398, which are located in the interglobular domain linking the G1 and G2 domains of the core protein of aggrecan. Further digestion of the proteoglycan aggregate with elastase resulted in the cleavage of the core protein within the chondroitin sulfate attachment domains.     

A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

Gold labeling of thrombin and ultrastructural studies of thrombin-gold conjugate binding by fibrin

Monodispersed thrombin-gold (T-Au) conjugates were prepared by the absorption of a monolayer (3.8 nm thick) of human alpha-thrombin around individual monodispersed colloidal gold particles (16.5 +/- 1.8 nm). Like free molecular thrombin, T-Au conjugates can cause platelet aggregation, plasma clotting, and the release of fibrinopeptides A and B from fibrinogen. At the same thrombin concentration, T-Au conjugates have only one-tenth the fibrinogen-clotting activity of free thrombin and one-third the amidolytic activity of free thrombin. Hirudin can completely inhibit the fibrinogen-clotting activity of both T-Au conjugates and free thrombin, but can inhibit only half of the amidolytic activity of the conjugates. Diisopropyl fluorophosphonate can completely inhibit the fibrinogen-clotting activity and the amidolytic activity of both T-Au conjugates and free thrombin. T-Au conjugates were further characterized by studying the mechanism of their binding to fibrin and the location of the binding site on fibrin. The results of electron microscopic studies showed that T-Au conjugates, but not albumin-Au conjugates, are bound by fibrin. Increasing T-Au conjugate concentrations are associated with an increase in the number of T-Au conjugates binding to fibrin. At 0.1 microM thrombin, 73% of the T-Au conjugates are bound to branch points of the fibrin network with 27% of the T-Au conjugates present in the fibrin strands. At higher thrombin concentration (e.g., 0.5 microM) the percentage of T-Au conjugates bound to locations other than branch points increases to 62%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Streptococcus salivarius strains carry either fibrils or fimbriae on the cell surface

Strains of Streptococcus salivarius were screened by negative staining for the presence of surface structures. Two structural subgroups were found, carrying either fibrils or fimbriae, projecting from the cell surface. Eight strains carried a very dense peritrichous array of fibrils of two distinct lengths. Long fibrils had an average length of 175 nm, and short fibrils had an average length of 95 nm. Two strains carried only long fibrils, one strain carried only short fibrils, and another strain carried a lateral tuft of very prominent fibrils of two lengths, with a fibrillar fuzz covering the remainder of the cell surface. In all the strains in which they were present, the long fibrils were unaffected by protease or trypsin treatment. In contrast, the short fibrils were completely digested by protease and partially removed by trypsin. Neither long nor short fibrils were affected structurally by mild pepsin digestion or by lipase. The Lancefield extraction procedure removed both long and short fibrils. These twelve fibrillar strains were therefore divisible into four structural subgroups. Extracts of all the fibrillar strains reacted with group K antiserum. The second main structural subgroup consisted of nine strains of S. salivarius, all of which carried morphologically identical, flexible fimbriae arranged peritrichously over the cell surface. The fimbriae were structurally distinct from fibrils and measured 0.5 to 1.0 micron long and 3 to 4 nm wide, with an irregular outline and no obvious substructure. There was no obvious reduction in the number of fimbriae after protease or trypsin treatment. Extracts of the fimbriated strains did not react with the group K antiserum. The two serological and structural subgroups could also be distinguished by colony morphology.     

Surface structures, haemagglutination and cell surface hydrophobicity of Bacteroides fragilis strains.

Nineteen strains of Bacteroides fragilis were examined by negative staining for surface structures. One strain (ATCC 23745) possessed peritrichous fibrils, 16 strains carried peritrichous fimbriae and two strains carried no surface structures. The fimbriae had a diameter of 2.1 +/- 0.25 nm and appeared to be 'curly'. Only a small proportion (4 to 41%, depending on the strain) of cells in a population carried fimbriae or fibrils. Strain A312 Showed phase variation of fimbriae as expression of fimbriae was repressed at 20 degrees C and in early exponential phase at 37 degrees C. The fibrils on strain ATCC 23745 did not exhibit phase variation in response to changes in incubation temperature, growth phase or growth in two different media. Capsules were demonstrated by the Indian ink method on 18 of the 19 strains, varying in size from strain to strain and within the same population. Cultures often contained both capsulate and noncapsulate cells. All strains possessed an electron dense ruthenium red staining layer between 7.9 and 23.9 nm in width attached to the outer membrane. Cell surface hydrophobicity quantified by the hexadecane partition assay gave low values ranging from 6.6 to 52.1%. Only a few strains were able to haemagglutinate and these were only weakly active. There was no correlation between cell surface hydrophobicity, haemagglutinating activity and surface structures.     

The extracellular processing and catabolism of hyaluronan in cultured adult articular cartilage explants.

Hyaluronan was shown to have the same turnover time as aggrecan in explant cultures of adult bovine articular cartilage. Inclusion of fetal calf serum in the culture medium resulted in a similar decrease in the rate of catabolism of both hyaluronan and proteoglycan. Less than 9% of the hyaluronan lost from the explants in the course of the experiment was recovered from the culture medium as hyaluronan, suggesting that the catabolism of hyaluronan involves the uptake of this glycosaminoglycan by the chondrocytes. Analysis of the molecular size of the newly synthesized hyaluronan in these cultures showed that the hyaluronan was initially synthesized as large macromolecules that were gradually depolymerized with time within the extracellular matrix. The resulting size distribution of newly synthesized hyaluronan molecules after 12 days in culture was similar to that determined for the endogenous hyaluronan. The kinetics of depolymerization of the newly synthesized hyaluronan was consistent with a random fragmentation of the macromolecule. The rate constants for the depolymerization of hyaluronan indicate that oxygen-derived radicals may be involved in the fragmentation of this macromolecule. Inclusion of either cycloheximide or proteinase inhibitors in the medium of the explant cultures resulted in a marked decrease in the rate of loss of hyaluronan from the tissue and in the inhibition of the depolymerization of the newly synthesized macromolecule. This suggests that both the catabolism and the depolymerization of hyaluronan are cell mediated and depend on metabolically active cells.     

The N-terminal sequence of the large proteoglycan of articular cartilage

A peptide with hyaluronic acid-binding properties was isolated from trypsin digests of bovine articular cartilage proteoglycan aggregate. This peptide originated from the N-terminus of the proteoglycan core protein, retained its function of forming complexes with hyaluronate and link protein and contained at least one keratan sulfate chain. Amino acid sequence data demonstrated that the first six amino acid residues of the N-terminus of bovine articular cartilage proteoglycan core protein differed from the same region from the rat chondrosarcoma proteoglycan. Further sequence data indicate areas of considerable sequence homology in the hyaluronic acid-binding regions of proteoglycans from the two species.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.