Antibody response of murine spleen cells with or without receptors for C3 to antigenic or mitogenic stimulation

Bone marrow-derived lymphocytes (B cells) with or without receptors for third component of complement (CR) were studies in their responsiveness to T-independent antigens and B-cell mitogens in terms of anti-DNP PFC response. Spleen cells were fractionated by centrifugation over a Ficoll-Hypaque density gradient after they were rosetted with antigen-antibody-complement complexes. The cells in interface fraction could not respond to any T-independent antigen tested, while they responded well to polyclonal stimulations by lipopolysaccharide and polymerized flagellin but not by keyhole limpet hemocyanin. Reduced response to T-independent antigen of the cells in interface fraction could not be explained by a shift of the kinetics, lack of number of B cells, or by the depletion of macrophages. Significance of CR in B-cell differentiation is discussed.     

Interaction of leucine enkephalin with (3H)naloxone binding in rat brain: Evidence for an opioid receptor complex

We recently presented evidence that distinct morphine and enkephalin receptors coexist in an opioid receptor complex (Mol. Pharmacol. 21:548-557, 1982). In this paper, we present data which demonstrate that in the presence of sodium leucine enkephalin noncompetitively inhibits the binding of [3H]naloxone to a crude particulate fraction of rat brain. Since the binding site labeled by [3H]naloxone in the presence of sodium may be an alternate conformation of the morphine receptor, these data provide further evidence that morphine and enkephalin receptors are allosterically coupled.     

Interaction of mitogenic lectins with lymphocyte plasma membranes]

It is shown that mitogenic stimuli from concanavalin A and phytohemagglutinin are summed up in the time. Under given experimental conditions each of the mitogens used did not induce mitogenesis when the interaction with lymphocytes continued for 14 and/or 20 hours. The results obtained are discussed from the point of view of the cell-to-cell interaction between the lymphocytes. It is assumed that the ability of the stimuli to sum up in the time underlies the nonspecific mechanism of defense from tolerance.     

The mitogenic effect of insulin isolated from the mammalian brain on Swiss 3T3 cells]

Mitogenic properties of the insulin derived from pig brain were compared with the action of pancreatic (standard) pig insulin and epidermal growth factor (EGF) using the culture of Swiss 3T3 cells. The brain insulin, likely as the pancreatic insulin, induced uptake of 14C-thymidine by resting cells in a dose-dependent manner at concentration of 0.5-2.0 micrograms/ml in culture medium. However, at equal concentrations of these hormones the proliferating effect of the brain insulin appeared to be 2-fold higher than the effect of the pancreatic hormone. At the same time the mitogenic action of both hormones was lower than that of EGF (10 ng/ml). The additive effect of the brain insulin and EGF, administered in combination, was more pronounced than the effect of the pancreatic insulin combined with EGF. The data obtained suggest a possible participation of brain insulin in the process of nerve cell proliferation.     

Interaction of [3H]dipyridamole with the nucleoside transporters of human erythrocytes and cultured animal cells

Summary Equilibrium binding of [³H]dipyridamole identified high-affinity (K _i 10nm) binding sites on human erythrocytes (5×10⁵ sites/cell) and on HeLa cells (5×10⁶ sites/cell). The equilibration of dipyridamole with these sites on human erythrocytes was compatible with a second-order process which proceeded at 22°C with a rate constant of about 6×10⁶ m ^–1 sec^–1. Binding of dipyridamole to these sites correlated kinetically with the inhibition of the equilibrium exchange of 500 m uridine in these cells and was inhibited in a concentration-dependent manner by nucleosides and other inhibitors of nucleoside transport, such as nitrobenzylthioinosine, dilazep and lidoflazine, but not by hypoxanthine, which is not a substrate for the nucleoside transporter of human erythrocytes. The results indicate that the substrate binding site of the transporter is part of the high-affinity dipyridamole binding site. Bound [³H]dipyridamole became displaced from these sites on human erythrocytes by incubation with an excess of unlabeled dipyridamole or high concentrations of nucleosides and inhibitors of nucleoside transport, but neither by hypoxanthine nor sugars. Dissociation of [³H]dipyridamole behaved as a simple first-order process, but the rate constant was about one order of magnitude lower (about 3×10^–3 sec^–1) than anticipated for typical ligand-protein binding on the basis of the measured association rate and equilibrium constants. The reason for this discrepancy has not been resolved. No high-affinity dipyridamole binding sites were detected on Novikoff rat hepatoma cells, P388, L1210 and S49 mouse leukemia cells or Chinese hamster ovary cells, and their absence correlated with a greater resistance of nucleoside transport in these cells to inhibition by dipyridamole. All cells expressed considerable low affinity (K _d>0.5 m) and nonspecific binding of dipyridamole.     

Interaction of leptospires with murine microglial cells

Leptospires, the agents of leptospirosis, exert tropism for the central nervous system, in the course of mammal infection. We evaluated the interaction between murine microglial cells and strains of pathogenic L. interrogans leptospires and non-pathogenic L. biflexa leptospires, mainly by flow cytometric assays. In the absence of opsonic conditions microglia are capable of ingesting--even quite slowly--the spirochetes and killing the non-pathogenic strain. The adhesion to microglia, which is quick and relevant for all the strains, does not involve the CR3 integrin receptor. These findings suggest that the murine microglia--in non opsonic conditions in vitro--do not effectively clear the pathogenic leptospires.     

Interaction of [3H] corticosterone from blood serum with rat liver cells during aging

It is shown that the consumption of glucocorticoids from the complexes with serum proteins by hepatocytes decreases with ageing. Transcortin-complexed decrease adenosine monophosphate binding by the liver cells but the degree of this inhibition decreases with an increase of animals' age, which is probably connected with the change in physicochemical properties of steroid-transport blood glycoprotein.     

Development of opiate ([3H] naloxone)-binding sites in female rabbit brain: correlation with prepubertal gonadotropin secretion

In neurochemical terms, little is known concerning the control of puberty onset in the female rabbit. In view of the established involvement of brain opiates in the sexual maturation of the rat, we have investigated the prepubertal development of opiate-binding sites in the hypothalamus and cerebral cortex of the rabbit. Binding of the opiate antagonist, [3H] naloxone, to thick (350 micron) slices of rabbit brain was found to be reversible, stereospecific, saturable, and of high affinity. In all respects these sites possessed the characteristics of opiate receptors. Specific binding (Bmax and KD) values were determined at 1, 8, 29, 40, 51, 100, and 168 days after birth in the hypothalamus and cerebral cortex. All all ages binding in the hypothalamus was higher, per mg of tissue, than in the cortex. Major differences in the pattern of development were also evident. In the cortex the Bmax slowly increased from a minimum at Day 1 to a maximum at about 100 days when puberty normally occurs. In contrast, binding in the hypothalamus rose rapidly to a maximum at 40 days and then fell abruptly, by about 40% at Day 51, after which a slow increase through puberty took place. This peak in the hypothalamic Bmax value correlates closely with the major prepubertal surge of gonadotropin secretion. It remains to be determined whether the coincidence of spontaneous gonadotropin secretion with the rapid appearance of hypothalamic opiate receptors is developmentally meaningful for the reproductive system.     

Labelling of murine epidermal Langerhans cells with H3-thymidine.

Epidermal Langerhans cells may be identified by light microscopy by their strongly positive reaction following incubation for ATPase activity. Intact sheets of epidermis from mice killed at various time intervals following a single pulse label of H3-thymidine were incubated to demonstrate ATPase activity and subsequently processed for autoradiography. In specimens taken one hour after labelling, many basal keratinocytes were labelled but very few ATPase-positive dendritic cells. At subsequent time periods a few pairs of labelled ATPase-positive cells were found but individually labelled cells were not observed. The findings suggest that epidermal Langerhans cells form a very stable (labelling index less than 0.01%) self-replicating population which divides to maintain cell spacing during growth. No evidence was found for migration and interchange of Langerhans cells with the connective tissue, or for an origin of Langerhans cells by transformation of another cell type.     

Distribution of the four flagellar (H) antigenic subserotypes of Bacillus thuringiensis H serotype 3 in Japan

A total of 302 strains of Bacillus thuringiensis flagellar (H) serotype 3, collected from sericultural environments and soils of the three main islands (Honshu, Shikoku and Kyushu) of Japan, were examined for their H antigenic subserotypes. Of these strains, 259 (85.8%) were identified as the H subserotype 3a : 3c (subsp. alesti ), 16 (5.3%) were referable to the subserotype 3a : 3b : 3c (subsp. kurstaki ), 26 (8.6%) belonged to the subserotype 3a : 3d : 3e (subsp. fukuokaensis ) and one strain (0.3%) was the subserotype 3a : 3d (subsp. sumiyoshiensis ). The subserotypes 3a : 3c and 3a : 3b : 3c were present in sericultural environments only; the former was distributed in Honshu and Shikoku Islands but not in Kyushu Island, and the latter was distributed in Honshu and Kyushu Islands but not in Shikoku Island. The subserotype 3a : 3d : 3e, mainly found in soils but occasionally in sericultural environments, was distributed in the three main islands. It also appeared that the subserotype 3a : 3d : 3e, a recently described subserotype, was distributed in Japan more widely than the globally disseminated subserotype 3a : 3b : 3c. The results clearly showed geographical and ecological differences in the distribution of subserotypes.     

Interaction of the radiolabelled high-affinity anti-oestrogen [3H]H1285 with the cytoplasmic oestrogen receptor.

The high-affinity triarylethylene anti-oestrogen H1285 [4-(NN-diethylaminoethoxy)-beta-ethyl-alpha-(p-hydroxyphenyl) -4'-methoxystilbene] was tritiated to high specific radioactivity (35 Ci/mmol). Competition experiments between [3H]H1285 and H1285 or oestradiol demonstrated that both compounds would compete with [3H]H1285 for oestrogen-specific binding sites in rat uterine cytosol. [3H]H1285 had at least 10 times the affinity for the receptor compared with oestradiol at the 50% competition level. [3H]H1285 appeared to have at least twice the association rate for the oestrogen receptor compared with [3H]oestradiol. In addition, the dissociation half-life (t1/2) of specific binding of [3H]H1285 to oestrogen receptors at 0 degrees C was about 220 h compared with a value of 60 h for [3H]oestradiol. Because of the extremely slow dissociation of [3H]H1285 from the oestrogen receptor, we were able to compare the sedimentation profiles of [3H]H1285-receptor complexes with those of [3H]oestradiol-receptor complexes in the presence of 0.4 M-KCl on 5-20% sucrose density gradients. [3H]Oestradiol-receptor complexes had a major peak at 4.4 S with a smaller peak at 5.6 S, whereas with [3H]H1285-receptor complexes the 5.6 S peak was always higher than the 4.4 S peak. There was significant variation between the dissociation behaviour at 20 degrees C of [3H]H1285-receptor complexes and [3H]oestradiol-receptor complexes pre-activated at 25 degrees C for 30 min in the presence and in the absence of 10 mM-sodium molybdate. The dissociation t1/2 of [3H]oestradiol-receptor complexes at 20 degrees C decreased from 1.5 h to 0.5 h when molybdate was present during heat treatment whereas the dissociation t1/2 for [3H]H1285-receptor complexes was 5 h for both conditions. These observations indicate that there are fundamental differences in the initial interaction of H1285 and oestradiol with the oestrogen receptor.     

Glucose regulates insulin mitogenic effect by modulating SHP-2 activation and localization in JAr cells

The glucose effect on cell growth has been investigated in the JAr human choriocarcinoma cells. When JAr cells were cultured in the presence of 6 mm glucose (LG), proliferation and thymidine incorporation were induced by serum, epidermal growth factor, and insulin-like growth factor 1 but not by insulin. In contrast, at 25 mm glucose (HG), proliferation and thymidine incorporation were stimulated by insulin, serum, epidermal growth factor, and insulin-like growth factor 1 to a comparable extent, whereas basal levels were 25% lower than those in LG. HG culturing also enhanced insulin-stimulated insulin receptor and insulin receptor substrate 1 (IRS1) tyrosine phosphorylations while decreasing basal phosphorylations. These actions of glucose were accompanied by an increase in cellular tyrosine phosphatase activity. The activity of SHP-2 in HG-treated JAr cells was 400% of that measured in LG-treated cells. SHP-2 co-precipitation with IRS1 was also increased in HG-treated cells. SHP-2 was mainly cytosolic in LG-treated cells. However, HG culturing largely redistributed SHP-2 to the internal membrane compartment, where tyrosine-phosphorylated IRS1 predominantly localizes. Further exposure to insulin rescued SHP-2 cytosolic localization, thereby preventing its interaction with IRS1. Antisense inhibition of SHP-2 reverted the effect of HG on basal and insulin-stimulated insulin receptor and IRS1 phosphorylation as well as that on thymidine incorporation. Thus, in JAr cells, glucose modulates insulin mitogenic action by modulating SHP-2 activity and intracellular localization.     

Different effect of testosterone on polypotential stem hematopoietic stem cells and immunocompetent B-lymphocytes]

A research was made to study the dynamics of the proliferative, colony-forming and migration capacity of stem hemopoietic cells in (CBA X C57Bl) F1 hybrid mice under the influence of testosterone propionate, 10 mg/100 g, as well as the migration of immunocompetent B lymphocytes from the bone marrow to the spleen and the accumlation of their progeny, antibody-producing cells, in the spleen. The immunodepressive effect of testosterone was manifested by a decrease in the migration of B cells and the number of antibody-producing cells in the spleen. On the contrary, testosterone had a stimulating effect on the functional activity of stem hemopoietic cells, increasing their proliferation and migration. Under conditions of the suppressed erythropoietic differentiation of multipotent stem hemopoietic cells the injection of testosterone resulted in an increase in the number of antibody-producing cells in the spleen. This suggests that the stimulation of erythropoiesis and immunosuppression, induced by testosterone, are interconnected and determined by the direct action of the hormone on the cellular cycle of the stem cells, as well as by their prevailing differentiation towards the erythroid series, resulting in the decrease of their differentiation into B cells.     

Interaction of proflavin with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT): thermodynamic analysis from (1)H NMR data]

Temperature relationships of chemical shifts of protons of proflavin mixed with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in water solution were investigated on impulse NMR spectrometer (500 MHz). Procedure is suggested for calculating values of mole fractions of various associates in solution as a function against temperature. Free energies of Gibbs, entalpy and entropy were determined in the reactions of complex formation 1:1, 1:2, 2:1 of proflavin with tetranucleotide. The results point to a significant role of hydrophobic interactions during the formation of dye--tetramere duplex complexes.     

Effect of regional (wound) revaccination with tetanus anatoxin on the functional activity of immunocompetent cells in the region]

The study made with the use of complex methods established that the local (wound) application of tetanus toxoid rapidly made the manifestation of the lysosomal apparatus more pronounced, increased the oxidizing activity (determined in the nitro blue tetrazolium test) and phagocytic activity of the mononuclear phagocytizing system in the wound and in the regional lymph nodes. The wound application of tetanus toxoid significantly increased blast transformation of T lymphocytes in guinea pigs simultaneously with tetanus wound infection. The study confirmed the pathogenetic expediency of the proposed method for the stimulation of anti-tetanus immunity by the application of tetanus toxoid on the wound which specifically inhibited the primary stage of the infectious process.     

Synthesis of 4(3H)quinazolinimines with selective cytotoxic effect on human acute promyelocytic leukemia cells

We synthesized a new family of six 4(3H)quinazolinimines based on the reaction between (E)-N-(2-cyanophenyl)benzimidoyl chloride and substituted anilines reaching the formation of their corresponding C2, N3-substituted quinazoliniminium chlorides. This method provides novel, direct and flexible access to diverse substituted 4(3H)quinazolinimines.New compounds obtained following the proposed synthesis were fully characterized and, including the thirteen 4(3H)quinazolinimines synthesized by this method and previously reported by us, were used to study its cytotoxic effect on neoplastic cell lines. The mechanism involved in cell toxicity was also studied. Results showed that these compounds were highly cytotoxic, in particular on Human Promyelocytic Leukemia cells (HL60) and Chronic Myelogenous Leukemia cells (K562) when compared with conventional antineoplastic drugs such as etoposide and cisplatin. The mechanism associated to cytotoxic effect was mainly apoptosis, which not was decreased by antioxidant addition, thereby suggesting that the compounds exert apoptotic death through a mechanism unrelated with oxidative stress.