6]-Gingerol inhibits nitric oxide synthesis in activated J774.1 mouse macrophages and prevents peroxynitrite-induced oxidation and nitration reactions

Reactive nitrogen species (RNS), such as nitric oxide (NO) and its derivatives, e.g. peroxynitrite (ONOO-), have been proposed as being able to influence signal transduction and cause DNA damage, contributing to carcinogenic processes. In this study, the effect of [6]-gingerol, a pungent phenolic compound present in ginger (Zingiber officinale Roscoe), on NO synthesis in lipopolysaccharide (LPS)-activated J774.1 macrophages was tested, and the protective ability of this compound against peroxynitrite-mediated oxidation and nitration reactions were evaluated. [6]-Gingerol exhibited dose-dependent inhibition of NO production and significant reduction of inducible NO synthase (iNOS) in LPS-stimulated J774.1 cells. Moreover, [6]-gingerol effectively suppressed peroxynitrite-induced oxidation of dichlorodihydrofluorescein, oxidative single strand breaks in supercoiled pTZ 18U plasmid DNA, and formation of 3-nitrotyrosine in bovine serum albumin (BSA) and J774.1 cells. Our results indicate that [6]-gingerol is a potent inhibitor of NO synthesis and also an effective protector against peroxynitrite-mediated damage.     

Autoimmune MRL/lpr mice sera contain IgG with interleukin 3-like activity

The spleen cells, thymocytes, and bone marrow cells of autoimmune MRL/MP-lpr/lpr (MRL/lpr) mice do not constitutively produce interleukin 3 (IL-3), but these mice had IL-3-like activity in their sera. MRL/lpr sera supported the growth of the IL-3-dependent cell lines FDC-P2 and DA-1 but not the growth of IL-2-dependent T-572 cells. This IL-3-like activity increased with age. Biochemical analysis of the MRL/lpr sera by anion-exchange chromatography, gel filtration on a Superose 12 column, the binding to protein-A and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the serum factor with the IL-3-like activity was not IL-3 itself but was associated with IgG. Flow cytometric analysis also showed that the serum level of the Ig capable of binding FDC-P2 cells was high in MRL/lpr but not in MRL/+ mice. We suggest that IL-3 is not responsible for lymphoid hyperplasia, contrary to a previous report; rather auto-antibodies directed toward the IL-3 receptor may act pathogenically in MRL/lpr mice.     

Which skeletal myoblasts and how to be transplanted for cardiac repair?

Clinical efficacy of skeletal myoblast (skMb) transplantation is controversial whether this treatment produces beneficial outcome in patients with dilated cardiomyopathy (DCM). Based on immunological tolerance between wild-type and DCM hamsters with the deletion of δ-sarcoglycan (SG) gene, skMb engraftment in TO-2 myocardium (3 × 10⁵ cells in ∼100 mg heart) was verified by the donor-specific expression of δ-SG transgene constitutively produced throughout myogenesis. At 5 weeks after the transplantation, the cell rates expressing fast-myosin heavy chain (MHC) exceeded slow-MHC in δ-SG⁺ cells. Fifteen weeks after (corresponding to ∼12 years in humans), fast MHC⁺ cells nullified, but the δ-SG⁺ and slow MHC⁺ cell number remained unaltered. These skMbs fused with host cardiomyocytes via connexin-43 and intercalated disc, modestly improving the hemodynamics without arrhythmia, when engrafted skMbs were sparsely disseminated in autopsied myocardium. These results provide us evidence that disseminating delivery of slow-MHC⁺ myoblasts is promising for repairing DCM heart using histocompatible skeletal myoblasts in future.     

Conjugative transfer of the IncP-7 carbazole degradative plasmid, pCAR1, in river water samples

The transfer of the IncP-7 carbazole degradative plasmid pCAR1 from Pseudomonas putida SM1443 (derived from strain KT2440) into bacteria of river water samples was monitored using a reporter gene encoding red fluorescent protein (RFP). The number of transconjugants drastically increased in the presence of carbazole, and most appeared to belong to the genus Pseudomonas. The results suggest that the presence of carbazole benefits the appearance of transconjugants belonging to the genus Pseudomonas. Intriguingly, we also detected the transfer of pCAR1 into non-Pseudomonas, Stenotrophomonas-like bacteria.     

Characterization of Fyn-mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B) subunit of the N-methyl-D-aspartate receptor

The N-methyl-d-aspartate (NMDA) receptors play critical roles in synaptic plasticity, neuronal development, and excitotoxicity. Tyrosine phosphorylation of NMDA receptors by Src-family tyrosine kinases such as Fyn is implicated in synaptic plasticity. To precisely address the roles of NMDA receptor tyrosine phosphorylation, we identified Fyn-mediated phosphorylation sites on the GluR epsilon 2 (NR2B) subunit of NMDA receptors. Seven out of 25 tyrosine residues in the C-terminal cytoplasmic region of GluR epsilon 2 were phosphorylated by Fyn in vitro. Of these 7 residues, Tyr-1252, Tyr-1336, and Tyr-1472 in GluR epsilon 2 were phosphorylated in human embryonic kidney fibroblasts when co-expressed with active Fyn, and Tyr-1472 was the major phosphorylation site in this system. We then generated rabbit polyclonal antibodies specific to Tyr-1472-phosphorylated GluR epsilon 2 and showed that Tyr-1472 of GluR epsilon 2 was indeed phosphorylated in murine brain using the antibodies. Importantly, Tyr-1472 phosphorylation was greatly reduced in fyn mutant mice. Moreover, Tyr-1472 phosphorylation became evident when hippocampal long term potentiation started to be observed, and its magnitude became larger in murine brain. Finally, Tyr-1472 phosphorylation was significantly enhanced after induction of long term potentiation in the hippocampal CA1 region. These data suggest that Tyr-1472 phosphorylation of GluR epsilon 2 is important for synaptic plasticity.     

A factor of inducing IgE from a filarial parasite prevents insulin-dependent diabetes mellitus in nonobese diabetic mice

Parasitic helminth infections are characterized by eosinophilia and markedly elevated levels of circulating antigen-nonspecific immunoglobulin E (IgE), responses from which concern helminth protection. We previously purified a factor from Dirofilaria immitis that induces antigen-nonspecific IgE in mice and rats. Recombinant DiAg (rDiAg) has various biological activities. It is also known that parasitic helminth infection generates tremendous Th2 responses. The nonobese diabetic (NOD) mouse spontaneously develops Th1 cell-dependent autoimmune diabetes. Here we investigated the effects of rDiAg on the initiation and progression of this disease. rDiAg treatment of 6-week-old NOD females (the age at which insulitis typically begins) completely prevented insulitis and diabetes. Thus, rDiAg impaired the islet Ag-specific Th1 cell response in vivo, and the prevention of diabetes by rDiAg was associated with switching of the response from a Th1 to a Th2 profile. Since rDiAg clearly prevented insulitis by inhibiting the development and further accumulation of pathogenic Th1 cells to islets of Langerhans, we conclude that DiAg is a native Th2 inducer in filarial helminth and that Th1 responses are required for early events in the development of spontaneous autoimmune diabetes. In conclusion, the presence of parasitic helminth infections may play an important role as an immunomodulator in some autoimmune diseases or allergies.     

Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1

A DNA fragment carrying the gene encoding poly(3-hydroxybutyrate) (P(3HB)) depolymerase was cloned from the genomic DNA of Marinobacter sp. DNA sequencing analysis revealed that the Marinobacter sp. P(3HB) depolymerase gene is composed of 1734 bp and encodes 578 amino acids with a molecular mass of 61,757 Da. A sequence homology search showed that the deduced protein contains the signal peptide, catalytic domain (CD), cadherin-type linker domain (LD), and two substrate-binding domain (SBD). The fusion proteins of glutathione S-transferase (GST) with the CD showed the hydrolytic activity for denatured P(3HB) (dP(3HB)), P(3HB) emulsion (eP(3HB)) and p-nitrophenylbutyrate. On the other hand, the fusion proteins lacking the SBD showed much lower hydrolytic activity for dP(3HB) compared to the proteins containing both CD and SBD. In addition, binding tests revealed that the SBDs are specifically bound not to eP(3HB) but dP(3HB). These suggest that the SBDs play a crucial role in the enzymatic hydrolysis of dP(3HB) that is a solid substrate.     

Lowering intracellular and extracellular calcium contents prevents cytotoxic effects of ethylene glycol-based vitrification solution in unfertilized mouse oocytes

We investigated the characteristics of the changes in intracellular calcium (Ca2+) concentration ([Ca2+](i)) and the viability of the unfertilized mouse oocytes exposed to various concentrations of ethylene glycol (EG)-containing solutions or vitrification solutions. Oocytes exposed to EG (1, 5, 10, 20, and 40% (v/v)) exhibited a rapid and dose-dependent increase in [Ca2+](i). The survival rate was 100% when oocytes were exposed to the EG concentration up to 5% through 5 min, while all oocytes were dead within 3 min when exposed to 10, 20, or 40% EG. When extracellular Ca2+ was removed, increase in [Ca2+](i) at 10 and 20% EG was less than that at the same concentrations of EG with extracellular Ca2+. The survival rates of the oocytes exposed to 10, 20, and 40% EG at 3 min were 100, 97, and 0%, respectively. In the presence of 20 microM 1,2-bis(o-aminopheoxy)ethane-N,N,N',N'-tetraacetic acid tetra acetoxymethyl ester (BAPTA-AM), a Ca2+ chelator, a small increase in [Ca2+](i) exposed to 10, 20, and 40% EG was observed until 4 min. Subsequently prolonged elevation of the [Ca2+](i) was observed in the oocytes exposed to 40% EG but not with 10 and 20% EG. The survival rate of the oocytes, in the presence of 20 microM BAPTA-AM, exposed to 10 and 20% EG was 100% throughout 5 min, while the oocytes exposed to 40% EG were alive only for 3 min. Treatment by the vitrification solution with various concentrations of EG (10, 20, and 40%) caused a smaller increase in [Ca2+](i), while the survival rates were higher compared to those without vitrification solution at the same concentrations of EG. These data suggested that the sustained [Ca2+](i) rises by EG in unfertilized mouse oocytes resulted in cell death. Therefore, the lowering of [Ca2+](i) in the oocytes exposed to the cryoprotectant may improve the viability of cryopreserved unfertilized oocytes.     

Comparison of post-translational modifications of alpha A-crystallin from normal and hereditary cataract rats

Summary. In order to investigate the relationship between lens opacities and the various modifications of lens proteins, we analyzed and compared the properties of lens proteins of 85-day old normal Wistar rats and the hereditary cataract model, ICR/f rats. The present study identified many differences between normal and mutant lens proteins. In the ICR/f mutant rats, the relative amounts of gamma-crystallin decreased and high molecular weight (HMW) protein increased. Racemization and isomerization of Asp-151 of alpha A-crystallin was observed in the mutant ICR/f rats, and Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. These modifications were not found in the age-matched normal rats. These tendencies are consistent with aged and cataractous human lenses.     

Enhancing effect of lipids and emulsifiers on the accumulation of quercetin metabolites in blood plasma after the short-term ingestion of onion by rats

The effects of co-ingested lipids and emulsifiers on the accumulation of quercetin metabolites in blood plasma after the short-term ingestion of onion by rats were investigated. Plasma extracts of rats that had been fed onion-containing diets for one and two weeks were analyzed by HPLC with electrochemical detection after a treatment with sulfatase/beta-glucuronidase. Almost all of the quercetin metabolites in the plasma were sulfate/glucuronide conjugates of quercetin and isorhamnetin. More than 4.6% (w/w) of soybean oil in the diets significantly enhanced the accumulation of quercetin metabolites in the plasma. Fish oil and beef tallow increased this to an extent similar to that with soybean oil, and lecithin was more effective than the other three lipids. Two emulsifiers, sodium caseinate and sucrose fatty acid ester, also showed an enhancing effect on the accumulation of quercetin metabolites. These results indicate that co-ingested lipids and emulsifiers could enhance the bioavailability of quercetin glucosides in onion.