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Biomedical subjects

J Rousseaux

Publications and source records attributed to J Rousseaux.

At least 37 records · Page 2Linked to original sources
PubMed1988

Nuclear protein transitions in cuttle-fish spermiogenesis: immunocytochemical localization of a protein specific for the spermatid stage.

The changes in basic nuclear proteins throughout cuttle-fish spermiogenesis were investigated both by immunocytochemical procedures and by isolation of late spermatid nuclei (by virtue of their resistance to sonication). Antibodies were raised in rabbits to a protein, named protein T, isolated from testis chromatin. The anti-protein T immune serum was found to recognize protein T and not histones from the testis. Immunoperoxidase staining of sections or of smears of testis with anti-protein T antibodies showed that protein T appears in the nuclei of round spermatids, is abundant in elongating spermatid nuclei, but cannot be detected in elongated spermatids. Nuclei from these elongated spermatids were isolated by sonication treatment of testis cells. A protein, named protein Sp, with the characteristic mobility of a protamine, was isolated from elongated spermatid nuclei. This protein has the same mobility as the protamine present in mature spermatozoa. Taken together, the results indicate that in cuttle-fish, nuclear protein transitions involve the replacement of histones by a spermatid-specific protein (protein T), which is replaced at the end of elongation of the nucleus by a protamine (protein Sp). Thus, spermiogenesis of the cuttle-fish (and perhaps of other cephalopods), shows two basic nuclear protein transitions, which are similar to the transitions observed in higher vertebrates such as mammals.

Animals↗
PubMed1987

Evidence for an increased glycation of IgG in diabetic patients.

The levels of non-enzymatically glycated total plasma proteins, albumin and IgG were determined in diabetic and non-diabetic patients using affinity chromatography on boronate-agarose gels. A significant increase in both glycated albumin and IgG, and in total glycated plasma proteins was demonstrated. A good correlation between glycated albumin and glycated hemoglobin was observed, while the correlation between glycated IgG and glycated albumin (or hemoglobin) was lower. This was found probably related to wide variations of glycated IgG for diabetics patients who have high glycated hemoglobin levels. These variations were inversely correlated with total serum IgG. Determination of fructosamine or 5-hydroxymethylfurfural content of IgG showed a significant increase in diabetics. Glycated IgG was found to contain approximately one mole of glucose. The main site of glycation was determined to be present in the Fab (more precisely the Fd) part of the IgG molecule.

Blood Glucose↗
PubMed1987

Studies of the IgE binding sites to rat mast cell receptor with proteolytic fragments and with a monoclonal antibody directed against epsilon heavy chain: evidence that the combining sites are located in the C epsilon 3 domain.

The binding sites of rat IgE to mast cell receptor were investigated by the use of proteolytic fragments and a monoclonal antibody to epsilon chain (MARE-1). Three main fragments were characterized by short-time papain digestion of IgE: F(ab')2-E, a fragment related to the C, 4 domain, and an asymmetric fragment corresponding probably to an IgE molecule with one proteolyzed C, 3 domain. Neither F(ab')2-E nor C, 4 could interfere with the binding of IgE to rat mast cells. These two fragments did not show significant polymerization upon heating at 56 degrees C, while large amounts of polymers were produced from whole IgE, MARE-1 monoclonal antibody was found to react neither with F(ab')2 nor with C, 4, thereby suggesting its interaction with the C, 3 domain. MARE-1 was found to inhibit partially (about 55%) the binding of IgE to its receptor. Taken together the results indicate that the binding sites of IgE to rat mast cell receptor are located within the C, 3 domain. In addition, isolation of the C, 4 domain will be useful to evaluate its participation in the affinity of IgE to receptors of other cells such as lymphocytes or macrophages.

Animals↗
PubMed1984

Isolation of two molecular populations of human complement factor H by hydrophobic affinity chromatography.

Human complement factor H was prepared in highly purified form from fresh serum by euglobulin precipitation, DEAE-Sephacel chromatography and Sephacryl S-300 gel filtration. This preparation allowed the recovery of 37% of the initial factor H. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that factor H was homogeneous both in reduced and non-reduced media and exhibited a molecular mass of 150 kDa. Charge-shift experiments clearly showed the presence of hydrophobic sites in the factor H molecule. Charge shifts were observed with two detergent systems (Triton/sodium deoxycholate and Triton/cetyltrimethylammonium bromide). Factor H was able to bind to phenyl-Sepharose. This property allowed us to study two populations of factor H. These two populations exhibited the same physicochemical parameters, but revealed differences in their ability to aggregate in low- and iso-ionic-strength media. The molecular basis and biological significance of this heterogeneity are discussed.

Amino Acid Sequence↗
PubMed1984

Differential reduction of the inter-chain disulfide bonds of rat immunoglobulin E: relation to biological activity.

Monoclonal rat IgE was reduced over a range of dithiothreitol (DTT) concns. The number of disulfide bonds reduced and their location in the IgE molecule were studied. One millimolar DTT was found to split the two inter-heavy-chain disulfide bonds of the C epsilon 2 domain while increasing DTT concn to 10 mM split the two inter-heavy-light-chain disulfide bridges. Therefore, the sensitivities to reduction of disulfide bonds in rat IgE were found to be the opposite of those in human IgE. In addition, the results indicated the absence, in rat IgE, of the intra-epsilon-chain labile disulfide bond of the C epsilon 1 domain, which is reduced by 2 mM DTT in human IgE. Circular dichroism studies showed significant modifications, mainly of tertiary structure, for rat IgE reduced with 10 mM DTT, but not for IgE reduced with 1 mM DTT. The ability to block passive sensitization with reaginic antibody was not modified when IgE was reduced with 1 mM DTT (which split the two inter-heavy-chain disulfide bonds), but was lost when inter-heavy-light-chain bridges were reduced with 10 mM DTT. In addition, a non-covalent epsilon-chain dimer was found to have the same blocking activity as native IgE (or IgE reduced with 1 mM DTT). Therefore, the results suggest that reduction of most or all the inter-chain disulfide bonds, in rat as in human IgE, induces changes in quaternary structure, more especially in the relationship between the Fab and Fc parts of the molecule, leading to steric blockade, by Fab, of the binding sites for mast cells present on Fc.

Amino Acid Sequence↗
PubMed1983

Optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal IgG of different rat IgG subclasses.

The optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal rat IgG of different subclasses are described. Digestion of IgG for 2-4 h at 37 degrees C with 1% (w/w) papaïn at pH 7.0 in the presence of 0.01 M cysteine leads to almost complete cleavage into Fab and Fc fragments. Fab fragments are isolated by sequential chromatography on Ultrogel AcA 44 and on DEAE-cellulose columns. In the case of IgG2c subclass, Fab fragment may be directly isolated by chromatography on Protein A-Sepharose. Production of F(ab')2 fragments from rat IgG1 and IgG2a is obtained with best yield by treatment at acid pH (pH 2.8) before incubation with 1% (w/w) pepsin at pH 4.5 for 4 h at 37 degrees C. For monoclonal IgG2b the best procedure is incubation with S. aureus V8 protease at pH 7.8 (4 h at 37 degrees C with an E/S ratio of 1/30 (w/w]. The best yield of F(ab')2 from monoclonal IgG2c is obtained by incubation for 4 h at 37 degrees C with 1% (w/w) pepsin. F(ab')2 fragments (or the F(ab)2-like fragment released by digestion of IgG2b with S. aureus V8 protease) are isolated by gel filtration on Ultrogel AcA 44.

Animals↗
PubMed1983

Proteolysis of rat IgG subclasses by Staphylococcus aureus V8 proteinase.

Monoclonal IgG belonging to the four rat IgG subclasses (IgG1, IgG2a, IgG2b, IgG2c) and some IgG subclasses from normal rat serum were subjected to enzymatic degradation with Staphylococcus aureus V8 proteinase. The results show that only one subclass, IgG2b, is significantly cleaved by the enzyme, with the release of two main products identified as F(ab)2 and Fc-like fragments. This unique susceptibility of the IgG2b subclass represents therefore an easy means of identification and also offers a simple procedure for a preparation of F(ab)2 fragments from monoclonal IgG2b antibodies.

Animals↗
PubMed1983

Formation of biologically inactive polymers is responsible for the thermal inactivation of rat IgE.

The structural changes induced by heating rat IgE at 56 degrees C and relationship with loss of cytotropic activity were examinated in the present study. Circular dichroism spectrum of IgE heated at 56 degrees C showed irreversible changes in the peptide bond spectral regions: increase in beta-sheet structure, but no significant modifications in the aromatic side chain region. Thus, circular dichroism studies did not suggest important perturbations of the tertiary structure of the IgE molecule. Parallel studies with F(ab')2-epsilon fragment did not show significant alterations of either peptide bond or aromatic side chain spectral regions. Analysis of IgE heated at 56 degrees C by polyacrylamide gradient gel electrophoresis showed the presence of large amounts of polymeric material. Polymerization of IgE was found to increase with time of heating at 56 degrees C and to depend on protein concentration; polymerization was decreased at temperatures lower than 56 degrees C. A relationship between loss of cytotropic activity and the proportion of polymeric material in the heated IgE solutions was observed. Isolated polymeric molecules produced by heating showed considerable decrease in cytotropic activity whereas monomer isolated from heated IgE was found biologically active. The ability to form polymers is an intrinsic property of the carboxy-terminal domains C epsilon 3 and C epsilon 4, as the F(ab')2-epsilon fragment did not polymerize upon heating at 56 degrees C. A model of thermal inactivation of rat IgE is proposed in which aggregation of the carboxy-terminal domains of the epsilon-chain does not allow interaction of these domains with the monovalent IgE receptor of mast cells.

Animals↗
PubMed1982

Studies of an abnormal serum albumin unstable upon storage.

A case of inherited bisalbuminemia was discovered in which electrophoretic controls showed that the proportion of the abnormal albumin decreased progressively during storage of the serum either at 4 degrees C or at room temperature. Such a decrease was also found when the serum was incubated with the proteolytic enzymes trypsin or plasmin. Studies with the isolated abnormal albumin showed that either during storage or after incubation with trypsin (or plasmin), its mobility became identical to that of normal serum albumin. Structural analyses showed that the albumin variant was identical to the previously described pro-albumin Christchurch that contains an additional N-terminal sequence: Arg-Gly-Val-Phe-Arg-Gln. It was therefore suggested that the progressive decrease of the abnormal albumin during storage by serum was related to the cleavage of the N-terminal abnormal sequence by plasmin already present in the serum. The decrease of the abnormal albumin during storage was inhibited by addition of cortisol to serum (1 mol per mol serum albumin). This was found related to a protective effect of cortisol bound to the albumin variant on proteolysis by plasmin.

Adolescent↗
PubMed1982

Comparative study of the conformational features of rat immunoglobulin G subclasses by circular dichroism.

Four subclasses of rat immunoglobulin G and some of their fragments were studied by circular dichroism. These studies were carried out in different conditions in order to determine whether conformational differences exist between each subclass. The results showed that in their native state the subclasses cannot be distinguished by characteristic differences. Moreover, differences between monoclonal immunoglobulins G of the same subclass were found, particularly in the 200-250 nm region, and their relationship to aromatic side chain contributions (especially those of tyrosine) has been investigated. However, the conformations of the immunoglobulin G were found to be differently affected by denaturing treatments and a relation to subclass has been demonstrated.

Animals↗
PubMed1982

Conformational studies of rat monoclonal immunoglobulin E.

Two monoclonal IgEs (IR 2 and IR 162) were studied in terms of their conformational features by circular dichroism and differential u.v. absorption. The studies were performed both on the native proteins and under different conditions known to affect the biological properties of IgE (heating at 56 degrees C, acid pH, and presence of reducing agents). Whereas IgE IR 162 appeared to undergo significant conformational changes upon heating and at acid pH, IgE IR 2 was found to be unaffected or less affected by these treatments. The two monoclonal IgEs were found to be equally effective in blocking passive sensitization by IgE antibodies. However, the blocking capacity of IgE IR 162 was affected much more by heating at 56 degrees C than that of IgE IR 2.

Animals↗
PubMed1982

Deleted IgG1 and IgG2 H chains in a patient with an IgG subclass imbalance.

The serum of a female patient studied over 7 months initially showed, in addition to normal residual IgG, two abnormal IgG proteins and in the last 3 months of the disease showed only one abnormal IgG protein. Gm typing and serological subclass determinations revealed an imbalance of allelic forms within the IgG1 subclass during the disease. The IgG2 level remained markedly elevated throughout the study. The two abnormal IgG-related proteins were devoid of light chains. The abnormal gamma cathodic immunoglobulin and the abnormal beta 2-immunoglobulin were Fc-like and covalently disulphide linked, with molecular weights of 60,000 daltons (N terminal, SER) and 72,000 daltons (N terminal, GLY) respectively. The first belonged to the IgG1 subclass and the second to the IgG2 subclass. No abnormal proteolytic activity was noted and plasma cells reacted with anti-gamma-chain antisera only. We hypothesize that the molecular defect leading to the deleted chains was an early event, preceding the differentiation into plasma cells which produced the two IgG1 and IgG2 deleted H chains.

Chromatography, Gel↗