The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α^−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α^−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α^−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α^−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α^−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.     

Toll-like receptor 3 contributes to spinal glial activation and tactile allodynia after nerve injury

Toll-like receptors (TLRs) play an essential role in innate immune responses and in the initiation of adaptive immune responses. Microglia, the resident innate immune cells in the CNS, express TLRs. In this study, we show that TLR3 is crucial for spinal cord glial activation and tactile allodynia after peripheral nerve injury. Intrathecal administration of TLR3 antisense oligodeoxynucleotide suppressed nerve injury-induced tactile allodynia, and decreased the phosphorylation of p38 mitogen-activated protein kinase, but not extracellular signal-regulated protein kinases 1/2, in spinal glial cells. Antisense knockdown of TLR3 also attenuated the activation of spinal microglia, but not astrocytes, caused by nerve injury. Furthermore, down-regulation of TLR3 inhibited nerve injury-induced up-regulation of spinal pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α. Conversely, intrathecal injection of the TLR3 agonist polyinosine–polycytidylic acid induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Indeed, TLR3-deficient mice did not develop tactile allodynia after nerve injury or polyinosine–polycytidylic acid injection. Our results indicate that TLR3 has a substantial role in the activation of spinal glial cells and the development of tactile allodynia after nerve injury. Thus, blocking TLR3 in the spinal glial cells might provide a fruitful strategy for treating neuropathic pain.     

Unique cell surface phenotypes of proliferating lymphocytes in mice homozygous for lpr and gld mutations, defined by monoclonal antibodies to MRL/Mp-lpr/lpr T cells

In mice bearing the autosomal recessive gene of either lpr or gld, generalized T-cell proliferation and autoimmunity occurs. The surface antigen profiles of these proliferating cells were analyzed using two-color flow cytometry analysis with two newly established rat monoclonal antibodies (ALP-1, ALP-2) directed to lpr cells. The Lp-1 antigen, defined by ALP-1, is expressed exclusively on approximately one-half of proliferating lpr and gld lymph node cells. The Lp-2 antigen, like B 220, is expressed on 80-90% of lpr and gld lymph node cells, the cells in B-cell lineage and a small population of Ly-2+ T cells from normal mice. Thus, the lpr and gld lymph node cells were classified into three subsets, Lp-1+/Lp-2+, Lp-1-/Lp-2+ and Lp-1-/Lp-2-. After stimulation with Con A or a combination of IL-2 and phorbol ester, a small population of T cells from normal mice became Lp-1+. The same treatment increased Lp-2+/Ly-2+ and induced Lp-2+/L3T4+ T-cell populations. Therefore, it seems likely that these phenotypically unique T cells are generated at some stage during the proliferation and differentiation of certain normal T-cell subpopulations. The aberrant T cells in mice with lpr and gld mutations may even be normal regulatory T cells, if they are not proliferating abnormally.     

Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Phenotypic Plasticity in Photosynthetic Temperature Acclimation among Crop Species with Different Cold Tolerances

While interspecific variation in the temperature response of photosynthesis is well documented, the underlying physiological mechanisms remain unknown. Moreover, mechanisms related to species-dependent differences in photosynthetic temperature acclimation are unclear. We compared photosynthetic temperature acclimation in 11 crop species differing in their cold tolerance, which were grown at 15°C or 30°C. Cold-tolerant species exhibited a large decrease in optimum temperature for the photosynthetic rate at 360 μL L⁻¹ CO₂ concentration [Opt (A₃₆₀)] when growth temperature decreased from 30°C to 15°C, whereas cold-sensitive species were less plastic in Opt (A₃₆₀). Analysis using the C₃ photosynthesis model shows that the limiting step of A₃₆₀ at the optimum temperature differed between cold-tolerant and cold-sensitive species; ribulose 1,5-bisphosphate carboxylation rate was limiting in cold-tolerant species, while ribulose 1,5-bisphosphate regeneration rate was limiting in cold-sensitive species. Alterations in parameters related to photosynthetic temperature acclimation, including the limiting step of A₃₆₀, leaf nitrogen, and Rubisco contents, were more plastic to growth temperature in cold-tolerant species than in cold-sensitive species. These plastic alterations contributed to the noted growth temperature-dependent changes in Opt (A₃₆₀) in cold-tolerant species. Consequently, cold-tolerant species were able to maintain high A₃₆₀ at 15°C or 30°C, whereas cold-sensitive species were not. We conclude that differences in the plasticity of photosynthetic parameters with respect to growth temperature were responsible for the noted interspecific differences in photosynthetic temperature acclimation between cold-tolerant and cold-sensitive species.The temperature dependence of leaf photosynthetic rate shows considerable variation between plant species and with growth temperature (Berry and Björkman, 1980; Cunningham and Read, 2002; Hikosaka et al., 2006). Plants native to low-temperature environments and those grown at low temperatures generally exhibit higher photosynthetic rates at low temperatures and lower optimum temperatures, compared with plants native to high-temperature environments and those grown at high temperatures (Mooney and Billings, 1961; Slatyer, 1977; Berry and Björkman, 1980; Sage, 2002; Salvucci and Crafts-Brandner, 2004b). For example, the optimum temperature for photosynthesis differs between temperate evergreen species and tropical evergreen species (Hill et al., 1988; Read, 1990; Cunningham and Read, 2002). Such differences have been observed even among ecotypes of the same species (Björkman et al., 1975; Pearcy, 1977; Slatyer, 1977).Temperature dependence of the photosynthetic rate has been analyzed using the biochemical model proposed by Farquhar et al. (1980). This model assumes that the photosynthetic rate (A) is limited by either ribulose 1,5-bisphosphate (RuBP) carboxylation (A_c) or RuBP regeneration (A_r). The optimum temperature for photosynthetic rate in C₃ plants is thus potentially determined by (1) the temperature dependence of A_c, (2) the temperature dependence of A_r, or (3) both, at the colimitation point of A_c and A_r (Fig. 1; Farquhar and von Caemmerer, 1982; Hikosaka et al., 2006).Open in a separate windowFigure 1.A scheme illustrating the shift in the optimum temperature for photosynthesis depending on growth temperature. Based on the C₃ photosynthesis model, the A₃₆₀ (white and black circles) is limited by A_c (solid line) or A_r (broken line). The optimum temperature for the photosynthetic rate is potentially determined by temperature dependence of A_c (A), temperature dependence of A_r (B), or the intersection of the temperature dependences of A_c and A_r (C). When the optimum temperature for the photosynthetic rate shifts to a higher temperature, there are also three possibilities determining the optimum temperature: temperature dependence of A_c (D), temperature dependence of A_r (E), or the intersection of the temperature dependences of A_c and A_r (F). Especially in the case that the optimum temperature is determined by the intersection of the temperature dependences of A_c and A_r, the optimum temperature can shift by changes in the balance between A_c and A_r even when the optimum temperatures for these two partial reactions do not change.In many cases, the photosynthetic rate around the optimum temperature is limited by A_c, and thus the temperature dependence of A_c determines the optimum temperature for the photosynthetic rate (Hikosaka et al., 1999, 2006; Yamori et al., 2005, 2006a, 2006b, 2008; Sage and Kubien, 2007; Sage et al., 2008). As the temperature increases above the optimum, A_c is decreased by increases in photorespiration (Berry and Björkman, 1980; Jordan and Ogren, 1984; von Caemmerer, 2000). Furthermore, it has been suggested that the heat-induced deactivation of Rubisco is involved in the decrease in A_c at high temperature (Law and Crafts-Brandner, 1999; Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004a; Yamori et al., 2006b). Numerous previous studies have shown changes in the temperature dependence of A_c with growth temperature (Hikosaka et al., 1999; Bunce, 2000; Yamori et al., 2005). Also, the temperature sensitivity of Rubisco deactivation may differ between plant species (Salvucci and Crafts-Brandner, 2004b) and with growth temperature (Yamori et al., 2006b), which may explain variation in the optimum temperature for photosynthesis (Fig. 1, A and D).A_r is more responsive to temperature than A_c and often limits photosynthesis at low temperatures (Hikosaka et al., 1999, 2006; Sage and Kubien, 2007; Sage et al., 2008). Recently, several researchers indicated that A_r limits the photosynthetic rate at high temperature (Schrader et al., 2004; Wise et al., 2004; Cen and Sage, 2005; Makino and Sage, 2007). They suggested that the deactivation of Rubisco at high temperatures is not the cause of decreased A_c but a result of limitation by A_r. However, it remains unclear whether limitation by A_r is involved in the variation in the optimum temperature for the photosynthetic rate (Fig. 1, B and E).A shift in the optimum temperature for photosynthesis can result from changes in the balance between A_r and A_c, even when the optimum temperatures for these two partial reactions do not change (Fig. 1, C and F; Farquhar and von Caemmerer, 1982). The balance between A_r and A_c has been shown to change depending on growth temperature (Hikosaka et al., 1999; Hikosaka, 2005; Onoda et al., 2005a; Yamori et al., 2005) and often brings about a shift in the colimitation temperature of A_r and A_c. Furthermore, recent studies have shown that plasticity in this balance differs among species or ecotypes (Onoda et al., 2005b; Atkin et al., 2006; Ishikawa et al., 2007). Plasticity in this balance could explain interspecific variation in the plasticity of photosynthetic temperature dependence (Farquhar and von Caemmerer, 1982; Hikosaka et al., 2006), although there has been no evidence in the previous studies that the optimum temperature for photosynthesis occurs at the colimitation point of A_r and A_c.Temperature tolerance differs between species and, with growth temperature, even within species from the same functional group (Long and Woodward, 1989). Bunce (2000) indicated that the temperature dependences of A_r and A_c to growth temperature were different between species from cool and warm climates and that the balance between A_r and A_c was independent of growth temperature for a given plant species. However, it was not clarified what limited the photosynthetic rate or what parameters were important in temperature acclimation of photosynthesis. Recently, we reported that the extent of temperature homeostasis of leaf respiration and photosynthesis, which is assessed as a ratio of rates measured at their respective growth temperatures, differed depending on the extent of the cold tolerance of the species (Yamori et al., 2009b). Therefore, comparisons of several species with different cold tolerances would provide a new insight into interspecific variation of photosynthetic temperature acclimation and their underlying mechanisms. In this study, we selected 11 herbaceous crop species that differ in their cold tolerance (Yamori et al., 2009b) and grew them at two contrasting temperatures, conducting gas-exchange analyses based on the C₃ photosynthesis model (Farquhar et al., 1980). Based on these results, we addressed the following key questions. (1) Does the plasticity in photosynthetic temperature acclimation differ between cold-sensitive and cold-tolerant species? (2) Does the limiting step of photosynthesis at several leaf temperatures differ between plant species and with growth temperature? (3) What determines the optimum temperature for the photosynthetic rate among A_c, A_r, and the intersection of the temperature dependences of A_c and A_r?     

Comparative Analysis of argK-tox Clusters and Their Flanking Regions in Phaseolotoxin-Producing Pseudomonas syringae Pathovars

DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181–10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064–11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named “tox islands.” The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437–457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Effects of various growth conditions in a chemostat on expression of virulence factors in Porphyromonas gingivalis

Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide-limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.     

Cell-to-cell movement of the CAPRICE protein in Arabidopsis root epidermal cell differentiation

CAPRICE (CPC), a small, R3-type Myb-like protein, is a positive regulator of root hair development in Arabidopsis. Cell-to-cell movement of CPC is important for the differentiation of epidermal cells into trichoblasts (root hair cells). CPC is transported from atrichoblasts (hairless cells), where it is expressed, to trichoblasts, and generally accumulates in their nuclei. Using truncated versions of CPC fused to GFP, we identified a signal domain that is necessary and sufficient for CPC cell-to-cell movement. This domain includes the N-terminal region and a part of the Myb domain. Amino acid substitution experiments indicated that W76 and M78 in the Myb domain are critical for targeted transport, and that W76 is crucial for the nuclear accumulation of CPC:GFP. To evaluate the tissue-specificity of CPC movement, CPC:GFP was expressed in the stele using the SHR promoter and in trichoblasts using the EGL3 promoter. CPC:GFP was able to move from trichoblasts to atrichoblasts but could not exit from the stele, suggesting the involvement of tissue-specific regulatory factors in the intercellular movement of CPC. Analyses with a secretion inhibitor, Brefeldin A, and with an rhd3 mutant defective in the secretion process in root epidermis suggested that intercellular CPC movement is mediated through plasmodesmata. Furthermore, the fusion of CPC to tandem-GFPs defined the capability of CPC to increase the size exclusion limit of plasmodesmata.     

Effects of Various Growth Conditions in a Chemostat on Expression of Virulence Factors in Porphyromonas gingivalis

Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide-limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.     

Factors controlling induction of commitment of murine erythroleukemia (TSA8) cells to CFU-E (colony forming unit-erythroid)

On addition of DMSO, the MEL cell line TSA8 becomes committed into erythroid progenitor cells (CFU-E) which can form differentiated colonies in the presence of erythropoietin. To understand the mechanism of cellular commitment, the number and the affinity of the receptors for erythropoietin were estimated. The affinity of the receptors did not change before or after induction. The number of receptors changed depending on the growth phase, but was not dependent on the addition of the inducer. Thus, the presence of the receptors for erythropoietin may be required, but are not essential for responsiveness to erythropoietin. Further examination of the optimum conditions for commitment suggests that the concomitant actions of induced factor(s) with the receptors may control commitment of TSA8 cells to CFU-E.