Dimeric sesquiterpene thioalkaloids with potent immunosuppressive activity from the rhizome of Nuphar pumilum: structural requirements of nuphar alkaloids for immunosuppressive activity

Potent immunosuppressive dimeric sesquiterpene thioalkaloids, 6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythionuphlutine B and 6'-hydroxythionuphlutine B, were isolated from the rhizome of Nuphar pumilum together with five inactive quinolizidine alkaloids, neothiobinupharidine, nupharidine, deoxynupharidine, 7-epideoxynupharidine and nupharolutine. These dimeric sesquiterpene thioalkaloids were found to significantly inhibit anti-sheep erythrocyte plaque forming cell formation in mouse splenocytes at 1 microM. At this concentration. 6-hydroxythiobinupharidine, 6-hydroxythionuphlutine B and 6'-hydroxythionuphlutine B did not show cytotoxic effects to mouse splenocytes, and 6,6'-dihydroxythiobinupharidine also showed only minor or minimal cytotoxicity. By comparison of the inhibitory activity of several Nuphar alkaloids on anti-sheep erythrocyte plaque forming cell formation, some structural requirements of Nuphar alkaloids for immunosuppressive activity were obtained. Namely, the 6- or 6'-hydroxyl group at the quinolizidine ring of dimeric sesquiterpene thioalkaloids is essential for the immunosuppressive effect. The number of hydroxyl groups appears to be related to the cytotoxicity, and the influence on splenocytes is greater with increasing numbers of hydroxyl groups.     

Anti‐inflammatory effects of activated protein C on human dendritic cells

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti‐apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF‐α and IL‐6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL‐12 and anti‐inflammatory cytokine IL‐10. Interestingly, treatment with 5 μg/mL, but not 25 μg/mL, of APC significantly enhanced production of IL‐10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co‐stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti‐inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF‐α and/or IL‐6 production.     

GHRP-6 mimics ghrelin-induced stimulation of food intake and suppression of locomotor activity in goldfish

Ghrelin was first identified and characterized from rat stomach as an endogenous ligand for the growth hormone secretagogue (GHS) receptor (GHS-R). Ghrelin also acts as an orexigenic factor and regulates energy balance in rodents. In goldfish, native ghrelin consists of 11 molecular variants, the major form being a 17-residue peptide with n-octanoic acid modification (n-octanoyl ghrelin17), and intraperitoneal (IP) administration of n-octanoyl ghrelin17 induces central actions such as stimulation of food intake and suppression of locomotor activity through capsaicin-sensitive afferents. Four types of GHS-Rs (1a-1, 1a-2, 2a-1 and 2a-2) have been identified in goldfish, and one GHS, GHRP-6, can activate only GHS-R2a-1 in vitro. However, there is no information about the effect of GHRP-6 on food intake and locomotor activity in goldfish in vivo. Therefore, in the present study, we examined whether IP-administered GHRP-6 would mimic the orexigenic action of n-octanoyl ghrelin17 and its suppression of locomotor activity. IP administration of GHRP-6 at 1pmol/g body weight (BW) stimulated food intake, and was equipotent to the orexigenic action of n-octanoyl ghrelin17 at 10 pmol/g BW. IP-injected GHRP-6 at 1 pmol/g BW also induced a significant decrease of locomotor activity, as was the case for IP-injected n-octanoyl ghrelin17 at 10 pmol/g BW. The action of GHRP-6 was blocked by IP-preinjected capsaicin at 160 nmol/g BW. These results suggest that the central action of GHRP-6 might be mediated via the GHS-R2a-1-signaling pathway, and subsequently through capsaicin-sensitive afferents in goldfish.     

Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.     

Focused differential glycan analysis with the platform antibody-assisted lectin profiling for glycan-related biomarker verification

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.     

Pre-treatment of recombinant mouse MFG-E8 downregulates LPS-induced TNF-α production in macrophages via STAT3-mediated SOCS3 activation

Milk fat globule-epidermal growth factor factor 8 (MFG-E8) regulates innate immune function by modulating cellular signaling, which is less understood. Herein, we aimed to investigate the direct anti-inflammatory role of MFG-E8 in macrophages by pre-treatment with recombinant murine MFG-E8 (rmMFG-E8) followed by stimulation with LPS in RAW264.7 cells and in peritoneal macrophages, isolated from wild-type (WT) or MFG-E8(-/-) mice. RAW264.7 cells and mouse peritoneal macrophages treated with rmMFG-E8 significantly downregulated LPS-induced TNF-α mRNA by 25% and 24%, and protein levels by 29% and 23%, respectively (P<0.05). Conversely, peritoneal macrophages isolated from MFG-E8(-/-) mice produced 28% higher levels of TNF-α, as compared to WT mice when treated with LPS. In in vivo, endotoxemia induced by intraperitoneal injection of LPS (5 mg/kg BW), at 4 h after induction, serum level of TNF-α was significantly higher in MFG-E8(-/-) mice (837 pg/mL) than that of WT (570 pg/mL, P<0.05). To elucidate the direct anti-inflammatory effect of MFG-E8, we examined STAT3 and its target gene, SOCS3. Treatment with rmMGF-E8 significantly induced pSTAT3 and SOCS3 in macrophages. Similar results were observed in in vivo treatment of rmMFG-E8 in peritoneal cells and splenic tissues. Pre-treatment with rmMFG-E8 significantly reduced LPS-induced NF-κB p65 contents. These data clearly indicated that rmMFG-E8 upregulated SOCS3 which in turn interacted with NF-κB p65, facilitating negative regulation of TLR4 signaling for LPS-induced TNF-α production. Our findings strongly suggest that MFG-E8 is a direct anti-inflammatory molecule, and that it could be developed as a therapy in attenuating inflammation and tissue injury.     

Magnetic and photo-magnetic properties of Co dinuclear complexes

The magnetic and photo-magnetic properties of Co dinuclear compounds were studied. The Co compounds, [{Co(tpa)}₂(dhbq)](BF₄)₃ · 2H₂O and [{Co(tpa)}₂(dhbq)](ClO₄)₃ · 2H₂O, have the structure [(tpa)Co^III-LS–(μ-dhbq)^3?–Co^III-LS(tpa)]^3? (where LS, H₂dhbq, tpa denote low-spin, 2,5-di-hydroxy-1,4-benzoquinone and tris(2-pyridylmethyl)amine, respectively) at room temperature. On heating, these compounds exhibit valence-tautomeric inter-conversion; [(tpa)Co^III-LS–(μ-dhbq)^3?–Co^III-LS(tpa)]^3? ? [(tpa)Co^II-HS–(μ-dhbq)^2?–Co^III-LS(tpa)]^3? (where HS denotes high-spin). Furthermore, both of these compounds exhibit photo-induced valence tautomerism at low temperature (<60 K). These results show that the electronic structures of these complexes can be controlled by modifying the counter anions.     

Teprenone, but not H2-receptor blocker or sucralfate, suppresses corpus Helicobacter pylori colonization and gastritis in humans: teprenone inhibition of H. pylori-induced interleukin-8 in MKN28 gastric epithelial cell lines

Background. The role of teprenone in Helicobacter pylori‐associated gastritis has yet to be determined. To investigate the effect of teprenone on inflammatory cell infiltration, and on H. pylori colonization of the gastric mucosa in H. pylori‐infected patients, we first compared the effect of teprenone with that of both histamine H2 receptor antagonists (H2‐RA) and sucralfate on the histological scores of H. pylori gastritis. We then examined its in vitro effect on H. pylori‐induced interleukin (IL)‐8 production in MKN28 gastric epithelial cells. Materials and Methods. A total of 68 patients were divided into three groups, each group undergoing a 3‐month treatment with either teprenone (150 mg/day), H2‐RA (nizatidine, 300 mg/day), or sucralfate (3 g/day). All subjects underwent endoscopic examination of the stomach before and after treatment. IL‐8 production in MKN28 gastric epithelial cells was measured by enzyme‐linked immunosorbent assay (ELISA). Results. Following treatment, the teprenone group showed a significant decrease in both neutrophil infiltration and H. pylori density of the corpus (before vs. after: 2.49 ± 0.22 vs. 2.15 ± 0.23, p = .009; 2.36 ± 0.25 vs. 2.00 ± 0.24, p = .035, respectively), with no significant differences seen in either the sucralfate or H2‐RA groups. Teprenone inhibited H. pylori‐enhanced IL‐8 production in MKN28 gastric epithelial cells in vitro, in a dose‐dependent manner. Conclusions. Teprenone may modify corpus H. pylori‐associated gastritis through its effect on neutrophil infiltration and H. pylori density, in part by its inhibition of IL‐8 production in the gastric mucosa.     

Delayed copulation as a means of female choice by the hermit crab Pagurus filholi

Male hermit crabs perform precopulatory mate-guarding behavior during their reproductive season. As females generally cannot reject guarding attempts by males, male guarding prevents females from inspecting and choosing other male mates. However, as guarding males are often replaced by other males through competition for females during the guarding phase, females may be able to select males by delaying their copulation. To examine the possibility of female choice by hermit crabs, we investigated whether female Pagurus filholi that were being guarded in the field were ready to copulate and spawn. We found that about 30% of females guarded in the field were ready to spawn, indicating that guarded females delayed copulation with their current male. Our results suggest that by delaying copulation females may exploit male–male competition to choose dominant males. However, delaying copulation reduced the spawning potential of females. Hence, there is a trade-off between waiting for the opportunity to mate with a dominant male and decreased spawning success if females exploit male–male competition.