Genetic diversity and differentiation of the endangered Japanese endemic tree Magnolia stellata using nuclear and chloroplast microsatellite markers

Genetic diversity and differentiation were analyzed in 11 populations of Magnolia stellata (Sieb. and Zucc.) Maxim. (Magnoliaceae) in the Tokai district, Japan. Variation at four nuclear microsatellite (nSSR) loci was examined, three chloroplast microsatellite (cpSSR) markers were developed and 13 haplotypes identified. The 11 populations were divided into three groups (A, B and C). Each population within the group was separated less than 40 km. Group B harbored the highest gene diversity (H) and allelic richness (Ar) for nSSR (H=0.74 and Ar=8.02). Group C had the highest diversity of chloroplast haplotypes (H=0.79 and Ar=6.8): 2.5 times more haplotypes than the other groups. Each population contributed differently to the total diversity, with respect to nSSR and cpSSR. AMOVA revealed that 58% of haplotypic and 15% of nSSR variation was partitioned among populations within groups. A Mantel test revealed significant correlations between population pairwise geographic ln(distance) and F_ST/(1−F_ST) for both nSSR (r=0.479; P=0.001) and cpSSR (r=0.230; P=0.040). Dendrograms of populations for nSSR, based on Nei’s genetic distance, were constructed using UPGMA and the neighbor-joining method. These results suggest that populations in group C have diverged from the other populations, while those in group B are similar to each other. For group B, fragmentation between populations should be avoided in order to maintain gene flow. For group C, the uniqueness of each population should be given the highest priority when planning genetic conservation measures for the species.     

An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4+ T cells

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation, induces proliferation of naive CD4+ T cells, and synergizes with IL-12 in IFN-gamma production. It has been recently reported that IL-27 induces T-bet and IL-12Rbeta2 expression through JAK1/STAT1 activation. In the present study, we further investigated the JAK/STAT signaling molecules activated by IL-27 and also the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. In addition to JAK1 and STAT1, IL-27-activated JAK2, tyrosine kinase-2, and STAT2, -3, and -5 in naive CD4+ T cells. The activation of STAT2 and STAT5, but not of STAT3, was greatly diminished in STAT1-deficient naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 hardly induced T-bet and subsequent IL-12Rbeta2 expression, and synergistic IFN-gamma production by IL-27 and IL-12 was impaired in STAT1-deficient naive CD4+ T cells. Moreover, IL-27 augmented the expression of MHC class I on naive CD4+ T cells in a STAT1-dependent manner. These results suggest that IL-27 activates JAK1 and -2, tyrosine kinase-2, STAT1, -2, -3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and subsequent IL-12Rbeta2 expression and MHC class I expression as well but not proliferation, while STAT3 presumably plays an important role in IL-27-induced proliferation.     

BCL-6 mutations in pulmonary lymphoproliferative disorders: demonstration of an aberrant immunological reaction in HIV-related lymphoid interstitial pneumonia

We used a PCR and sequence procedure to analyze the Ig V(H) gene and the mutations in the 5' regulatory regions of BCL-6 genes in pulmonary lymphoproliferative disorders (mucosa-associated lymphoid tissue (MALT) lymphoma, HIV-related, EBV-related, and virus-negative lymphocytic interstitial pneumonia (LIP)). Eight of 20 (40%) pulmonary MALT lymphoma and 10 of 20 LIP (5 of 5 (100%) HIV-related, 2 of 5 (40%) EBV-related, and 3 of 10 (30%) virus-negative LIP) cases showed BCL-6 gene mutations. Intraclonal heterogeneity of the BCL-6 mutations was observed only in pulmonary MALT lymphoma cases whose Ig V(H) genes also showed intraclonal heterogeneity. Ongoing BCL-6 mutations might reflect re-entry into a germinal center pathway to further mutations. BCL-6 mutations in pulmonary MALT lymphoma and HIV-negative LIP showed some features (high transition to transversion ratio, standard polarity, and RGYW/WRCY bias) of Ig V(H) gene hypermutation, leading to the view that pulmonary MALT lymphomas and HIV-negative LIP are under the influence of germinal center hypermutation mechanisms. Because BCL-6 mutations in HIV-related LIP cases did not demonstrate features of Ig V(H) gene hypermutation, immunological reactions in HIV-related LIP are the result of a process different from that found in HIV-negative pulmonary lymphoproliferative disorders.     

2,2-Functionalized analogues of 1alpha,25-dihydroxyvitamin D3, the potent inducers of cell differentiation

All four possible A-ring stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin D(3) (4) were designed and convergently synthesized. Nine-step conversion of methyl hydroxypivalate 6 provided the desired A-ring enyne synthon (13a,b) in good overall yield. Cross-coupling reaction of the A-ring synthon 13a,b with the CD-ring portion in the presence of palladium catalyst, followed by deprotection, gave the vitamin analogues (4a-d). We also synthesized four stereoisomers of 2,2-ethano-1,25-dihydroxyvitamin D(3) (5), as novel spiro-ring analogues having cyclopropane fused at the C2 position. Biological potencies of the synthesized compounds were assessed in terms of the vitamin D receptor (VDR) binding affinity, as well as the HL-60 cell differentiation-inducing activity. The 2,2-ethano analogue 5a showed a comparable activity to the natural hormone 1, while the 2,2-dimethyl analogue 4a exhibited one-third of the activity of 1 in cell differentiation, with the reduced VDR binding affinity.     

Synthesis of an immunosuppressant SQAG9 and determination of the binding peptide by T7 phage display

SQAG9, a new class of immunosuppressive sulfoquinovosylacylglycerol, and its biotinylated derivatives have been synthesized. A T7 Phage library, composed of random cDNA fragments from Drosophila melanogaster, displayed a possible binding peptide of 14 amino acids. The immobilized synthetic peptide on a sensor chip showed a dissociation constant of K(D)=1.5 x 10(-6) against SQAG9 in a surface plasmon resonance experiment.     

Dopamine Receptor 2 Regulates L-Type Voltage-Gated Calcium Channel in Primary Cultured Mouse Midbrain Neural Network

1. Synchronous oscillation of intracellular Ca²⁺ in the central nervous system is essential for neural development. We previously reported that endogenous dopamine was involved with synchronous Ca²⁺ oscillation of primary cultured midbrain neurons, and that regulation of dopamine in synchronous oscillation was distinctly different through dopamine receptor 1 (D1R) and 2 (D2R): the action of dopamine through D1R or D2R was facilitative or suppressive, respectively, to the Ca²⁺ influx of synchronous oscillation.2. In the present study, we confirmed that the suppressive effects of D2R were mediated by the regulation of the L-type voltage-gated Ca²⁺ channel, not by the regulation of NMDA receptor on the Ca²⁺ influx in the midbrain neural network showing synchronous oscillation.3. This evidence promotes better understanding of the regulation of neural activity by endogenous dopamine in networked neurons.     

Effect of aeration on gliotoxin production by Aspergillus fumigatus in its culture filtrate

Gliotoxin, one of the mycotoxins produced by Aspergillus fumigatus, has various, potent bioactivities. However, it has not been considered to be a toxic (or virulence) factor because of its slow production. The aim of the present study was to investigate the effects of aeration on the cytotoxicity of A. fumigatus culture filtrate, and to determine the optimal condition for the rapid production of gliotoxin from this fungus. Fungal culture filtrates were made in three different containers under various conditions of aeration and O2 concentration. These filtrates were compared in terms of their cytotoxicity on murine macrophages and analyzed by gas chromatography. The culture filtrate showed high cytotoxicity when it was made under highly aerated conditions, but it was significantly less cytotoxic when prepared under non-aerated conditions. The cytotoxic activity became evident within 15 h of culture at 20% O2, when the fungus had already started producing gliotoxin. The culture filtrates also contained some other as yet unidentified substances that might also to some extent contribute to the cytotoxicity. In light of these results, the authors propose that a highly aerated condition is responsible for the rapid production of gliotoxin, and that gliotoxin might play an important role in the respiratory infection by A. fumigatus, with other toxic substances acting additively or synergistically.     

Induction of IgG2a class switching in B cells by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. However, its role in B cells remains unexplored. We here show a role for IL-27 in the induction of T-bet expression and regulation of Ig class switching in B cells. Expression of WSX-1, one subunit of IL-27R, was detected at the mRNA level in primary mouse spleen B cells, and stimulation of these B cells by IL-27 rapidly activated STAT1. IL-27 then induced T-bet expression and IgG2a, but not IgG1, class switching in B cells activated with anti-CD40 or LPS. In contrast, IL-27 inhibited IgG1 class switching induced by IL-4 in activated B cells. Similar induction of STAT1 activation, T-bet expression and IgG2a class switching was observed in IFN-gamma-deficient B cells, but not in STAT1-deficient ones. The induction of IgG2a class switching was abolished in T-bet-deficient B cells activated with LPS. These results suggest that primary spleen B cells express functional IL-27R and that the stimulation of these B cells by IL-27 induces T-bet expression and IgG2a, but not IgG1, class switching in a STAT1-dependent but IFN-gamma-independent manner. The IL-27-induced IgG2a class switching is highly dependent on T-bet in response to T-independent stimuli such as LPS. Thus, IL-27 may be a novel attractive candidate as a therapeutic agent against diseases such as allergic disorders by not only regulating Th1 differentiation but also directly acting on B cells and inducing IgG2a class switching.     

IL-6 regulates in vivo dendritic cell differentiation through STAT3 activation

Dendritic cells (DCs) orchestrate immune responses according to their state of maturation. In response to infection, DCs differentiate into mature cells that initiate immune responses, while in the absence of infection, most of them remain in an immature form that induces tolerance to self Ags. Understanding what controls these opposing effects is an important goal for vaccine development and prevention of unwanted immune responses. A crucial question is what cytokine(s) regulates DC maturation in the absence of infection. In this study, we show that IL-6 plays a major role in maintaining immature DCs. IL-6 knockout (KO) mice had increased numbers of mature DCs, indicating that IL-6 blocks DC maturation in vivo. We examined this effect further in knockin mice expressing mutant versions of the IL-6 signal transducer gp130, with defective signaling through either Src homology region 2 domain-containing phosphatase 2/Gab/MAPK (gp130(F759/F759)) or STAT3 (gp130(FxxQ/FxxQ)), and combined gp130 and IL-6 defects (gp130(F759/F759)/IL-6 KO mice). Importantly, we found STAT3 activation by IL-6 was required for the suppression of LPS-induced DC maturation. In addition, STAT3 phosphorylation in DCs was regulated by IL-6 in vivo, and STAT3 was necessary for the IL-6 suppression of bone marrow-derived DC activation/maturation. DC-mediated T cell activation was enhanced in IL-6 KO mice and suppressed in gp130(F759/F759) mice. IL-6 is thus a potent regulator of DC differentiation in vivo, and IL-6-gp130-STAT3 signaling in DCs may represent a critical target for controlling T cell-mediated immune responses in vivo.