Vα14 NKT cells activated by alpha-galactosylceramide augment lipopolysaccharide-induced nitric oxide production in mouse intra-hepatic lymphocytes

Vα14 natural killer T (Vα14 NKT) cells activated by α-galactosylceramide (α-GalCer) secrete a large amount of Th1 and Th2 cytokines. IFN-γ plays a crucial role in the inflammation response, and is also known as an activator of nitric oxide (NO) production. We previously reported that lipopolysaccharide (LPS)-induced NO production is augmented by α-GalCer in mouse peritoneal cells. Since the liver is susceptible to LPS stimulation via the portal vein, we examined the effect of α-GalCer on LPS-induced NO production in murine intra-hepatic lymphocytes (IHLs). Although IHLs augmented LPS-induced NO production by α-GalCer administration, such an augmentation was not observed in non-treated mice. Furthermore, α-GalCer did not augment LPS-induced NO production in IHLs from IFN-γ knockout mice. In flow cytometry analysis of IHLs from α-GalCer-treated mice, the ratio and number of F4/80- and TLR4-positive cells rose as compared with non-treated mice. The liver injury may be induced by LPS and NO under the condition where Vα14 NKT cells were activated.     

Rapid sequencing gel electrophoresis using glycerol-tolerant sodium taurine medium

We describe a new glycerol-tolerant sodium taurine (ST) medium for rapid sequencing gel electrophoresis by substituting the standard conductive media of Tris-boric acid-ethylenediaminetetraacetic acid (EDTA) (TBE) and Tris-taurine-EDTA (TTE) and other low-ionic-strength media of sodium boric acid (SB). Low-ionic-strength and cost-effective ST media gave glycerol tolerance up to 50% (v/v) glycerol-containing DNA sample solution, shorter running time, and better resolution to separate small DNA oligonucleotides (20-45 mer) in 12% denaturing sequencing gel electrophoresis.     

Effects of tidal fluctuations on CO2 and CH4 fluxes in the littoral zone of a brackish-water lake

We examined the effects of tidal fluctuations on CO₂ and CH₄ fluxes from sediment or soil to the atmosphere in the littoral zone of a brackish-water lake during the growing seasons in 2004 and 2005. The dominant plants at the study site formed three sub-zones (Phragmites zone, Juncus zone and Miscanthus zone) across a topographic gradient on the shoreline. In the Phragmites and Juncus zones, we observed a positive correlation between hourly changes in CO₂ and CH₄ fluxes and changes in the water table. In particular, the magnitude and pattern of daily variation in CO₂ and CH₄ fluxes were different on days during spring tide and neap tide in the Phragmites and Juncus zones. Variations in CO₂ and CH₄ fluxes in the Phragmites and Juncus zones over the course of a day during spring tide were correlated with water-table level. We found that the rate of change of the water table, as distinguished from just differences in the water table, was a major environmental factor controlling the CO₂ and CH₄ fluxes. In the Miscanthus zone during spring tide, soil temperature was the main factor affecting daily variation in CO₂ and CH₄ fluxes.     

Hepcidin Revisited, Disulfide Connectivity, Dynamics, and Structure

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys¹–Cys⁸, Cys³–Cys⁶, Cys²–Cys⁴, and Cys⁵–Cys⁷. NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.Regulation of iron levels is critical to the survival of species that live in an oxygen-rich environment (1). In mammals, iron homeostasis is principally regulated by hepcidin, a 25-residue peptide hormone containing a complex network of four disulfide bonds. Hepcidin was discovered by three groups investigating either novel anti-microbial peptides or iron regulation (2–4), and subsequent genetic evidence has shown that mutation of the hepcidin gene can lead to systemic iron overload or hemochromatosis (5). Similarly, mutations in upstream control proteins HFE and hemojuvelin or mutation of the gene for ferroportin, the hepcidin receptor, cause forms of hemochromatosis of varying clinical severity (6–9). Genetic studies in mice have confirmed these relationships, identifying the hepcidin pathway as a critical component in the control of iron metabolism (10–12). Dysfunction of the hepcidin pathway and the resulting iron imbalance may play a role in multiple diseases such as anemia of inflammation (13), atherosclerosis (14), and neurodegenerative disorders (15). In anemia of inflammation, suppression of hepcidin constituted a successful treatment, suggesting that it may be an appropriate therapeutic target in the treatment of disease.³The human hepcidin gene encodes an 84-residue prepropeptide that contains a 24-residue N-terminal signal peptide that is subsequently cleaved to produce pro-hepcidin. Pro-hepcidin is then processed to produce a mature 25-amino acid hepcidin that is detectable in both blood and urine. Mass spectrometry and chemical analysis have revealed that all eight cysteines in hepcidin are involved in disulfide bonds (3) suggesting a highly constrained structure containing a precise disulfide bonding pattern.The NMR solution structure of hepcidin first reported by Hunter et al. (16) revealed a compact fold with β-sheet and β-hairpin loop elements. From structure calculations and dynamic signatures in NMR spectra, the authors inferred a disulfide connectivity of Cys¹–Cys⁸, Cys²–Cys⁷, Cys³–Cys⁶,⁴ and a rare vicinal disulfide bond at Cys⁴–Cys⁵. A later study of bass hepcidin (17) determined essentially the same fold and confirmed the same disulfide connectivity. Both studies, however, were based on incomplete NMR data because the resonances from two adjacent cysteines, Cys-13 and Cys-14 of hepcidin, were not detected, presumably due to exchange broadening.Here we demonstrate a new pattern of disulfide connectivity obtained independently from chemical and spectroscopic analysis. In addition, we present the first complete solution NMR structure of hepcidin and x-ray structure of the peptide in complex with an anti-hepcidin Fab. NMR data obtained at different temperatures reveal that hepcidin exhibits significant conformational dynamics in solution, a problem that likely occluded previous NMR studies. Data presented here show that these dynamics can be almost completely resolved by temperature variation, yielding two distinct structures of hepcidin, one at 325 K and one at 253 K in supercooled water. In addition to inferring disulfide bonds from structure calculations, we present an argument based on probabilistic interpretation of NMR data, which unequivocally establishes the same connectivity as obtained from chemical analysis.Because of the complexity of the disulfide network, hepcidin production is prone to misfolding artifacts. We demonstrate this through biophysical and biological activity characterization of hepcidin samples obtained from different sources. This information is essential for establishing accurate standards for quantitation of hepcidin levels in humans. In our experience, the highest quality material appeared to be critical for the structural studies presented here.     

Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture

Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.     

The outer membrane fraction of Flavobacterium psychrophilum induces protective immunity in rainbow trout and ayu

Flavobacterium psychrophilum is the causative agent of coldwater disease, which is responsible for serious losses in fish aquaculture in several parts of the world. No commercial vaccines are currently available for the prevention of coldwater disease. The present study sought to assess the efficacy of a F. psychrophilum vaccine based on the antigenic outer membrane fraction (OMF). This fraction induced significantly higher protection against coldwater disease in both rainbow trout (Oncorhynchus mykiss) and ayu (Plecoglossus altivelis) compared to inactivated whole cell F. psychrophilum bacterin. Coincident with higher protection, sera of fish immunised with the OMF vaccine had higher agglutination titres than those of fish immunised with inactivated whole cell F. psychrophilum.     

Essential role of IkappaB kinase alpha in thymic organogenesis required for the establishment of self-tolerance

IkappaB kinase (IKK) alpha exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-kappaB activation pathway. The in vivo function of IKKalpha, however, still remains elusive. Because a natural strain of mice with mutant NF-kappaB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKalpha axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKalpha-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKalpha-dependent manner was associated with defective processing of NF-kappaB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKalpha in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.