Murine spleen lymphocytes bearing receptors for peanut agglutinin: VII. Separation and functional characterization

Murine spleen lymphocytes having receptors for peanut agglutinin (PNA) were visualized by a specific rosetting technique. The number of lymphocytes forming PNA rosettes (PNA_R⁺) is dependent on the PNA concentration used. T lymphocytes are the primary PNA-rosetting lymphocytes for low PNA concentration (2.5 μg/ml), while both T and B lymphocytes are involved in high PNA concentration (10 μg/ml). Functional studies show that when spleen lymphocytes are separated at a low PNA concentration, the PNA_R⁺ do not respond to T mitogens and suppress antigen-specific immune responses. However, when spleen cells are separated at a high PNA concentration, the PNA_R⁺ do not exhibit differential functional properties as compared to non-PNA-rosetting lymphocytes. The implication of these findings is discussed.     

Conductimetric study of the binding of Mn2+ to bovine pancreatic deoxyribonuclease

The binding of Mn2+ on bovine pancreatic deoxyribonuclease has been studied by a conductimetric method. At low ionic strengths, a high-affinity single binding site is demonstrated. The association constant value (K = 1.2 x 10(5) M-1 at pH 8) is high enough to conclude that, in standard experimental conditions for DNA hydrolysis, the reacting species is the DNAase-cation complex. Competitive binding studies in presence of Mg++ and Ca++ show that these cations do not bind on the Mn++ site.     

In vitro spleen cell responsiveness to various analogs of MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine), a synthetic immunoadjuvant, in MDP high-responder mice

N-Acetylmuramyl-l-alanyl-d-isoglutamine (MDP), a synthetic immunoadjuvant, was incubated with spleen cells of DBA/2 or Balb/c mice and optimal responses were obtained after 4 or 5 days of culture in a serum-free medium supplemented with 2-mercaptoethanol. In contrast, lymphocytes of (C57B1/6 × AKR)F₁ hybrids responded weakly under the same conditions. The results reported here show that like in the case of DBA/2 and Balb/c strains, spleen cells of Swiss mice and of inbred AKR and CBA mice could be stimulated in vitro whereas C57B1/6 and LPS-refractory C3H/He mice did not respond. Fourteen synthetic MDP analogs (eight known to be adjuvant active and six devoid of activity) were tested in DBA/2 high-responder mice. A good correlation was observed between in vitro stimulation and the presence or absence of adjuvant activity in vivo of these compounds.     

Changes in calmodulin level after fertilization and during first cleavage in the egg of the urodelan amphibian Pleurodeles waltlii

We report three significant calmodulin rises related to Pleurodeles waltlii egg fertilization and following developmental events. These elevations are correlated to the major obvious Ca2+-dependent events: Na+-H+ exchange, activation of NAD kinase, triggering of cortical reaction, resumption of meiotic division II, initiation of DNA synthesis and regulation of cell division. Therefore, it is suggested that alterations in calmodulin level in fertilized egg may be part of the Ca2+-dependent regulatory mechanisms which turn on metabolisms, initiate development and govern cell cleavages.     

The tight junctions of rabbit choroid plexus and ciliary body epithelia. A comparative study using the double replica freeze-fracture technique

The spatial arrangement of tight junctions in choroid plexus and ciliary body rabbit epithelia has been determined by studying freeze-fracture complementary replicas. In the choroid plexus epithelium, the interruptions of the junctional P-face fibrils were measured to be 14% of their total length. In the ciliary body epithelium, where the fibrils were found to be more fragmented than in the choroid plexus, the P-face fibril interruptions accounted for 12 % of the total length of the zonulae occludentes sealing the non-pigmented cells and 30% in the focal linear tight junctions connecting the non-pigmented and pigmented cells at their apices. In both epithelia, the interruptions of the ridges are precisely complemented by particles or short bars of similar length found in the E-face furrows. Consequently, it is possible to conclude that the junctional fibrils are continuous in these two epithelia. For the zonulae occludentes, this continuity appears to be inconsistent with the ‘leaky’ properties of these epithelia shown by some physiological investigations.     

Transferrin binding to K562 cell line : Effect of heme and sodium butyrate induction

Experiments demonstrating the existence of receptors for iron-saturated transferrin on K562 cells are described. Binding of ¹²⁵I-labelled transferrin is rapid, saturable and reversible, and can be specifically inhibited by unlabelled transferrin, but not by other proteins. The number of receptors determined by Scatchard analysis significantly decreased when K562 cells moved from the exponential to the quiescent phase of growth. Induction by hemin or sodium butyrate resulted in a marked reduction of transferrin binding. This phenomenon was due entirely to reduction in the number of receptors and was without effect on the affinity of interaction. The effect of butyrate and hemin on the number of transferrin receptors in other hematopoietic cell lines was investigated. Butyrate on the various cell lines was variable in its effect, whereas hemin constantly elicited a significant reduction in the number of transferrin receptors.     

Trace metal requirements in total parenteral nutrition (TPN). 5. Formation constants for the copper(II)--histidine ternary complexes with threonine, lysine, glycine, phenylalanine, valine, and cystine, and discussion of their implications regarding the copper distribution in blood plasma during TPN and the evaluation of the daily dose of copper

Specific metal deficiencies have been reported to affect patients receiving total parenteral nutrition (TPN). Our previous studies on the topic were devoted to the computer-based interpretation of the extra urinary excretion of zinc; a theoretical approach was also proposed, with a view to compensating for the extra losses of this metal. Similarly, the present work deals with the problem of TPN-induced copper deficiency and its remedy. As is the case for zinc, the TPN-induced excretion of copper clearly stems from the relative mobilization of the plasma protein-bound pool of this metal into its diffusable low-molecular-weight fraction; this phenomenon being due to the competitive complexation of copper by the amino acids of the nutritive solution. The computer simulation of this effect thus required that first the equilibrium constants be experimentally determined for the main complexes of copper that might form in the solution as well as in plasma during the infusion. Accordingly, complex formation in the copper-histidine ternary systems with threonine, lysine, glycine, phenylalanine, valine, and cystine was investigated by potentiometry at 37 degrees C in NaCIO4 0.15 mol X dm-3. The implications of the results obtained are discussed with regard to the interpretation of the copper excretion and the estimation of the desirable daily dose of this metal for the TPN mixture under consideration.     

Formation of copper-bleomycin complexes: evidence of a three-step process

The formation of Cu(II)-bleomycin complexes as a function of pH has been studied using circular dichroism, absorption, electron paramagnetic resonance spectroscopy, and potentiometric titration. Our data support the following points: the formation of Cu(II)-bleomycin complexes occurs in a three-step process: a first complex (I) is formed at pH 1.2, which most probably involves the pyrimidine nitrogen, the secondary amine nitrogen, and two water molecules as the four in-plane ligands of copper. A second complex (II) is formed at pH 2.5, through the further coordination of the peptide nitrogen of histidine residue, and histidine imidazole nitrogen giving rise to the release of two protons. The fixation, in apical position, of the alpha-amino nitrogen of beta-aminoalanine occurs in a last step through the release of one additional proton. A value of 2.7 has been obtained for the pK of formation of this third complex, which is the species present at physiological pH. In the Cu(II)-depbleomycin system only one complex (II') has been detected.     

A sensitive method for the routine determination of plasma nicotine by flame ionization gas-liquid chromatography

A gas chromatographic procedure for the analysis of nicotine in plasma, which uses quinoline as an internal standard, is reported. The nicotine is extracted with diethyl ether, concentrated without any evaporation, thus avoiding losses, and analyzed without derivatization. The recovery is 83.2 +/- 6.1% (n = 6). Although the analysis is carried out with a classical flame ionization detector, the detection limit is 0.1 ng/ml. Linearity is observed up to 100 ng/ml. The results of the precision analysis performed in the working range indicate a good reproducibility: a coefficient of variation of 5.2% is obtained for within-run analysis and 10.5 to 4.5% for nicotine values from 2.9 to 19.1 ng/ml for day analysis. Since a single run (the limitative step) lasts less than 15 min this improved procedure allows a great number of samples to be processed per day.     

Kinetics of development of the inhibitory activity of mixed lymphocyte culture supernatants on macrophage migration: effect of presensitization

Supernatants of mixed cultures of Lewis and August rat lymphocytes show a spontaneous release of MIF-like activity beginning at the 20th hr of the culture. If the Lewis rat has received an August skin allograft 2 wk earlier, the release begins sooner and remains stronger throughout the 3 days of the mixed lymphocyte culture. The difference begins to be statistically significant at the 10th hr (p < 0.001) and is still highly significant with the 72-hr supernatant (p < 0.001). Such a comparison of the kinetics of MIF-like activity release in mixed lymphocytes cultures between donor-recipient pairs and similar nongrafted pairs may be of both practical and theoretical interest.     

Complexation of the fungal metabolite tenuazonic acid with copper (II), iron (III), nickel (II), and magnesium (II) ions

Tenuazonic acid (TA) is a phytotoxin produced by a fungal pathogen of rice, Pyricularia oryzae. We have synthesized and characterized the metal complexes of TA with copper (II), iron (III), nickel (II), and magnesium (II). The stoichiometry of the complexes determined by microanalysis and mass spectroscopy (D/CI) are Cu(II)TA2, Fe(III)TA3, Ni(II)TA2, and Mg(TA)2. Voltammograms of Fe(III)TA3, and Cu(II)TA2 in methanolic solutions confirmed this stoichiometry. Ni(II)TA2 paramagnetism and visible absorption data suggest an octahedral geometry. Fe(III)TA3 showed a characteristic visible absorption at 450 nm. Addition of Fe(III)Cl3 and Mg(II)Cl2 did not reverse the toxicity of NaTA to rice and bacterial cells, showing that this toxicity is not due to the privation of the cells of these metals essential for cell growth.     

Specific triggering by concanavalin A of a secondary T killer cell-mediated anti-tumor immune response.

Spleen cells from C57 B1/6 mice which rejected a Moloney sarcoma virus (MSV)-induced tumor elicited a strong cytolytic reaction against MSV-associated antigens when incubated for 7 days with a mitogenic dose (5 μg/ml) of concanavalin A (Con A). The cytolytic activity evaluated in a chromium release test was shown to be mediated by T lymphocytes, by anti-Thy 1–2 serum treatment, and independent of remaining Con A, by treatment with d-[α-methyl]mannopyranoside. A similar reactivity was obtained with phytohemagglutinin (PHA) at mitogenic doses but not with the B-cell mitogen lipopolysaccharide (LPS). This “secondary-like” response was regenerated up to 50 days post-MSV inoculation but decreased regularly. The cytolytic activity had an antigenic pattern indistinguishable from that of the relevant tumor cell reactivation and was directed by the same H-2 restriction.     

Role of magnesium cations in the yeast orotate phosphoribosyltransferase catalyzed reaction. Mechanism of the inhibition by Cu++ and Ni++ ions

The magnesium chelate of the N(3)H tautomer of orotate, L₃Mg, is the true substrate in the biosynthesis of orotidine 5′-monophosphate (OMP) catalyzed by yeast orotate phosphoribosyltransferase (OPRTase, E.C. 2.4.210) with a Michaelis constant K_m^L₃Mg equal to 12(2) μM. It is postulated that Mg⁺⁺ cations activate the transport of orotate to the active site by neutralizing the orotate charges; the ligand N(3)H is then exchanged between the incoming cation and the cation bound to the enzyme, thus ensuring the stabilization of the appropriate isomeric structure of orotate. This scheme, together with kinetic and thermodynamic data on orotate complexation by Mg⁺⁺ and Ca⁺⁺, accounts for the role of Ca⁺⁺ cations that neither activate nor inhibit OMP synthesis.Cu⁺⁺ and Ni⁺⁺ inhibiting properties arise from the formation of inert complexes of orotate. Ni⁺⁺ complexes have a poor affinity for the protein, whereas Cu⁺⁺ complexes have a Michaelis constant similar to that of the L₃Mg active species. The inertness of these complexes is tentatively understood in terms of low phosphoribosyl transfer rates as postulated from the kinetic study of the protonation of the complexes in water.     

Distinctive adjuvanticity of synthetic analogs of mycobacterial water-soluble components.

Synthetic N-acetyl-muramyl-l-alanyl-d-isoglutamine represents the minimal structure capable of duplicating the activity of mycobacteria in Freund's complete adjuvant. In contrast to mycobacterial adjuvant preparations, that function only in the form of water-in-oil emulsions, this compound and a second synthetic analog (N-acetyl-muramyl-l-alanyl-d-isoglutamic acid) augment the humoral immune responses of mice equally as well as aqueous solutions. Whereas N-acetyl-muramyl-l-alanyl-d-isoglutamic acid administered to guinea pigs in water-in-oil emulsion has no effect, N-acetyl-muramyl-l-alanyl-d-isoglutamine induces delayed hypersensitivity to ovalbumin and azobenezene-arsonate N-acetyl-l-tyrosine and increases the level of antibody against ovalbumin. Under these conditions, challenge with the synthetic adjuvants themselves evokes no skin responses. Moreover, Freund's complete adjuvant sensitizes guinea pigs to tuberculin and to native mycobacterial water-soluble adjuvant but not to the synthetic analogs.     

Biochemical research on oogenesis. RNA accumulation during oogenesis of the dogfish Scyliorhinus caniculus

Four biochemical mechanisms have been shown to operate in the oocytes of amphibians and teleosts: (1) amplification of the 28 S and 18 S genes, (2) noncoordinate accumulation of 5 S RNA and 28 S + 18 S RNA, (3) storage of 5 S and transfer RNA made in excess by small oocytes within nucleoprotein particles, (4) expression of different 5 S genes in oocytes and somatic cells. We have tried to extend these observations to another group of vertebrates, i.e., selacians (Chondrichthya). Our data suggest that ribosomal gene amplification is low or absent in the oocytes of the dogfish Scyliorhinus caniculus. However, previtellogenic oocytes of this species accumulate more 5 S RNA than needed for ribosome assembly. Transfer and 5 S RNA present in small oocytes are probably not free in the cell sap. A substantial fraction of these RNAs sediments at 10 S when homogenates of immature ovaries are centrifuged in sucrose density gradients. In contrast to what we observed in amphibians and teleosts, 5 S RNA from ovaries of S. caniculus is identical in sequence to 5 S RNA from liver. Among the four mechanisms mentioned above, the second and probably the third one are used by the oocytes of S. caniculus. Mechanism (4) is absent in this species. No definitive conclusion can be drawn concerning mechanism (1), i.e., ribosomal gene amplification.     

Cytophilic binding of IgE to the macrophage. III. Involvement of cyclic GMP and calcium in macrophage activation by dimeric or aggregated rat myeloma IgE

Discharge of lysosomal enzymes, measured by release of β-glucuronidase, was studied in uninduced rat macrophages stimulated in vitro with rat monoclonal IgE (IR 162) in different states of aggregation. Monomeric IgE showed negligible activity, while dimeric and aggregated IgE were shown to induce a rapid and selective release of β-glucuronidase as well as new synthesis of the enzyme, without change in the cytoplasmic marker, leucine aminopeptidase. Lysosomal enzyme release is related to the dose of dimeric IgE, becoming maximal above 2.5 μg/ml. β-Glucuronidase release from macrophages by dimers is competitively inhibited by monomeric IgE but only at high ratios, approximately 100-fold greater than those needed to block mast cell release of the same enzyme. The difference in inhibitability is consistent with the difference in binding affinity of macrophages and mast cells for monomeric IgE. This observation rules out the participation of the few remaining mast cells contained in the macrophage monolayer in β-glucuronidase release. Dimeric or aggregated IgE produced a rise in cyclic GMP coincident with the peak fixation of IgE by macrophages. Elevation of cyclic GMP by pharmacological means also stimulated β-glucuronidase release and new synthesis, as well as enhancing the effect on these of aggregated IgE. Enzyme release by IgE did not occur in the absence of extracellular calcium. We conclude that IgE, which has been cross-linked to form dimers before binding to specific macrophage receptors, triggers the cell and that cyclic GMP (and perhaps calcium) modulates the early step of macrophage activation.     

In vitro viability of lymphoid cells from lines of mice genetically selected for high or low responsiveness to phytohemagglutinin

The kinetics of viability of lymph node and spleen cells of mice genetically selected for "high" or "low" in vitro lymphocyte responsiveness to PHA were studied in PHA or PPD-stimulated short-term cultures. Lo/PHA cells were found to be less viable than Hi/PHA cells in unstimulated control cultures. PHA improved the viability of Lo/PHA cells while inducing proliferation of Hi/PHA cells with the appearance of more and larger lymphoblasts in the latter. PPD only improved the viability of spleen cell cultures, more so for the Hi/PHA line. The interline difference in thymidine uptake was smaller after PPD than after PHA stimulation. Modifications of culture conditions designed to decrease the interline difference in cell viability lessened but did not abolish the separation between the two lines for the PHA response as measured by thymidine uptake.