T cell-replacing activity of C8-derivatized guanine ribonucleosides

The capacity of the C8-substituted guanine ribonucleosides to provide T cell-like signals to cultures of splenic B cells was evaluated. We showed previously that these low m.w. nucleoside derivatives traverse the cell membrane and induce their effects from an intracellular location. The current studies clearly demonstrate that 8 mercaptoguanosine (8MGuo), when added to cultures of B cells and macrophages in the presence of antigen, is capable of supplying a "second signal" for B cells, enabling them to generate high numbers of specific plaque-forming cells against the immunizing antigen. This effect is duplicated in cultures of spleen cells from congenitally athymic mice. Inhibition of interleukin 2 (IL 2) generation by cyclosporin A, such that the antibody response of normal spleen cells is entirely abrogated, has minimal effects on the T cell-replacing activity of 8MGuo. Additivity studies with MLC supernatants as well as kinetic analyses with IL 2-associated lymphokines substantiate that these factors act by a mechanism distinct from that of 8MGuo and 8BrGuo. These observations establish these nucleoside activators as exciting new probes for T helper cell activity and an effective non-T cell source of T cell-like signals.     

Enhancement of the human antibody response by C8-substituted guanine ribonucleosides in synergy with interleukin 2

The antigen-specific primary antibody response of human lymphocytes in vitro was studied with respect to dependency upon the lymphokine interleukin 2 (IL 2) and its subsequent modulation by C8-substituted guanine ribonucleosides. The specific response to sheep erythrocytes was shown to be dependent on the presence of IL 2 in culture. However, addition of optimal concentrations of the nucleoside, 7-methyl-8-oxoguanosine (7m8oGuo), to cultures containing antigen and IL 2 resulted in marked amplification of the underlying antibody response. This synergistic effect between 7m8oGuo and IL 2 was antigen dependent and could not be accounted for by summation of the independent antigen-specific and nonspecific (polyclonal) components. That IL 2 itself was in fact responsible for both the specific response to antigen and the synergistic interaction with 7m8oGuo was confirmed in experiments with purified IL 2 produced by recombinant DNA technology. The response to antigen was enhanced by 7m8oGuo in a dose-dependent fashion. The results of kinetic studies demonstrated that this nucleoside is fully effective within the context of an ongoing immune response, because addition of 7m8oGuo could be delayed up to 3 days of the 6-day culture period without loss of subsequent immunoenhancement. Lymphocyte populations largely depleted of T cells were capable of mounting vigorous responses to antigen in the presence of 7m8oGuo so long as IL 2, either partially purified or purified recombinant material, was added to culture.     

Distinct effects of dual substitution on inductive and differentiative activities of C8-substituted guanine ribonucleosides

The current studies compare the inductive and differentiative properties of 8-mercaptoguanosine with those of 7-methyl-8-oxoguanosine. 7-Methyl-8-oxoguanosine (7m8oGuo) is a new member of the family of C8-substituted guanine ribonucleosides, the first such biologically active compound described that differs from guanosine other than by the specific substituent at the 8 position. Like 8MGuo, 7m8oGuo stimulates proliferation selectively in B lymphocytes. However, 7m8oGuo possesses greater activity than 8MGuo as a mitogen and greater potency as an adjuvant for humoral immune responses. Thus, as a B-lymphocyte mitogen, 7m8oGuo induces quantitatively greater [3H]TdR uptake than does 8MGuo, but with the same concentration optimum. As an adjuvant for in vitro antibody responses, however, 7m8oGuo achieves the same degree of immunoenhancement as 8MGuo but at approximately 10-fold lower concentrations, that is, the dose-response profile has been shifted to the left. Moreover, whereas the mitogenic responses to 8MGuo and 7m8oGuo exhibit parallel kinetic profiles, the adjuvant activity of 7m8oGuo arises earlier and persists later than does that of 8MGuo. These results are interpreted in terms of two distinct intracellular pathways: one mediating mitogenesis and the other adjuvanticity.     

Dissociation of inductive from differentiative signals transmitted by C8-substituted guanine ribonucleosides to B cells from SJL mice

A hitherto unknown defect in the immune responsiveness of B lymphocytes from SJL mice has enabled us to distinguish two qualitatively distinct classes of signal delivered to B cells by C8-substituted guanine ribonucleosides. This defect renders B cells from SJL mice unresponsive to the inductive (early acting) signal of 8-mercaptoguanosine (8MGuo) that culminates in mitogenesis and nonspecific secretion of immunoglobulin. Unresponsiveness is not attributable to a shift in either the dose-response or kinetic profiles, nor can the presence of suppressor cells be demonstrated. In striking contrast, however, SJL B cells exhibit normal responsiveness to the differentiative (T cell-like, or late acting) signal provided by the substituted nucleoside. This signal enables SJL B cells, depleted of T cells, to respond to T cell-dependent antigens, and synergizes with T cell-derived lymphokines. These data suggest 1) that nonspecific secretion of immunoglobulin is dependent on both inductive and differentiative signals, 2) that antigen alone can supply an effective inductive signal for antigen-specific responses, and 3) that the SJL mouse will provide a useful model for selective study of inductive vs differentiative events.     

Synthesis and immunostimulatory activity of 8-substituted amino 9-benzyladenines as potent Toll-like receptor 7 agonists

Several 9-benzyl adenine derivatives bearing various substituted amines at the 8-position have been prepared and evaluated for interferon induction in peripheral blood mononuclear cells (PBMC) from healthy human donors. The 8-bromoadenine derivative 5 was used as a versatile intermediate for all substitutions. The most active 8-substituted amino compound was found to be the 8-morpholinoethylamino derivative 19 which had an EC(50) in the submicromolar range.     

Cytokinin activity of O6-substituted guanine and hypoxanthine derivatives

O⁶-Substituted guanine and hypoxanthine derivatives were prepared and tested for their cytokinin activity by the tobacco callus, radish cotyledons and lettuce seed bioassay systems. The results indicated that some derivatives of both types possess cytokinin activity.     

Mechanism of synergy between T cell signals and C8-substituted guanine nucleosides in humoral immunity: B lymphotropic cytokines induce responsiveness to 8-mercaptoguanosine

B lymphocytes require a source of T cell-like help to produce antibody to T cell-dependent antigens. T cell-derived lymphokines and C8-substituted guanine ribonucleosides (such as 8-mercaptoguanosine; 8MGuo) are effective sources of such T cell-like help. Addition of T cell-derived lymphokines to antigen-activated B cells together with 8MGuo results in synergistic B cell differentiation, amplifying the sum of the individual responses twofold to four-fold. Lymphokine activity is required at initiation of culture for optimal synergy with 8MGuo, whereas the nucleoside can be added up to 48 hr after the lymphokines with full synergy. 8MGuo provides a perceived T cell-like differentiation signal to B cells from immunodeficient xid mice, thereby distinguishing a subset of Lyb-5- nucleoside-responsive B cells from those activated by soluble anti-mu followed by B cell stimulatory factor-1, interleukin 1, and B cell differentiation factors, which are Lyb-5+. Moreover, at least a subset of the B cells recruited by the synergistic interaction of lymphokines and nucleoside is distinct from that responsive to 8MGuo + antigen, insofar as Sephadex G-10 nonadherent xid B cells fail to respond to either 8MGuo or lymphokines alone, but do respond to the combination. A distinct subpopulation can also be demonstrated among normal B cells by limiting dilution analysis in which the precursor frequency of antigen-reactive B cells in the presence of lymphokines or nucleoside alone increases substantially when both agents are present together. In concert with the kinetic data, these observations suggest that synergy derives at least in part from the ability of lymphokines to induce one or more elements the absence of which limits the capacity of a distinct B cell subpopulation to respond to 8MGuo.     

Studies on the conformation and dynamics of the C8-substituted guanine adduct of the carcinogen acetylaminofluorene; model for a possible Z-DNA modified structure

The structure of an adduct between guanine and the carcinogen acetylaminofluorene has been examined in the solid state by X-ray crystallography, and in solution by NMR techniques. The observed conformations have been compared with predictions from energy calculations and their relevance to models of adducts with DNA has been examined.     

Synthesis and anti-protozoal activity of C2-substituted polyazamacrocycles

A focused library of C2-substituted-1,4,7,10-tetraazacyclododecanes was synthesised and the compounds were tested for their ability to kill trypanosome and malaria parasites. Several compounds showed significant in vitro activity and were selectively active against the parasites over human embryonic kidney cells used as a counter screen.     

NMR and FT-IR conformational studies of 8-substituted guanine nucleosides and nucleotides and their metal adducts and cancer

NMR and FT-IR Studies of the conformational changes of guanosine and guanosine-5′-monophosphate upon substitution of the H8 of guanine by a heavy, large atom, such as bromine, are presented. The conformational forms, syn, anti, C2′-endo and C3′-endo and gg, gt and tg rotamers of the above molecules are compared to those of their metal (Mg²⁺ and P^t2+) adducts, where the metal is fixed to the N7 nitrogen atom of guanine. The antitumor activity of cisplatin is discussed with relation to the conformational form and the effect of cisplatin is compared to the effects of the Mg²⁺ ion and carcinogens.     

Role of immunostimulatory molecules in poultry vaccines

Immunization by vaccination is the most suitable and safest method for preventing infectious diseases in the poultry worldwide. Vaccines alone cannot effectively protect birds from variety of pathogens under field conditions. The combined use of potent immunostimulants in vaccines is an alternative to increase the efficacy of vaccines that can be achieved by the development of better adjuvant. One such adjuvant is cytokine; cytokines have been used extensively as adjuvant in vaccines and are responsible for the type and extent of an immune response following vaccination. Although the innate immune system in birds is not fully characterized but their immune system is very much similar to that of mammals, and moreover with the recent discovery of a number of avian cytokine genes it is now possible to study their effectiveness in enhancing the immune response during vaccination. This review focuses on the recent studies and developments involving the role of immunomodulating agents especially cytokines of avian origin in poultry vaccines.     

Demonstration of binding components specific for 7,8-disubstituted guanine ribonucleosides in murine B lymphocytes

7,8-Disubstituted guanine ribonucleosides are known to be potent intracellular modulators of immune responses. These compounds trigger and modulate a wide variety of lymphocyte responses including effects exerted directly on B cells. However, little is known about their mechanism of action. The current paper describes studies undertaken to evaluate whether binding components specific for these bioactive molecules exist in splenic B lymphocytes. After exposure of cells to labeled nucleoside, two different pools of nucleoside can be distinguished: a rapidly exchangeable nucleoside pool and a slowly exchangeable pool. The material in the latter pool consists of authentic unaltered nucleoside that is complexed to a relatively hydrophobic cellular component with an apparent Mr of 30,000-40,000; binding appears to interfere with free interaction of the nucleoside's cis hydroxyls with a boronate affinity resin. The slowly exchangeable nucleoside pool is seen to localize predominantly to the nucleus in electron microscopic autoradiographs. This pool is maximally bound by 30 min of incubation. Specific, saturable binding is demonstrable, with an apparent Kd of approximately 7 microM. This value correlates well with concentrations at which half-maximal biological activity occurs and suggests that the binding component likely mediates antigen-dependent immunomodulatory activity. Splenic B cells express approximately 2 x 10(4) binding sites/cell, whereas thymic lymphocytes, which do not respond functionally to nucleosides, do not display a measurable number of nucleoside binding sites. Ligand specificity of the binding interaction is confirmed by binding inhibition studies, in which binding inhibitory activity of unlabeled agonistic structural analogs recapitulate their degree of immunobiological activity. These data are most consistent with the existence of a saturable binding component with apparent specificity for 7,8-disubstituted guanine ribonucleosides in splenic B cells.     

Role of protein phosphorylation in regulation of brainL-glutamate decarboxylase activity

In the brain, the -aminobutyric acid (GABA) level is primarily controlled by the activity of its synthesizing enzyme,L-glutamate decarboxylase (GAD). At present, mechanisms responsible for regulation of GAD activity remain largely unknown. Here we report that GAD activity is inhibited by conditions favoring protein phosphorylation, and this inhibition can be reversed by phosphatase treatment. Furthermore, this inhibition appears to result from the suppression of a Ca²⁺-dependent phosphatase. Phosphorylation of GAD is demonstrated by direct incorporation of³²P into the GAD protein. These results suggest that GAD activity in the brain is inhibited by phosphorylation and activated by dephosphorylation. A model for regulation of GABA synthesis related to neuronal excitation is discussed.     

Effects of site-specific guanine C8-modifications on an intramolecular DNA G-quadruplex

Understanding the fundamentals of G-quadruplex formation is important both for targeting G-quadruplexes formed by natural sequences and for engineering new G-quadruplexes with desired properties. Using a combination of experimental and computational techniques, we have investigated the effects of site-specific substitution of a guanine with C8-modified guanine derivatives, including 8-bromo-guanine, 8-O-methyl-guanine, 8-amino-guanine, and 8-oxo-guanine, within a well-defined (3 + 1) human telomeric G-quadruplex platform. The effects of substitutions on the stability of the G-quadruplex were found to depend on the type and position of the modification among different guanines in the structure. An interesting modification-dependent NMR chemical-shift effect was observed across basepairing within a guanine tetrad. This effect was reproduced by ab initio quantum mechanical computations, which showed that the observed variation in imino proton chemical shift is largely influenced by changes in hydrogen-bond geometry within the guanine tetrad.     

Role of a guanine nucleotide regulatory protein in the activation of phospholipase C by different chemoattractants

It is well established that formyl peptide chemoattractants can activate a phospholipase C in leukocytes via a pertussis toxin (PT)-sensitive guanine nucleotide regulatory (G) protein. Whether this pathway is similarly used by chemoattractant receptors as a class has been unclear. We now report that lipid and peptide chemoattractants in direct comparative studies induced similar amounts of initial (less than or equal to 15 sec) inositol trisphosphate (IP3) release in human polymorphonuclear leukocytes, but the response to lipid chemoattractants was more transient. Production of IP3 by all chemotactic factors was inhibited by treatment of the cells with PT, indicating that chemotactic factor receptors as a class are coupled to phospholipase C via a G protein that is a substrate for ADP ribosylation by PT. The peptide and lipid factors had comparable chemotactic activity, which was also inhibitable by PT. However, transient activation of phospholipase C is apparently an insufficient signal for full cellular activation, since the lipid chemotactic factor leukotriene B4 and platelet-activating factor were poor stimuli for O2- production and lysosomal enzyme secretion compared with N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Nonetheless, treatment with PT inhibited O2- production and enzyme secretion in response to all chemoattractants, but as previously noted, did not affect Ca2+ ionophores, lectins, or phorbol myristate acetate. Formyl peptide and lipid chemotactic factors induced similar levels of Ca2+ mobilization when monitored by Quin 2 or chlortetracycline (CTC) fluorescence. Although these responses to fMet-Leu-Phe were blocked by PT, the Quin 2 and initial CTC response to the lipid factors were only partially susceptible. Thus, the lipid factors apparently utilize an additional PT-resistant mechanism for redistributing intracellular Ca2+. This latter process requires extracellular Ca2+ and may be independent of the PT-sensitive G protein.     

Role of the carboxyl terminal di-leucine in phosphorylation and internalization of C5a receptor

The carboxyl tail of G protein-coupled receptors contains motifs that regulate receptor interactions with intracellular partners. Activation of the human neutrophil complement fragment C5a receptor (C5aR) is terminated by phosphorylation of the carboxyl tail followed by receptor internalization. In this study, we demonstrated that bulky hydrophobic residues in the membrane-proximal region of the C5aR carboxyl tail play an important role in proper structure and function of the receptor: Substitution of leucine 319 with alanine (L319A) resulted in receptor retention in the endoplasmic reticulum, whereas a L318A substitution allowed receptor transport to the cell surface, but showed slow internalization upon activation, presumably due to a defect in phosphorylation by both PKC and GRK. Normal agonist-induced activation of ERK1/2 and intracellular calcium release suggested that the L318A mutation did not affect receptor signaling. Binding of GRK2 and PKCbetaII to intracellular loop 3 of C5aR in vitro indicated that mutagenesis of L318 did not affect kinase binding. Limited proteolysis with trypsin revealed a conformational difference between wild type and mutant receptor. Our studies support a model in which the L318/L319 stabilizes an amphipathic helix (Q305-R320) in the membrane-proximal region of C5aR.     

Role of the C8 gem-dimethyl group of bryostatin 1 on its unique pattern of biological activity

The role of the C(8) gem-dimethyl group in the A-ring of bryostatin 1 has been examined through chemical synthesis and biological evaluation of a new analogue. Assays for biological function using U937, K562, and MV4-11 cells as well as the profiles for downregulation of PKC isozymes revealed that the presence of this group is not a critical determinant for the unique pattern of biological activity of bryostatin.