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Biomedical subjects

A Scheid

Publications and source records attributed to A Scheid.

At least 37 records · Page 2Linked to original sources
PubMed1989

Histomorphological and lectin-histochemical confirmation of the antidegenerative effect of diclofenac in experimental osteoarthrosis.

The integrity of cartilage matrix depends on the homeostasis of synthetic and degradative processes. Any disturbance of the rate of synthesis and catabolism may alter the amount of matrix components (e.g. proteoglycans). Based upon a biochemically induced osteoarthrosis (OA) in the knee joints of rats we investigated the histomorphological alterations under therapy with diclofenac sodium by histological-histochemical grading. Lectin-binding techniques using labelled wheat germ agglutinin (WGA), concanavalin A (Con A), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), and peanut agglutinin (PNA) were applied to analyze the cellular as well as the extracellular glycoconjugates in situ. Lectin-binding patterns quantitatively describe the topographical localization of structural components of the cartilage matrix which carry certain sugar residues. The therapy of the experimental OA with diclofenac sodium (2.0 mg/kg s.c.) led to a marked reduction of cartilage degenerations. Our results indicate antidegenerative properties of this compound. These findings are consistent with the fluorescent analytical data which show a stimulating effect on the anabolic activity of chondrocytes in the osteoarthritic joints under the treatment with diclofenac sodium. Fluorescein isothiocyanate labelled lectins are useful histochemical tools to determine alterations in the integrity of cartilage by their specific binding patterns, because zonal differentiation in physiological function and morphological structure of cartilage tissue as well as cellular, pericellular, and interterritorial local events are characterized.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Protease activation mutants of Sendai virus: sequence analysis of the mRNA of the fusion protein (F) gene and direct identification of the cleavage-activation site.

Trypsin cleaves the fusion protein (F) of wild-type Sendai virus into two disulfide-linked polypeptides, F1 and F2, and thereby activates the membrane fusion activity of the virus. A. Scheid and P.W. Choppin [1976). Virology, 265-277) selected mutant viruses of which the F protein could be activated by different proteases, either elastase, chymotrypsin, or plasmin. Herein, we have further characterized five of these mutants. Sequencing of each mutant mRNA encoding the 60-70 amino acids surrounding the cleavage site revealed one or two amino acid changes near or at the cleavage sites. Virions cleaved in vitro by the appropriate proteases were assayed of their fusion activity by hemolysis, and the cleavage sites were determined by amino acid sequencing. In three cases, the change of protease specificity can be accounted for by changed amino acids right at the cleavage site, whereas several other mutations that potentiate cleavage at new sites by new proteases are somewhat removed from the actual cleavage site. We surmise that such mutations might alter local polypeptide conformation, thereby allowing the proteases access to existing sites. Cleavage at new sites produced fusion proteins with novel F1 NH-termini. We found that a mutant with a charged residue at the third position of this normally hydrophobic NH-terminal sequence retains activity in the hemolysis assay, whereas a mutant with a charged residue at the first position does not.

Amino Acid Sequence↗
PubMed1983

Fusion of Sendai virus with liposomes: dependence on the viral fusion protein (F) and the lipid composition of liposomes.

The characteristics of fusion of the membrane of Sendai virus with that of liposomes has been investigated using two different methods to monitor the fusion reaction. The first method, which permits quantitation of lipid fused with virus, depends on separation by centrifugation of unfused liposomes from those fused with virus. The second involves the digestion after fusion of internal viral proteins by trypsin contained in liposomes; this assay is completely independent of exchange of lipid between liposomal and viral membranes in the absence of fusion. A fusion-inactive mutant virus, pa-cl, with an uncleaved F protein served as the appropriate control in these experiments. It was found that fusion of the virus with liposomes that contained no protein required cleavage of the F protein; such cleavage was previously shown to be required for fusion of the virus with cell membranes. This indicates the relevance of this model system for studies of fusion. Kinetic studies indicated that at neutral pH fusion was 88% complete in 10 min at 37 degrees. Investigation of the effects of liposomal lipid composition indicated that the presence of cholesterol in the liposomal membrane was required for fusion; a 0.3-0.4-mole fraction of cholesterol was optimal. The presence of neuraminic acid in the membrane was not essential for fusion. The results obtained are compatible with previous evidence suggesting a hydrophobic interaction between the cleaved F protein and the target membrane during fusion.

Chemical Phenomena↗
PubMed1982

Enhancement of membrane-fusing activity of sendai virus by exposure of the virus to basic pH is correlated with a conformational change in the fusion protein.

The effect of pH on the membrane-fusion activity of Sendai virus was examined (pH 5.0-9.5) by using, as assays of activity, hemolysis of chicken erythrocytes and the fusion of baby hamster kidney (BHK-21) cells. Exposure of virus to basic pH increased fusion activity; the optimum pH was found to be approximately equal to 9.0. All assays were carried out at pH 7.0, and the virus retained enhanced fusion activity after it was exposed to basic pH and returned to neutral pH. The enhanced fusion activity was correlated with an irreversible conformational change in the fusion protein (F protein) of the virus, as demonstrated by a change in the circular dichroism spectrum of the protein.

Animals↗
PubMed1981

Activation of the Sendai virus fusion protein (f) involves a conformational change with exposure of a new hydrophobic region.

The F protein of paramyxoviruses is actively involved in the induction of membrane fusion. This fusion may be between viral and cellular membranes, as in the initiation of infection or in virus-induced lysis of erythrocytes, or between the plasma membranes of different cells. The F protein is activated by proteolytic cleavage to yield two disulfide-linked polypeptides (F1 and F2); however, its mechanism of action is not clear. In the present study, the conformations of the inactive, uncleaved precursor of glycoprotein (F0), and the active, cleaved form (F1,2) have been compared. The UV circular dichroism spectra of the two forms of the F protein indicate that cleavage results in a conformational change. Detergent-binding studies by velocity sedimentation analysis of Triton X-100-protein complexes revealed an increase in exposed hydrophobic surface of the protein on cleavage. The inactive F0 bound an estimated 27 molecules of Triton X-100/F polypeptide; these molecules are presumably bound to the hydrophobic region of the glycoprotein that anchors the spike-like protein in the virus membrane and that is common to both forms of F. The active form, F1,2, bound 67 molecules of Triton X-100. This increase in the number of detergent binding sites upon F protein activation indicates the presence of a hydrophobic region that is peculiar to the active form, and that may be of functional significance in the membrane fusion reaction.

Centrifugation, Density Gradient↗
PubMed1981

Functions of surface glycoproteins of myxoviruses and paramyxoviruses and their inhibition.

Two glycoproteins, HN and F, are present on the surface of paramyxoviruses. HN has receptor-binding amd neuraminidase activities. F is involved in viral penetration, cell fusion and haemolysis and is activated by proteolytic cleavage by a host enzyme into two disulphide-bonded subunits (f1 and F2). The ability of the virus to initiate infection and undergo multiple cycle replication depends on the presence of an activating protease in the host; thus cleavage of F is a major determinant of pathogenesis. The new N-terminus generated on F1 by cleavage is involved in biological activity, and the amino acid sequence of this region of F1 by cleavage is involved in biological activity, and the amino acid sequence of this region of F1 is hydrophobic and highly conserved among para-myxoviruses. In an attempt to design specific inhibitors, oligopeptides and analogous to this region were synthesized and found to be highly active, specific inhibitors of viral penetration, cell fusion and haemolysis. Inhibition is amino-acid-sequence-specific and affected by peptide length, steric configuration and addition of groups to the n-terminal and C-terminal amino acids. Replication of influenza virus was also specifically inhibited by oligopeptides resembling the N-terminus of the HA2 polypeptide. Like that of F1 protein the N-terminus of HA2 is generated by a proteolytic cleavage that activates infectivity. These results have provided information on the action of proteins in viral penetration and membrane fusion and they suggest a possible new approach to chemical inhibition of viral replication. Studies with specific antibodies to each of the paramyxovirus glycoproteins have shown that antibodies to the F protein are essential for effective prevention of the spread of infection. Antibodies to the HN protein, although capable of neutralizing released virus, do not prevent spread to adjacent cells through membrane fusion mediated by the F protein. These findings have implications for the design of effective vaccines against paramyxoviruses and also provided additional insight into the mechanisms involved in the atypical and severe infections observed in individuals who received inactivated paramyxovirus vaccines and were later infected.

Amino Acid Sequence↗
PubMed1981

The functions and inhibition of the membrane glycoproteins of paramyxoviruses and myxoviruses and the role of the measles virus M protein in subacute sclerosing panencephalitis.

The F glycoprotein of paramyxoviruses is responsible for cell fusion and hemolysis and for virus penetration via fusion of viral and cell membranes. These functions are activated by specific proteolytic cleavage of an inactive precursor (F0) into two disulfide-linked polypeptides (F1 and F2). The susceptibility of the F0 protein to cleavage by a host protease is a major determinant of virus host range and virulence. Synthetic oligopeptides that mimic the N-terminal region of the F1 polypeptide are specific inhibitors of paramyxoviruses, and oligopeptides that mimic the N-terminus of the HA2 polypeptide of influenza virus, also generated by cleavage, specifically inhibit that virus. Antibodies to F protein prevent the spread of paramyxovirus infection via membrane fusion, but antibodies to HN protein do not, although they neutralize released virus. These results and previous findings that formalin-treated virus does not induce antibodies to F protein provide an explanation for atypical measles. The HN protein has both receptor-binding and neuraminidase activities, and Cl- inhibition of neuraminidase may modulate these antagonistic activities. Studies in patients with subacute sclerosing panencephalitis (SSPE) suggest that there is a host restriction of synthesis of the M protein of measles virus in brain cells which is involved in the abortive, persistent infection that causes SSPE.

Antibodies, Viral↗
PubMed1980

Importance of antibodies to the fusion glycoprotein of paramyxoviruses in the prevention of spread of infection.

The effects of monospecific antibodies to the viral glycoprotein with hemagglutinating and neuraminidase activity (HN) and the viral glycoprotein with membrane-fusing activity (F) of the paramyxovirus simian virus 5 (SV5) on the spread of infection in two cell types have been investigated. In CV-1 cells, infection can spread by either released progeny virus adsorbing to and infecting other cells, or by fusion of an infected cell with an adjacent cell as a result of the cell-fusing activity of the F glycoprotein. In these cells, antibodies specific for the HN glycoprotein prevented the dissemination of infection by released infectious virus, but spread by cell fusion was not inhibited. Antibodies to the F glycoprotein completely prevented the spread of infection in these cells. In Madin-Darby bovine kidney cells, which are relatively resistant to SV5-induced fusion, antibodies to either the HN or F glycoproteins were capable of preventing the dissemination of infection. These results indicate that effective immunological prevention of the spread of paramyxovirus infection requires the presence of antibodies that inactivate the F glycoprotein. This requirement for anti-F antibodies has obvious implications for the design of effective paramyxovirus vaccines and provides an explanation for previous failures of formalin-inactivated paramyxovirus vaccines as well as additional insight into the possible immunopathological mechanisms involved in the atypical and severe infections that have occurred in individuals who received inactivated paramyxovirus vaccines and were subsequently infected by the virus.

Animals↗
PubMed1980

Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changes in salt concentration.

The conformations of the helical nucleocapsids of the paramyxoviruses Sendai virus and simian virus 5, and of a rhabdovirus, vesicular stomatitis virus, have been found to vary extensively with changes in salt concentration. In 10 mM sodium phosphate buffer at pH 7.2, the nucleocapsids are loosely coiled or almost completely extended; with increasing concentrations of NaCl they become more tightly coiled and less flexible. Under isotonic conditions (150 mM) the Sendai virus nucleocapsid is moderately tightly coiled but still curved and apparently flexible, whereas at 400 mM or higher it is very tightly coiled, with the appearance of a rigid rod. These salt-dependent changes in conformation were also found with nucleocapsids composed of proteolytically cleaved protein subunits. Because of the effect of salt concentration, and the fact that it may change during the preparation of negatively stained samples of electron microscopy, it was necessary to fix that nucleocapsids before negative staining to preserve their original conformation. The striking changes in nucleocapsid conformation in response to the ionic milieu indicate the plasticity of its helical structure and suggest that changes in the microenvironment of the nucleocapsid could influence its conformation during viral RNA transcription and replication or during virus assembly by budding, processes in which changes in the coiling of the nucleocapsid or its flexibility could be important.

Capsid↗
PubMed1980

Role of paramyxovirus glycoproteins in the interactions between viral and cell membranes.

Interactions of paramyxoviruses with cell membranes are mediated by two virus-coded glycoproteins of the virus membrane: HN and F. The HN protein is responsible for the attachment of virions to the membrane of the target cell. The F protein mediates fusion between the virus membrane and the cell membrane, and this step is essential for the infection process as well as for the expression of other biological activities of paramyxoviruses, i.e., cell fusion and hemolysis. The activity of F is dependent on the proteolytic processing of a precursor (F0) to yield two disulfide-linked subunits (F1 and F2), and unless this cleavage occurs, the virions are inactive and not infectious. Viruses differ in their susceptibility to specific proteases, as demonstrated with Sendai virus and its protease activation (pa) mutants. Findings with these viruses indicate that the host range, tissue tropism, and the ability of paramyxoviruses to spread within a host and to cause disease are dependent on the availability of an appropriate protease capable of cleaving the viral glycoprotein. Experiments with reconstituted particles that contain purified F protein and phosphatidylcholine indicate that to be active the F protein must be inserted in a lipid bilayer, and that no other viral function is needed for the membrane fusion process. Attachment of the F-protein-containing particles to the target membrane is a prerequisite for fusion; however, this function can be supplied by wheat germ agglutinin as well as by the virus protein responsible for attachment of intact virus, i.e., HN. Studies on the structure of the paramyxovirus glycoproteins indicate that they are inserted with a hydrophobic region of the molecule in the lipid bilayer of the virus membrane. Analysis of the primary structure of the F protein at the cleavage site revealed striking sequence homology among three paramyxoviruses, indicating a requirement for cleavage at a sharply defined site and the importance of a specific primary sequence for biological activity. This region is highly hydrophobic, suggesting that it may interact with the lipid biylayer of the target cell membrane during virus penetration, cell fusion, or hemolysis. Further studies of this system should provide knowledge regarding the mechanisms involved in viral penetration and in membrane fusion in general.

Amino Acid Sequence↗