Jacques Panijel

Immunochemical characterization of polyribonucleotides.

The degree of organization of polyribonucleotides determines their modalities of reaction with antibodies (NG-I) which are present in serums of animals hyperimmunized with ribosomes. The immunochemical behavior of the highly helical two-stranded complex of polyadenylic acid and polyuridylic acid and the corresponding three-stranded complex of one molecule of polyadenylic acid and two molecules of polyuridylic acid can be determined from examination of the associatinge and nonassociating mixtures of polyadenylic and polyuridylic acids. The immunochemical characteristics of various forms of polyinosinic acid in solution are described.

Correlation between the injection of specific proteins in starfishes and the appearance of binding sites for the same proteins in cell populations of their axial organ

Abstract Sea stars were injected with two different proteins (bovine serum albumin and whale myoglobin). Cell suspensions were prepared from the axial organ of injected and untreated animals. After exposure of these suspensions to soybean agglutinin (SBA), agglutinable and nonagglutinable cell subpopulations were obtained and compared as to their binding to fluorescent conjugates of the same proteins: FITC-serum albumin and FITC-myoglobin. The SBA-agglutinable subpopulation from injected animals showed a significantly higher number of membrane fluorescent cells than the nonagglutinable subpopulation. No significant membrane fluorescence was observed in the cell subpopulations obtained from untreated animals. The membrane fluorescence appeared mainly when the protein of the fluorescent conjugate was the same as that used for injecting the animal.

Immunochimie des polyribonucléotides: I. Homopolymères à une ou plusieurs chaînes

Abstract Immunochemistry of polyribonucleotides. I. Single or multiple chain homopolymers Anti-RNA antibodies NG 1 were prepared in solutions containing no electrolytes. Precipitation reactions of the polyribonucleotides were performed in media of very low ionic strength or in saline solutions of varying concentrations. At zero ionic strength, all the polyribonucleotides precipitated equally, which shows that the nature of the base does not determine, neither qualitatively nor quantitatively, the precipitation reaction. When the concentration of electrolytes was increased, the immunochemical behaviour varied according the level of organization of the polyribonucleotides used. In the case of poly (U), it was seen that the immunochemical characteristics are linked to its randomly coiled conformation. On the other hand, the modifications in behaviour of poly (A) or poly (C) at neutral pH are attributed to intramolecular organization. Finally, this method of immunochemical analysis allowed us to show that poly (I) in solution takes on a variable number of strands, depending on the concentration and nature of the electrolytes used.

In vitro study of IgM polymerization

Abstract The polymerization of 7S IgMs from normal rabbit lymphoid cells, stimulated either with antigen or with mitogen (Con A), has been studied. The process was analyzed by characterizing the various molecular forms by sucrose gradient sedimentation and susceptibility to anti-μ serum and 2-mercaptoethanol. It has been shown that native J chain and an enzyme are both required for the proper assembly of IgM pentamer. The enzyme preparation (PMF) is active only if it is extracted from spleen cells stimulated to IgM production. When the extract is prepared from nonstimulated lymphoid cells, or from liver cells, incubation of IgMs with PMF does not lead to the formation of 19S IgM, but to molecules of intermediate size and to various aggregates. It is shown that antibody activity of IgMs and of these heterogeneous polymers are not susceptible to treatment with 2-mercaptoethanol. In contrast, antibody activity of the pentameric IgM is completely inhibited by 2-mercaptoethanol. A PMF inhibitory substance was present in the postmicrosomal supernatant. When added in the incubation medium, this substance prevented the proper polymerization. Its eventual role in IgM biosynthesis in nonstimulated, and specifically stimulated cells is discussed compared with mitogen stimulated cells, and tumor lymphoid cells.

On the mechanism of early recovery of specifically depleted lymphoid cell populations by nonspecific activation of T cells.

Abstract Specific depletion from normal CBA mouse spleen cells of those bound on pigeon erythrocyte (PRBC) immunoabsorbent columns before transfer of the depleted population into irradiated syngeneic recipients resulted in elimination of the anti-PRBC responsiveness as assessed by rosette (RFC) and hemolytic plaque (PFC) formation. The anti-sheep erythrocyte (SRBC) responses of cell populations treated in the same manner remained unimpaired. When, however, these populations were stimulated with both PRBC and muramyl dipeptide (MDP), an early recovery of specific anti-PRBC responsiveness was produced. PFC response in particular, suddenly increased between the fourth and fifth day after transfer and stimulation thus exhibiting a doubling time of only 4 to 6 hr. This effect of MDP was T-cell dependent since treatment of the depleted population with anti-θ antigen serum and complement hindered early recovery. Depleted populations stimulated with PRBC alone resumed their T-dependent RFC (but not PFC) responsiveness after the eighth day. In spite of the existence of these educated T cells, a second stimulation on the tenth day with PRBC was unable to elicit a specific PFC response. On the other hand stimulation with MDP alone on the day of cell transfer (Day 0) followed by stimulation with PRBC on Day 10 resulted in a specific PFC response on Day 15. Thus, MDP appeared to do more than simply promote education of T cells by antigen. In vitro cultures of depleted populations also recovered their specific reactivity when stimulated by antigen and MDP.

In vitro induction of IgM synthesis and secretion in rabbit spleen cells by soluble concanavalin A.

Abstract Rabbit spleen cells are not activated by Concanavalin A (Con A) conjugated to Sepharose 4B but are stimulated by soluble Con A which induces DNA and protein synthesis. At optimal concentration (5 μg/ml) one notes an increased intracellular protein and IgM synthesis and then secretion. This increase in protein synthesis is seen at all phases of the culture. At the intracellular level, IgM is found in the form of 7S molecules and a significant proportion of polymers with a centrifugation constant smaller than 19S. Fully assembled 19S polymers are found in the fluid phase. These results are compatible with a model of cellular cooperation, the basis of which is not the presentation of the inducer (mitogen or antigen) by a cell type to another, but rather the secretion of mediators by one of the cell populations making other cells responsive to the inducer.

Studies on precursors of hemolytic plaque-forming cells: Effect of the specific removal of natural PFC by erythrocyte-conjugated Sepharose beads on the responsiveness of depleted spleen cells

Abstract Influenza virus particles, inactivated with formalin, have been covalently bound to cyanogen bromide-activated Sepharose beads (Se-vi beads). Preservation of the hemagglutination properties of the viral particles enabled a strong binding of pigeon or human group O erythrocytes (PRBC or HoRBC) to these Se-vi beads. The conditions for preparation of PRBC- or HoRBC-Se-vi columns are described. Spleen cell suspensions from mice immunized with the above erythrocytes were considerably depleted of cells forming hemolytic plaques (PFC) against the corresponding erythrocytes after passage through these columns. In the case of cells from nonimmunized mice, the depletion is still greater and reaches up to 95–100%. However, the number of PFC reactive to unrelated erythrocytes is not affected in the filtered population. Specifically attached cells recovered from the Se-vi-RBC columns passed with normal spleen cells are considerably enriched in the number of PFC against homologous erythrocytes. Syngeneic irradiated hosts transferred with filtered cells are able to give a normal primary PFC response against heterologous, but not against homologous RBC up to the 12th day after immunization. These results are discussed in relation to the problem of precommitment of specific PFC precursor cells.

Use of Anti-RNA Antibodies in the Study of the Structure of Polynucleotides and Ribosomal Particles

Immunization of an animal with bacterial ribosomes generally elicits the formation of antibodies capable of precipitating not only the homologous ribosomes used for immunization but also ribosomes of different origin (Barbu et al., 1961; Panijel and Barbu, 1961). However, for a given antiserum, the qualitative aspects of the immune reaction vary with the species of bacteria being tested. In addition, as shown in Figure 1 (A, B, C), the amount of antibody precipitable by a given bacterial ribosome varies with the antiserum used. It is evident that ribosomes even from distant species possess common antigenic determinants, thus explaining the cross-reactions observed. With a given antiserum, such as the E. coli K12 ribosome antiserum, one can distinguish the following: (a) homologous ribosomes, e.g., those from various strains of E. coli or even other enteric bacteria; (b) close heterologous ribosomes, e.g., ribosomes of Proteus vulgaris; and (c) distant heterologous ribosomes, e.g., ribosomes of Clostridia. Such a “classification” of ribosomes relative to a given antiserum seems to be genetically significant. Indeed, McCarthy and Bolton (1963), who used the hybridization technique to study the relationship between messenger RNAs extracted from E. coli and DNAs of other origin, subsequently arrived at a classification in agreement with ours.

Immunochimie des polyribonucléotides: II. Complexes d'homopolymères différents

Abstract The immunochemistry of polyribonucleotides. II. Complexes of different homopolymers The results of the precipitation of poly (A + U), poly (A + 2U) and poly (C + I) in 5 · 10 −4 M Mg 2+ with NG 1 (anti-RNA) antibodies are compared with theoretical curves representing the expected precipitation, in the same medium, of the corresponding non-associating mixture of polynucleotides. The formation of the poly (A + U) complex demonstrates: 1. (a) a masking of antigenic sites, as indicated by a comparison with the theoretical curves, which corresponds to no organization, 2. (b) a precipitation of nucleotide material quantitatively twice that obtained with a single strand devoid of order, and 3. (c) a continuous decrease in the ratio: antibody precipitated/antigen precipitated in excess antigen. The poly (A + 2U) complex precipitated three times as much nucleotide as a single strand. No additional masking of antigenic sites was noted. Conversely, the formation of the complex poly (C + I) brought about an unmasking of antigenic sites as compared with the theoretical curve, which is indicative of a higher level of organization due to the tetra-stranded structure of poly (I) in Mg 2+ . These various results represent a number of principles in the immunochemical analysis of polyribonucleotides which are discussed.

Action des anticorps anti-RNA sur les ribosomes et les phages

Abstract Action of anti-RNA antibodies on ribosomes and phages A study was made of the precipitation of ribosomes and phages by anti-RNA antibodies. The group of antibodies, “NG I”, capable of precipitating various RNA and polyribonucleotides, was obtained under conditions of solubility in which the ribosomes were stable. “NG I” precipitated 90–100 % of the bacterial as well as of the animal ribosomes. In contrast, these antibodies did not react with the DNA and RNA phages. This property makes it possible to obtain, without inactivation, virus preparations from which most of the subcellular contaminants are removed. The importance of these results in the structural investigation of ribosomes is discussed.