Structural characterization and immunosuppressive activity of a new pregnane glycoside from Epigynum cochinchinensis.

Phytochemical investigation on the ethyl acetate fraction of the leaves of Epigynum cochinchinensis led to the isolation of a new C21 pregnane glycoside, epigycoside B (1), together with three known analogues. Their structures were elucidated on the basis of extensive spectroscopic techniques, including UV, MS, and NMR experiments, as well as the chemical methods. Compound 1 displayed in vitro immunosuppressive activity against concanavalin A (Con A)/Lipopolysaccharides (LPS)-stimulated proliferation of mice splenocyte. The activity was significant as compared with control group at 50 µM concentration.

In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates concanavalin-A-induced acute liver injury in mice.

Oxidative stress contributes to the progression of acute liver failure (ALF). Transcription factor nuclear factor-erythroid 2-related factor (Nrf2) serves as an endogenous regulator by which cells combat oxidative stress. We have investigated liver damage and the balance between death and survival signaling pathways in concanavalin A (ConA)-mediated ALF using in vivo siRNA delivery targeting Keap1 in hepatocytes. For that goal, mice were injected with Keap1- or luciferase-siRNA-containing liposomes via the tail vein. After 48 hours, ALF was induced by ConA. Liver histology, pro-inflammatory mediators, antioxidant responses, cellular death, and stress and survival signaling were assessed. Keap1 mRNA and protein levels significantly decreased in livers of Keap1-siRNA-injected mice. In these animals, histological liver damage was less evident than in control mice when challenged with ConA. Likewise, markers of cellular death (FasL and caspases 8, 3 and 1) decreased at 4 and 8 hours post-injection. Nuclear Nrf2 and its target, hemoxygenase 1 (HO1), were elevated in Keap1-siRNA-injected mice compared with control animals, resulting in reduced oxidative stress in the liver. Similarly, mRNA levels of pro-inflammatory cytokines were reduced in livers from Keap1-siRNA-injected mice. At the molecular level, activation of c-jun (NH2) terminal kinase (JNK) was ameliorated, whereas the insulin-like growth factor I receptor (IGFIR) survival pathway was maintained upon ConA injection in Keap1-siRNA-treated mice. In conclusion, our results have revealed a potential therapeutic use of in vivo siRNA technology targeted to Keap1 to combat oxidative stress by modulating Nrf2-mediated antioxidant responses and IGFIR survival signaling during the progression of ALF.

Structural features of an immunostimulating and antioxidant acidic polysaccharide from the seeds of Cuscuta chinensis.

Three crude polysaccharide fractions, named CS-A, CS-B and CS-C, were prepared from the seeds of Cuscuta chinensis by hot water extraction and diluted alkali extraction subsequently, then EtOH precipitation, and tested for lymphocyte proliferation activity. CS-A showed a stimulating effect on concanavalin A or lipopolysaccharide induced mitogenic activity of lymphocytes. An acidic polysaccharide (CS-A-3beta) was purified from CS-A by anion exchange and gel filtration chromatography. The polysaccharide showed potent stimulating effects on lymphocyte proliferation and antibody production, but did not significantly influence the serum IgG level. Its structural features were elucidated by methylation analysis, partial acid hydrolysis, 1D and 2D NMR and ESI-mass spectroscopy. The data obtained indicated that CS-A-3beta had a backbone consisting of alpha-D-1,4-linked GalpA residues and beta-L-1,2-linked Rhap residues with branches at C-4 of Rhap residues and C-3 of GalpA residues, composed of arabinogalactan and glucobiose. This structure is typical for a pectic polysaccharide of the rhamnogalacturonan type. In addition, the effect of CS-A, CS-B, CS-C and CS-A-3beta on hydrogen peroxide induced cell lesion in rat pheochromocytoma line PC 12 was investigated. The results indicated that, besides its immunostimulating activity, CS-A-3beta had a protective effect against free radical-induced cell toxicity.

Impaired beta-adrenergic control of immune function in patients with chronic heart failure: reversal by beta1-blocker treatment.

BACKGROUND: In patients with chronic heart failure (CHF) and heightened sympathetic activity alterations in immune function have been described. OBJECTIVES: To find out whether, in CHF patients, beta-blocker treatment might beneficially affect immune function. METHODS: We studied activation of circulating lymphocytes (assessed as concanavalin A (CON A)-induced inositol phosphate (IP) formation and proliferation ([3H]-thymidine incorporation) from 8 CHF patients on standard medication (Group A, mean age 54 +/- 6 yrs, NYHA class II - IV, mean 3.1 +/- 0.3) and in 9 CHF patients on standard medication and additional treatment with the beta1-blocker metoprolol (Group B, mean age: 56 +/- 3 yrs, NYHA class II - IV, mean 2.9 +/- 0.2); 8 age-matched healthy volunteers (mean age 49 +/- 3 yrs) served as controls. RESULTS: Compared to controls, in group A isoprenaline-induced lymphocyte cyclic AMP-increase was reduced, CON A-evoked IP formation significantly enhanced and isoprenaline-induced inhibition of CON A-evoked IP formation and proliferation almost abolished. In group B, however, all these parameters were not significantly different from controls. CONCLUSION: In CHF patients lymphocyte cyclic AMP response to beta-adrenoceptor stimulation is blunted and the inhibitory effect of cyclic AMP on lymphocyte activation is almost abolished; this could result in a non-regulated increased production and release of proinflammatory cytokines that might contribute to the progression of the disease. Chronic treatment of CHF patients with the beta1-blocker metoprolol (at least partly) restores lymphocyte cyclic AMP responses to beta-adrenoceptor stimulation and the inhibitory effects of cyclic AMP on lymphocyte activation; the resulting "normalization" of the immune function might contribute to the beneficial effects of beta1-blockers in treatment of CHF.

Interaction between inflammatory cells and heparin-surface-modified intraocular lens.

PURPOSE: To investigate the interaction and adherence of inflammatory cells to a heparin-surface-modified intraocular lens (HSM IOL). SETTING: Department of Ophthalmology, Tokyo Medical University Hospital, Tokyo, Japan. METHODS: Splenic mononuclear leukocytes from rats with experimental autoimmune uveitis were cultured with the optic of an HSM IOL for 96 hours. The number of adherent cells on the HSM IOL surface was measured with and without the addition of interphotoreceptor retinoid-binding protein and concanavalin A (ConA) to the culture medium. The adherent cells were observed under a light microscope or a scanning electron microscope. RESULTS: Interphotoreceptor retinoid-binding protein and ConA increased the number of adherent cells on the HSM IOL relative to the control. Adherent cells on the HSM IOL were small and round, considered to be mainly lymphocytes. CONCLUSION: Activated lymphocytes tended to adhere to the surface of the HSM IOL.

Sequential structural changes upon zinc and calcium binding to metal-free concanavalin A.

The lectin concanavalin A (ConA) sequentially binds a transition metal ion in the metal-binding site S1 and a calcium ion in the metal-binding site S2 to form its saccharide-binding site. Metal-free ConA crystals soaked with either Zn2+ (apoZn-ConA) or Co2+ (apoCo-ConA) display partial binding of these ions in the proto-transition metal-binding site, but no further conformational changes are observed. These structures can represent the very first step in going from metal-free ConA toward the holoprotein. In the co-crystals of metal-free ConA with Zn2+ (Zn-ConA), the zinc ion can fully occupy the S1 site. The positions of the carboxylate ligands Asp10 and Asp19 that bridge the S1 and S2 sites are affected. The ligation to Zn2+ orients Asp10 optimally for calcium ligation and stabilizes Asp19 by a hydrogen bond to one of its water ligands. The neutralizing and stabilizing effect of the binding of Zn2+ in S1 is necessary to allow for subsequent Ca2+ binding in the S2 site. However, the S2 site of monometallized ConA is still disrupted. The co-crystals of metal-free ConA with both Zn2+ and Ca2+ contain the active holoprotein (ConA ZnCa). Ca2+ has induced large conformational changes to stabilize its hepta-coordination in the S2 site, which comprise the trans to cis isomerization of the Ala207-Asp208 peptide bond accompanied by the formation of the saccharide-binding site. The Zn2+ ligation in ConA ZnCa is similar to Mn2+, Cd2+, Co2+, or Ni2+ ligation in the S1 site, in disagreement with earlier extended x-ray absorption fine structure results that suggested a lower coordination number for Zn2+.

Cyclosporin elicits a non-responsive state and a shift in K+ fluxes in the early phase of activation of human lymphocytes with anti-CD3.

The effect of cyclosporin A on the initial phase of activation of human peripheral blood lymphocytes (PBL) by anti-CD3 was studied in a two-step incubation process. Cyclosporin A treatment in the initial phase of activation blocked the second phase activation of a cell population by anti-CD3, IL-2, or concanavalin A (ConA). In contrast, similar treatment with the calcium ionophore, ionomycin, enhanced the activation of anti-CD3. Only a marginal synergistic increase of intracellular [Ca2+] was elicited by cyclosporin A during anti-CD3 stimulation and this drug prevented activation-induced depolarization of lymphocytes by its ability to hyperpolarize cells. The hyperpolarization effect of cyclosporin A is related to the K+ flux but not the Na+ or Ca2+ flux and is unlikely to be mediated through calmodulin and protein kinase C. We postulate that the K+ flux-modulating ability of cyclosporin A renders T cells non-responsive in the initial phase of activation.

Single-sweep polarographic investigation of concanavalin A and its interaction with polysaccharide.

A simple, rapid, and sensitive single-sweep polarographic method has been developed for investigation of concanavalin A (con A) and its interaction with selected polysaccharides. In a solution containing 0.001 M 2,2'-bipyridine, 0.015 M hexamethylenetetramine, and 0.1 M sodium chloride, con A exhibits a single-sweep polarographic wave, and the cathodic peak potential is -1.50 V (vs SCE). The peak current varies linearly with con A concentration over a range of 1.0 x 10(-8) to 1.2 x 10(-7) M by derivative single sweep polarography. A preliminary discussion on properties of the con A polarographic wave has been made. In addition, it has been demonstrated that single sweep polarography can be a useful method for studies on interactions of con A with its complementary polysaccharides in solution.

[Inhibitory effect of aclarubicin, an anthracycline antibiotic, on the activity of suppressor cells in mice]. / Ingibiruiushchii éffekt aklarubitsina na aktivnost' suppressornykh kletok u myshei.

Antisuppressant activity of aclarubicin, an anthracycline antibiotic, was studied on models of immunodepression induced in vivo by concanavalin A or associated with progressive tumor growth. In vivo in a dose of 2.5 mg/kg aclarubicin markedly lowered or completely eliminated the ability of concanavalin A to induce activity on the suppressor cells in the mouse spleen. In vitro in doses of 0.0001 to 0.01 micrograms/ml aclarubicin decreased the suppressing activity of the splenocytes from mice with solid tumors in regard to spontaneous proliferation of the lymphoid cells.