Intraspecific Sequence Variation of Chloroplast DNA Reflecting Variety and Geographical Distribution of <Emphasis Type="Italic">Polygonum cuspidatum</Emphasis> (Polygonaceae) in Japan

Polygonum cuspidatum in Japan, we analyzed the chloroplast DNA sequences of a region from the rbcL to the accD gene (ca. 1,420 bp), and found nucleotide variations at 22 sites in 68 samples. The phylogenetic relationship deduced from the sequence variations revealed the existence of at least five groups. The first group consisted of P. cuspidatum var. cuspidatum in the central part of Honshu; in Nagano, Yamanashi, and Shizuoka. The second, a sister of the first, consisted of those plants in Shizuoka-Itoigawa Line. The third group consisted of plants in the northern part of Japan including P. sachalinense in Hokkaido, P. cuspidatum var. cuspidatum in Aomori and var. uzensis in Akita. The fourth consisted of var. uzensis in the Tohoku District. The fifth consisted of var. terminalis in the Izu Islands. P. cuspidatum are differentiated according to their distribution, and two varieties, var. terminalis and var. uzensis, are differentiated genetically. Polygonum sachalinensis, a distinct species morphologically, fell into the accessions of P. cuspidatum on the phylogenetic tree obtained in the present study. Received 9 July 2000/ Accepted in revised form 11 October 2000     

Staphylococcus aureus protein A triggers T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands

B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. Conclusion: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation.     

Human blood monocyte activation byNocardia rubra cell wall skeleton for productions of interleukin 1 and tumor necrosis factor-α

Human blood monocytes were obtained from peripheral blood of healthy donors by counter-flow centrifugal elutriation. Functional integrity of monocytes for production of interleukin 1 (IL-1) and tumor necrosis factor (TNF-) in response toNocardia rubra cell wall skeleton (N-CWS) was examined by bioassay and enzyme immunoassay. Monocytes treated with N-CWS at more than 0.5 g/ml produced IL-1 and TNF- extracellularly. Extracellular TNF activity appeared within 4 h, and maximally, 16 h after N-CWS stimulation, whereas longer time was needed for IL-1 activity to appear, the peak production being at 24 h. The neutralizing experiment also showed that anti TNF- antibody did not affect IL-1 production by the monocytes treated with N-CWS, suggesting independen cy of IL-1 production of TNF-.These results suggest that the therapeutic antitumor effect of N-CWS is due, in part at least, to the augmented production of these monokines.     

Phylogenetic analyses of <Emphasis Type="Italic">Phragmites</Emphasis> spp. in southwest China identified two lineages and their hybrids

The species/lineage delimitation and possible hybridization/introgression are prerequisites in the management of invasive organism. Phragmites australis invaded diverse habitats and displaced the native lineages in North America as a consequence of the introduction from the Eurasia. Such species threatened the biodiversity safety of the invaded regions, in particular the biodiversity hot spots. Southwest (SW) China is a biodiversity hot spot with the occurrence of Phragmites species, both native and introduced. However, the genetic identity of Phragmites species in this biodiversity hot spot remains unclear, hampering effective ecological managements. In this study, we explored the phylogenetic lineages of Phragmites species in SW China. A total of 44 accessions sampled across SW China were analyzed using two chloroplast DNA (cpDNA) markers and amplified fragment length polymorphisms. Two genetic lineages were recovered, i.e., (1) the tropical lineage which primarily consisted of native Phragmites species represented by cpDNA haplotypes I, Q, and U in relatively low altitude and (2) the common lineage including native species at higher elevations in the Hengduan Mountains as well as artificially planted species represented by cpDNA haplotype P. The between-lineage hybridization was suggested for five analyzed accessions collected from either natural or artificial habitats. The putative hybrids might have originated from the maternal native tropical lineages and paternal introduced common lineage. Our results suggest the likelihood of introgressive hybridizations in SW China and thus provided implications for future research and ecological management.     

Enhancement of translation elongation in neurons by brain-derived neurotrophic factor: implications for mammalian target of rapamycin signaling

The effects and signaling mechanisms of brain-derived neurotrophic factor (BDNF) on translation elongation were investigated in cortical neurons. BDNF increased the elongation rate approximately twofold, as determined by measuring the ribosomal transit time. BDNF-accelerated elongation was inhibited by rapamycin, implicating the mammalian target of rapamycin (mTOR). To explore the mechanisms underlying these effects, we examined the protein phosphorylation cascades that lead to the activation of translation elongation in neurons. BDNF increased eukaryote elongation factor 1A (eEF1A) phosphorylation and decreased eEF2 phosphorylation. Whereas eEF2 phosphorylation levels altered by BDNF were inhibited by rapamycin, eEF1A phosphorylation was not affected by rapamycin or PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor. BDNF induced phosphorylation of eEF2 kinase (Ser366), as well as decreased its kinase activity. All these events were inhibited by rapamycin. Furthermore, mTOR siRNA, which reduced mTOR levels up to 50%, inhibited the BDNF-induced enhancement in elongation rate and decrease in eEF2 phosphorylation. These results strongly suggest that BDNF enhances translation elongation through the activation of the mTOR-eEF2 pathway.     

Response to ADP-Ribose by Activation of TRPM2 in the CRI-G1 Insulinoma Cell Line

The response to intracellular ADP-ribose in the rat CRI-G1 insulinoma cell line was studied using a patch-clamp method. Dialysis of ADP-ribose into cells induced a response in a dose-dependent manner. The reversal potentials in various solutions showed that the ADP-ribose-gated channel was a Ca2+-permeable nonselective cation channel. In inside-out recordings, ADP-ribose and b-NAD induced responses in the same patch. The single-channel current-voltage relationships for ADP-ribose- and b-NAD-induced responses were almost identical, indicating that ADP-ribose and b-NAD activated the same channel. The physiological properties of the ADP-ribose-gated channel are similar to those we reported previously for the cloned transient receptor potential channel TRPM2. Moreover, RT-PCR analysis showed that TRPM2 was abundantly expressed in CRI-G1 cells, suggesting that the ADP-ribose-gated channel represents the native TRPM2 channel in CRI-G1 cells. These results suggest that ADP-ribose can be an endogenous modulator of Ca2+ influx through the TRPM2 channel into CRI-G1 cells.     

Human peptidoglycan recognition protein-L is an N-acetylmuramoyl-L-alanine amidase

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules coded by up to 13 genes in insects and 4 genes in mammals. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. In mammals one PGRP is an antibacterial neutrophil protein. We report that human PGRP-L is a Zn2+-dependent N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28), an enzyme that hydrolyzes the amide bond between MurNAc and l-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no direct bacteriolytic activity. The other members of the human PGRP family, PGRP-Ialpha, PGRP-Ibeta, and PGRP-S, do not have the amidase activity. The C-terminal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal delta1-343 deletion mutant) is reduced. The Zn2+ binding amino acids (conserved in PGRP-L and T7 amidase) and Cys-419 (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas three other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the "active" configuration does not convert PGRP-S into an active amidase. In conclusion, human PGRP-L is an N-acetylmuramoyl-l-alanine amidase and this function is conserved in prokaryotes, insects, and mammals.     

Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease

NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-L-Ala-D-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of L-Ala for D-Ala or D-isoGln for L-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development.