Defective thymocyte development and perturbed homeostasis of T cells in STAT-induced STAT inhibitor-1/suppressors of cytokine signaling-1 transgenic mice

Previous experiments have shown that STAT-induced STAT inhibitor-1 (SSI-1; also named suppressors of cytokine signaling-1 (SOCS-1) or Janus kinase binding protein) is predominantly expressed in lymphoid organs and functions in vitro as a negative regulator of cytokine signaling. To determine the function of SOCS-1 in vivo, we generated SSI-1 transgenic mice using the lck proximal promoter that drives transgene expression in T cell lineage. In thymocytes expressing SSI-1 transgene, tyrosine phosphorylation of STATs in response to cytokines such as IFN-gamma, IL-6, and IL-7 was inhibited, suggesting that SSI-1 suppresses cytokine signaling in primary lymphocytes. In addition, lck-SSI-1 transgenic mice showed a reduction in the number of thymocytes as a result of the developmental blocking during triple-negative stage. They also exhibited a relative increase in the percentage of CD4+ T cells, a reduction in the number of gammadelta T cells, as well as the spontaneous activation and increased apoptosis of peripheral T cells. Thus, enforced expression of SSI-1 disturbs the development of thymocytes and the homeostasis of peripheral T cells. All these features of lck-SSI-1 transgenic mice strikingly resemble the phenotype of mice lacking common gamma-chain or Janus kinase-3, suggesting that transgene-derived SSI-1 inhibits the functions of common gamma-chain-using cytokines. Taken together, these results suggest that SSI-1 can also inhibit a wide variety of cytokines in vivo.     

Structural analysis of pituitary adenylate cyclase-activating polypeptides bound to phospholipid membranes by magic angle spinning solid-state NMR

PACAP (pituitary adenylate cyclase-activating polypeptide) is a member of the VIP/secretin/glucagon family, which includes the ligands of class II G-protein coupled receptors. Since the recognition of PACAP by the receptor may involve the binding of PACAP to membranes, its membrane-bound structure should be important. We have carried out structural analysis of uniformly 13C,15N labeled PACAP27 and its C-terminal truncated form PACAP(1-21)NH2 (PACAP21) bound to membranes with high resolution solid-state NMR. Phosphatidylcholine bilayers and phosphatidylcholine/phosphatidylglycerol bilayers were used for PACAP27 and PACAP21, respectively. Most backbone signals were assigned for PACAP27 and PACAP21. TALOS analysis revealed that both peptides take on extended conformations on the membranes. Dilution of PACAP21 did not change the conformation of the major part. Selective polarization transfer experiment confirmed that PACAP27 is interacting with the membranes. It was concluded that the interaction of PACAP with the membrane surface causes their extended conformation. PACAP27 is reported to take an alpha-helical conformation in dodecylphosphocholine micelles and membrane-binding peptides usually take similar conformations in micelles and in membranes. Therefore, the property of PACAP27 changing its conformation in response to its environment is unique. Its conformational flexibility may be associated with its wide variety of functions.     

An in vivo dual-reporter system of cyanobacteria using two railroad-worm luciferases with different color emissions

In vivo genetic reporter systems using luciferase enzymes enable the real-time monitoring of gene expression in living cells. We have challenged concurrent monitoring of two independent promoter activities within the same cells to precisely compare their characteristics in vivo. In this report, we describe a simple dual-reporter system capable of simultaneously monitoring two promoter activities in living cyanobacterial cells. Two railroad-worm luciferases catalyzing the bioluminescent emissions of different colors served as the dual reporters; each emission was successfully separated by interference filters to estimate the individual bioluminescence signals using photomultiplier tubes. Using this system, we clearly demonstrated the difference in the expression profiles between promoters in the same cells.     

Isolation of Key Methanogens for Global Methane Emission from Rice Paddy Fields: a Novel Isolate Affiliated with the Clone Cluster Rice Cluster I

Despite the fact that rice paddy fields (RPFs) are contributing 10 to 25% of global methane emissions, the organisms responsible for methane production in RPFs have remained uncultivated and thus uncharacterized. Here we report the isolation of a methanogen (strain SANAE) belonging to an abundant and ubiquitous group of methanogens called rice cluster I (RC-I) previously identified as an ecologically important microbial component via culture-independent analyses. To enrich the RC-I methanogens from rice paddy samples, we attempted to mimic the in situ conditions of RC-I on the basis of the idea that methanogens in such ecosystems should thrive by receiving low concentrations of substrate (H₂) continuously provided by heterotrophic H₂-producing bacteria. For this purpose, we developed a coculture method using an indirect substrate (propionate) in defined medium and a propionate-oxidizing, H₂-producing syntroph, Syntrophobacter fumaroxidans, as the H₂ supplier. By doing so, we significantly enriched the RC-I methanogens and eventually obtained a methanogen within the RC-I group in pure culture. This is the first report on the isolation of a methanogen within RC-I.     

Identification of Two-pore Channel 2 as a Novel Regulator of Osteoclastogenesis

Osteoclast differentiation is one of the critical steps that control bone mass levels in osteoporosis, but the molecules involved in osteoclastogenesis are still incompletely understood. Here, we show that two-pore channel 2 (TPC2) is expressed in osteoclast precursor cells, and its knockdown (TPC2-KD) in these cells suppressed RANKL-induced key events including multinucleation, enhancement of tartrate-resistant acid phosphatase (TRAP) activities, and TRAP mRNA expression levels. With respect to intracellular signaling, TPC2-KD reduced the levels of the RANKL-induced dynamic waving of Ca²⁺ in RAW cells. The search for the target of TPC2 identified that nuclear localization of NFATc1 is retarded in TPC2-KD cells. Finally, TPC2-KD suppressed osteoclastic pit formation in cultures. We conclude that TPC2 is a novel critical molecule for osteoclastogenesis.     

Effect of organic and inorganic fertigation on yields, δ15N values, and δ13C values of tomato (Lycopersicon esculentum Mill. cv. Saturn)

We examined the effects of fertilizer application, especially the effects of fertigation and types of fertilizer (inorganic and organic) on yields and ¹⁵N and ¹³C values of tomato (Lycopersicon esculentum Mill. cv. Saturn). Fertigation is a method in which an appropriate diluted liquid fertilizer is applied to the plants each time they are drip-irrigated. We developed a method of organic fertigation using corn steep liquor (CSL) as the liquid fertilizer, because it is an industrial byproduct of cornstarch manufacture and can be used very effectively. We compared fruit yield, mineral content, ¹⁵N value, and ¹³C value of tomatoes grown under three different fertilizer treatments, basal dressing: basal dressing with granular chemical fertilizer; inorganic fertigation: fertigation with liquid chemical fertilizer; and organic fertigation: fertigaion with CSL. Mineral contents of tomatoes grown with basal dressing were generally lower than those grown under either fertigation treatment. These results indicated that yields and mineral contents were influenced more by the method of fertilizer application than by whether the fertilizers were inorganic or organic. There were, however, significant differences in the ¹⁵N values of tomato fruits grown under different types of fertilizer applications, especially between inorganic and organic fertilizers. The ¹⁵N value of the chemical fertilizer used for basal dressing was 0.81 ± 0.45{}, that of the chemical fertilizer for fertigation was 0.00 ± 0.04{}, and that of CSL was 8.50 ± 0.71{}. The ¹⁵N values of the soils reflected the ¹⁵N values of the fertilizers. Moreover, the ¹⁵N values of the fruits corresponded to the ¹⁵N values of the applied fertilizers. The ¹⁵N values were 3.18 ± 1.34{} in the fruits grown with a basal dressing of chemical fertilizer, 0.30 ± 0.61 in those grown under inorganic fertigation, and 7.09 ± 0.68 in those grown under organic fertigation. On the other hand, although the ¹³C values in the soil also reflected the ¹³C values of the applied fertilizers, there was no significant difference in the ¹³C values of fruits among the different treatments. In conclusion, because the ¹⁵N values of fertilizers correlated well with those of the fruits, it may be possible to use ¹⁵N values as an indicator of organic products.     

CXCL10 DNA vaccination prevents spontaneous diabetes through enhanced beta cell proliferation in NOD mice

CXCL10, a chemokine for Th1 cells, is involved in the pathogenesis of various Th1-dominant autoimmune diseases. Type 1 diabetes is considered to be a Th1-dominant autoimmune disease, and a suppressive effect of CXCL10 neutralization on diabetes development has been reported in a cyclophosphamide-induced accelerated diabetes model through induction of beta cell proliferation. However, intervention in a diabetes model might bring about opposite effects, depending on the timing, amount, or method of treatment. In the present study, we examined the effect of CXCL10 neutralization in a "spontaneous diabetes" model of NOD mice, using CXCL10 DNA vaccination (pCAGGS-CXCL10). pCAGGS-CXCL10 treatment in young NOD mice induced the production of anti-CXCL10 Ab in vivo and suppressed the incidence of spontaneous diabetes, although this treatment did not inhibit insulitis or alter the immunological response. pCAGGS-CXCL10 treatment enhanced the proliferation of pancreatic beta cells, resulting in an increase of beta cell mass in this spontaneous diabetes model as well. Therefore, CXCL10 neutralization is suggested to be useful for maintaining beta cell mass at any stage of autoimmune diabetes.     

Return of inactivated whole-virus vaccine for superior efficacy

The swine, influenza, H1N1 outbreak in 2009 highlighted the inadequacy of the currently used antibody-based vaccine strategies as a preventive measure for combating influenza pandemics. The ultimate goal for successful control of newly arising influenza outbreaks is to design a single-shot vaccine that will provide long-lasting immunity against all strains of influenza A virus. A large amount of data from animal studies has indicated that the cross-reactive cytotoxic T (Tc) cell response against conserved influenza virus epitopes may be the key immune response needed for a universal influenza vaccine. However, decades of research have shown that the development of safe T-cell-based vaccines for influenza is not an easy task. Here, I discuss the overlooked but potentially highly advantageous inactivation method, namely, γ-ray irradiation, as a mean to reach the Holy Grail of influenza vaccinology.     

Graph-based clustering for finding distant relationships in a large set of protein sequences

MOTIVATION: Clustering of protein sequences is widely used for the functional characterization of proteins. However, it is still not easy to cluster distantly-related proteins, which have only regional similarity among their sequences. It is therefore necessary to develop an algorithm for clustering such distantly-related proteins. RESULTS: We have developed a time and space efficient clustering algorithm. It uses a graph representation where its vertices and edges denote proteins and their sequence similarities above a certain cutoff score, respectively. It repeatedly partitions the graph by removing edges that have small weights, which correspond to low sequence similarities. To find the appropriate partitions, we introduce a score combining the normalized cut and a locally minimal cut capacities. Our method is applied to the entire 40,703 human proteins in SWISS-PROT and TrEMBL. The resulting clusters shows a 76% recall (20,529 proteins) of the 26,917 classified by InterPro. It also finds relationships not found by other clustering methods. AVAILABILITY: The complete result of our algorithm for all the human proteins in SWISS-PROT and TrEMBL, and other supplementary information are available at http://motif.ics.es.osaka-u.ac.jp/Ncut-KL/     

Tryptamine-based human beta3-adrenergic receptor agonists. Part 2: SAR of the methylene derivatives

A series of tryptamine derivatives with modified sulfonamide were designed, synthesized, and evaluated for their ability to stimulate cAMP accumulation in CHO cells expressing the cloned human β₃-adrenergic receptor (AR). For this series of compounds, our objective was to symmetrize the -position of the tryptamine moiety maintaining its activity and reducing the cost of production. Compound 11h, having m-aminobenzene, exhibited excellent agonistic activity for β₃-AR with excellent subtype selectivity.