Fibronectin glycosylation modulates fibroblast adhesion and spreading.

The role of the carbohydrate residues of fibronectin concerning the specificities of that glycoprotein to interact with fibroblastic cell surfaces, gelatin, and heparin was examined. Tunicamycin was used to produce carbohydrate-depleted fibronectin; it was synthesized by cultured fibroblasts. Unglycosylated and glycosylated fibronectins were analyzed for their ability to bind gelatin and heparin, using affinity columns. Fibronectin-coated surfaces were used to quantitatively measure cell adhesion and spreading. The results showed that the lack of carbohydrates significantly increased the interaction of the protein with gelatin and markedly enhanced its ability to promote adhesion and spreading of fibroblasts. In contrast, the binding of fibronectin to heparin was not influenced by glycosylation. The composite data indicate that the Asn-linked oligosaccharides of fibronectin act as modulators of biological functions of the glycoprotein.

Distribution in caesium chloride gradients of proteoglycans secreted by bovine aorta smooth muscle cells and study of their aggregation with hyaluronate and cartilage proteoglycan.

Smooth muscle cells from media of bovine aorta were cultured with [35S] sulphate and [3H]glucosamine. The 4 M guanidinium chloride extract of cell layer had a greater proportion of its glycosaminoglycans as hyaluronic acid, heparan sulphate and dermatan sulphate than did medium, which contained relatively more chondroitin sulphate. Fractionation of medium and cell layer extract by caesium chloride density gradient centrifugation, under associative and dissociative conditions, respectively, established that heparan sulphate and dermatan sulphate proteoglycans had lower buoyant densities than chondroitin sulphate proteoglycans. Chromatography on Sepharose CL2B showed that chondroitin sulphate-rich proteoglycan from medium bottom fraction contained no high molecular weight aggregate but underwent partial reaggregation with hyaluronic acid. A slight shift to higher molecular weight occurred if cartilage proteoglycan monomer was added to medium bottom fraction, but if both hyaluronic acid and cartilage proteoglycan monomer were added, a high degree of aggregation took place. These observations could be explained if medium bottom fraction contained a proteoglycan-deficient aggregate with a low molecular weight hyaluronate species. Bottom fraction from cell layer extract showed none of these properties.

Structures of the asparagine-linked sugar chains of laminin.

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-beta-galactosidase, endo-beta-N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: bi-, tri- and tetraantennary complex-type oligosaccharides; polylactosaminyl side chains containing Gal(beta 1----4)GlcNAc(beta 1----3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, alpha-galactose, beta-galactose and beta-N-acetylglucosamine; alpha-galactosyl residues linked to N-acetyllactosamine sequences; high-mannose-type oligosaccharides. These results, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.

Effect of protein deficiency on the metabolism of glycoproteins and glycosaminoglycans in albino rat skin.

1. The total content of neutral sugars in skin of the weanling albino rats kept on the protein-deficient diet was increased by about 40%; this was mainly due to the increased concentration of galactose. The content of sialic acid was increased by about 20%. The collagen nitrogen was decreased significantly, with a concomitant increase of non-collagen nitrogen. At the same time, the content of sulphated glycosaminoglycans in skin was significantly decreased and that of non-sulphated glycosaminoglycans was increased. 2. Protein-deficient diet enhanced the activities of the protein-bound carbohydrate-degrading lysosomal hydrolases, viz. cathepsin D (EC 3.4.4.23), N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) and beta-D-glucuronidase (EC 3.2.1.31) both in liver and skin. The activity of liver hyaluronidase (EC 3.2.1.35) was also increased upon limitation of protein supply. 3. The changes observed in skin were accompanied by increased concentration of the protein-bound hexoses, hexosamines and sialic acids in serum, and of hexosamine and uronic acid in urine. The serum fucose remained unchanged.

Effect of indomethacin and naproxen on collagen cross-linking.

The influence of indomethacin and naproxen on rat dermal collagen cross-linking was investigated in vivo. The analyses of skins showed that the naproxen group had a decrease in percent reversibility of neutral salt-soluble collagen gel, susceptibility of insoluble collagen to denaturing agents and pronase and alpha/beta ratio of neutral salt-soluble collagen whereas the aldehyde content was significantly increased. Indomethacin was ineffective for all these parameters. The results indicated that both the inter and intra-molecular cross-linking of collagen was increased in the naproxen treated group while indomethacin did not influence the collagen cross-linking.

Glycohydrolases and lysosomal stability in adjuvant induced arthritis.

Adjuvant induced arthritis in rats was studied by the changes in serum and urinary protein-bound carbohydrate metabolites, changes in serum and tissue lysosomal glycohydrolases and lysosomal fragility. From the second week onwards the urinary excretion of hexosamine and uronic acid is increased. Serum levels of protein bound hexose, hexosamine, sialic acid and fucose are increased significantly in both the acute and chronic phases of the disease. There is no change in the total activity of lysosomal glycohydrolases, viz., beta-glucuronidase, beta-N-acetyl glucosaminidase and cathepsin D in the tissues of liver, kidney and spleen except that of liver enzymes in the chronic phase which are elevated significantly. The free activities of lysosomal glycohydrolases investigated, viz., beta-glucuronidase, beta-N-acetyl glucosaminidase, beta-galactosidase, alpha-mannosidase and cathepsin D are increased in liver and spleen in the acute phase. The free activities of beta-glucuronidase, beta-N-acetyl glucosaminidase and cathepsin D of kidney showed no change whereas those of beta-galactosidase and alpha-mannosidase are increased. In the chronic phase of the disease the free activities of all glycohydrolases are significantly increased in all tissues. Serum glycohydrolases are significantly increased in both acute and chronic phases. Studies on lysosomal preparations showed increased fragility of lysosomes derived from liver and kidney of arthritic rats in both phases of the disease.

Behavioural rehabilitation of head injured patients.

Behavioural changes constitute the most common disturbances following head injury. They are closely related to neurological damage, neuropsychological deficits and psychosocial maladjustments In an explorative study of behavioural rehabilitation inthe native setting, 101 patients with behavioural problems were included. For each individual a treatment programme including pharmacological, behavioural and psychotherapeutic intervention was planned and executed by a team of neurosurgeon, psychiatrist psychologist and social workers. The problem of non-compliance also was studied in the same group. Improvement was good in atypical psychoses, affective psychosis, alcohol dependency, phobias, sexual dysfunctions and undue somatic concern. Irritability, amotivation and disinhibited behaviour did not register adequate improvement. The study revealed the usefulness of multi-disciplinary model, deficiencies in the present approach, the need for adapting the techniques in conformity with native needs and the reason for non-compliance.

Psychopathology of confabulations in head injury.

Confabulations observed during head injury recovery were of two types ; momentary and fantastic. Both occurred in relation to either the dysmnestic phase of early recovery or the post traumatic amnesic syndrome. In a follow-up of 174 head injured patients, all 12 patients evincing confabulations had suffered from acceleration injuries. In comparison to controls, they had a longer post traumatic amnesia period. Clinical and psychometric lateralization of the deficits pointed to left sided impairment. Their memory scores were not qualitatively or quantitatively different from those of equivalent controls. Patients differed from the controls in certain personality dimensions. Relative contribution of clinical deficits, memory impairment and personality dimensions to the occurrence of confabulations and its dynamic significance in maintaining the personal identity system of the patient are discussed.

Neuroanatomical correlates of delusions in head injury.

Twelve patients with organic delusions during recovery from head injury were studied in comparison to a control of non-deluded head injured patients. Clinical data such as duration of unconsciousness, length of post traumatic amnesia and occurrence of brain-stem signs pointed to the presence of subcortical functional disruption in these patients. Clinical and psychometric data indicated that left hemispheric functions were more impaired than those of the right. Recent concepts in the biomechanics of head injury indicated that subcortical and left sided dysfunction following head injury was significantly associated with the occurrence of delusions.

Alcohol dependence, head injury and memory impairment.

In a follow-up of 52 alcoholic head injured patients for a period of 18 months, 14 patients were found to abstain from alcohol totally. The rest resumed alcohol consumption between three and six months. Leaving out three patients with other complications. 11 patients in the abstainer group were compared with equivalent groups of persistent abusers, and non-alcoholic head injured patients, using PG1 Memory Scali. The performance of the groups ^indicated that persistent abusers wire the poorest and that abstinence were followed by welcome change in memory. Qualitative analysis of the results and their implications for the rehabilitation of the alcoholic head injured patient are discussed.

Clinical indices of head injury and memory impairment.

In a prospective follow-up of memory functions after head injury, 61 patients were tested with P.G.I. Memory Scale at the end of 18 months. Patients with acceleration injuries showed a poor performance in comparison to those with contact injuries. Memory was found to be related to indices of severity of injury, particularly post traumatic amnesia (PTA). Presence of fracture of skull or early neurological deficits was not associated with poor performance. Among contact injury patients, lateralization and location of the injury were not found to be discriminatory. Behaviour changes during follow-up were not significantly related to memory impairment.

Effect of indomethacin and naproxen on the metabolism of glycosaminoglycans.

Indomethacin and naproxen were examined regarding their effects on glycosaminoglycan metabolism in normal albino rats. The biosynthesis of sulphated glycosaminoglycans as evaluated by the uptake of [35S-]sulphate and the content of glycosaminoglycans were measured in specimens of skin, liver and kidney. The results indicated that the biosynthesis of sulphated glycosaminoglycans was significantly inhibited by both these drugs, thus reflecting their anti-inflammatory properties. The catabolism of glycosaminoglycans was followed by estimating the activities of lysosomal glycohydrolases, viz., beta-glucuronidase, beta-N-acetyl glucosaminidase and cathepsin D in liver, kidney and spleen and urinary excretion of hexosamine and uronic acid. The results, considered collectively, indicated that both indomethacin and naproxen depressed the metabolism of glycosaminoglycans.

Abnormal glycosylation of human cellular fibronectin in the presence of swainsonine.

The relationship between post-translational modifications of macromolecules and their intracellular routing is of fundamental importance. The availability of the indolizidine alkaloid, swainsonine, which interferes with glycoprotein processing, provides a new probe for studying relationships between glycosylation of proteins and their cellular routing. Using fibronectin as a model glycoprotein, we have explored the effect of swainsonine upon oligosaccharide structure, glycoprotein synthesis, and secretion. Confluent human fibroblasts were labeled with radioactive mannose or glucosamine in the presence or absence of swainsonine. Fibronectin was secreted into the medium of swainsonine-treated cultures and found to contain endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides, instead of the normal complex endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides. Pronase-released glycopeptides and hydrazine-released oligosaccharides of isolated fibronectin from the culture media were analyzed using endo-beta-N-acetylglucosaminidase H and specific exoglycosidase digestions in conjunction with calibrated gel filtration chromatography. The structure of the swainsonine-modified endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide was found to be a hybrid type, Gal beta leads to GlcNAc beta leads to Man alpha leads to [Man alpha leads to (Man alpha leads to) Man alpha leads to]Man beta leads to GlcNAc beta leads to (+/- Fuc alpha leads to)-GlcNAc. Other experiments showed that the synthesis and secretion of fibronectin were not affected by its change in glycosylation. The results also infer that swainsonine inhibits Golgi mannosidase II in intact cells, and that removal of two mannose residues by that mannosidase is not a prerequisite for addition of galactose to the existing peripheral nonreducing GlcNAc or for the addition of fucose to the innermost reducing GlcNAc. The composite results indicate that significant changes in oligosaccharide structure had little effect upon the routing and cellular release of a typical N-asparagine-linked glycoprotein.

Influence of prostaglandins E1 and E2 on collagen metabolism in adjuvant induced arthritis.

In rats with adjuvant induced arthritis, the ihnfluence of PGE1 and PGE2 on collagen metabolism was investigated after an injection of 3H-proline, both the specific and total radioactivity of 3H-ydroxyproline being determined in skin collagen fractions and in urine. In untreated animals with adjuvant induced arthritis, a decrease in the collagen synthesis accompanied by a retardation in the conversion of soluble to insoluble collagen was observed. It was also found that the catabolism of collagen is intensified. Subcutaneous administration of PGE1 (1 mg/kg body weight twice daily) to arthritic animals accelerated the conversion of soluble to insoluble collagen and inhibited the catabolism of collagen, but remained ineffective on the decreased synthesis of collagen observed in arthritic animals. PGE2 normalised on the changes in collagen metabolism - accelerated the synthesis of collagen and the conversion of soluble to insoluble collagen and inhibited the catabolism of collagen.

Swainsonine inhibits macrophage receptor-mediated uptake and degradation of a mannosyl-oligosaccharide.

Rat pulmonary macrophages were incubated in the presence of a radiolabeled mannosyl-oligosaccharide obtained from ovalbumin. Receptor-mediated endocytosis and degradation of this ligand by the cells was followed in the presence or absence of swainsonine, an inhibitor of alpha-mannosidases. The results indicated that at higher concentrations (greater than 1 microgram/ml) of swainsonine, both the internalization and degradation of the radiolabeled ligand were inhibited. At a concentration of 0.1 microgram/ml of swainsonine, only the degradation was inhibited while the uptake was unaltered. The degradation of the oligosaccharide was blocked due to the inhibition of lysosomal alpha-mannosidase. However, the inhibition of lysosomal alpha-mannosidase was reversible upon withdrawal of swainsonine.

Effect of indomethacin and naproxen on the metabolism of collagen in adjuvant-induced arthritis.

The effect of indomethacin and naproxen on collagen metabolism was studied in arthritic animals after an injection of [3H]-proline, both the specific and total radioactivity of [3H]-hydroxyproline being determined in skin collagen fractions and in urine. It was found that collagen synthesis and conversion of soluble to insoluble collagen are diminished, accompanied by an increased collagen degradation in untreated arthritic animals. Administration of indomethacin and naproxen to arthritic animals accelerated the synthesis of collagen and conversion soluble to insoluble collagen and inhibited the catabolism of collagen.

Interprotein disulfide bonding between F and G glycoproteins of human respiratory syncytial virus.

The envelope glycoprotein G, of human respiratory virus was purified by immunoaffinity chromatography using a monoclonal antibody reacting with G glycoprotein. The purified material was analyzed for its protein patterns and by western blot for its reactivity with specific monoclonal antibodies. In addition to the G specific proteins at 90 and 55 kilodalton (kDa) range, high molecular weight species were coeluted with G protein. Three high molecular weight species were noticed: one (140 kDa) reacting with fusion protein (F) monoclonal antibody and two other species (230 and 195 kDa) reacting with both fusion protein and G protein monoclonal antibodies. The protein reacting only with F monoclonal antibody consists of fusion protein dimer. Western blot and two dimensional gel electrophoretic analysis revealed that each of the other two complexes is composed of two moles of F protein and one mole of G protein. These two complexes differ in their molecular sizes depending on whether G is in the form of 90 or 55 kDa. Upon heat denaturation, fusion protein monomer (70 kDa) is released from the complex, leaving the two complexes, consisting of one mole of F protein and one mole of G protein (160 and 125 kDa species respectively). Disulfide-reducing agents are required to break the monomers of F and G complexes. These results provide a direct evidence for the presence of envelope glycoprotein complexes linked by interprotein disulfide bonding. This may have implications on the structural and functional properties of envelope glycoproteins.

Evidence that the fusion protein of respiratory syncytial virus exists as a dimer in its native form. Brief report.

The quaternary structure of respiratory syncytial virus (RSV) fusion protein has been studied. Crosslinking studies were done to stabilize the noncovalently associated proteins. These stable, heat-resistant, covalently linked complexes were analyzed by sodium dodecyl sulfate-polyacrylamide electrophoresis. In situ crosslinking studies demonstrated that the fusion protein of RSV exists as a dimer in its native form on the surface of infected cells. The purified protein was also found to be present predominantly as a dimer. In addition, the results suggest that F1 subunits may play a role in the dimerization of the fusion protein.

Mapping of a fusion related epitope of the respiratory syncytial virus fusion protein.

The region of the fusion glycoprotein of respiratory syncytial virus which reacts with a neutralizing and fusion inhibiting monoclonal antibody, was mapped using a deductive method derived from analysis of Western blot reactivity of proteolytic fragments. Reaction of the whole fusion protein was found to be so conformationally dependent, that complete digestion of the protein with a variety of proteases resulted in fragments which were not sufficiently reactive to permit mapping. For this reason, polyclonal antibodies to synthetic peptides which spanned the fusion protein sequence, were used to map the position of large peptides derived from partial digests, and these peptides were then analysed for their ability to react with the monoclonal antibody. Comparison of the peptides which were reactive with the monoclonal antibody to those which were not, identified a region of non-overlap between residues 283 and 327 in the F1 subunit of the fusion protein. Synthesis of a peptide within this region confirmed the placement of the epitope.