We have used oligonucleotide microarrays to detect Arabidopsis gene expression during early flower development. Among the 22,746 genes represented on the Affymetrix ATH1 chip, approximately 14,660 (approximately 64.5%) genes were expressed with signal intensity at or more than 50 in each of the six organs/structures examined, including young inflorescences (floral stages 1-9), stage-12 floral buds, developing siliques, leaves, stems, and roots. 17,583 genes were expressed with an intensity at or above 50 in at least one tissue, including 12,245 genes that were expressed in all the six tissues. Comparison of genes expressed between young inflorescence or stage-12 floral buds with other tissues suggests that relatively large numbers of genes are expressed at similar levels in tissues that are related morphologically and/or developmentally, as supported by a cluster analysis with data from two other studies. Further analysis of the genes preferentially expressed in floral tissues has uncovered new genes potentially involved in Arabidopsis flower development. One hundred and four genes were determined to be preferentially expressed in young inflorescences, including 22 genes encoding putative transcription factors. We also identified 105 genes that were preferentially expressed in three reproductive structures (the young inflorescences, stage-12 floral buds and developing siliques), when compared with the vegetative tissues. RT-PCR results of selected genes are consistent with the results from these microarrays and suggest that the relative signal intensities detected with the Affymetrix microarray are reliable estimates of gene expression.

Background:The development of platelet concentrated biomaterials has gained increasing awareness for regenerative medicine. With different protocol, derivatives such as advanced platelet-rich fibrin (A-PRF), injected platelet-rich fibrin, and concentrated growth factor (CGF) have been demonstrated effectively in preclinical and clinical studies. The aim of this study was to compare the level of growth factors releasing from A-PRF and CGF, and their clinical efficacy in the regenerative management of intrabony defects (IBDs).Methods: Thirty-two blood samples were collected from eight healthy donors and assessed for platelet-derived growth factor-, vascular endothelial growth factor, bone morphogenetic protein-2, and transforming growth factor-1 release at indicated times. In addition, the clinical records of 45 patients (15 per group) who had undergone guided tissue regeneration (GTR) with or without A-PRF/CGF were retrieved. The probing depth (PD) and clinical attachment level (CAL) were recorded preoperatively and 6 months postoperatively. Intrabony component (IC) depth, radiographic bone level (RBL), and bone defect filling were assessed radiographically. Results:A-PRF had a looser fibrin network than the CGF but presented larger amounts of growth factors with a more sustained release period. Although there was no difference in PD reduction, CAL gain, RBL height change and defect filling (%) between A-PRF and CGF group, both achieved a more favorable clinical result in IC height reduction and defect filling (%) than the control. Conclusions:A-PRF and CGF have the ability to stimulate a continual and steady release of total growth factors over a 14-day period. A-PRF and CGF show a similar effectiveness in periodontal bone regeneration with a potential benefit of improving GTR outcomes in IBD treatment. K E Y W O R D Sbone substitutes, growth factors, guided tissue regeneration, periodontitis, platelet-rich fibrin 462

Erythroid homeostasis depends critically upon erythropoietin (Epo) and stem cell factor cosignaling in late progenitor cells. Epo bioresponses are relayed efficiently by minimal receptor forms that retain a single Tyr-343 site for STAT5 binding, while forms that lack all cytoplasmic Tyr(P) sites activate JAK2 and the transcription of c-Myc plus presumed additional target genes. In FDCER cell lines, which express endogenous c-Kit, the signaling capacities of such minimal Epo receptor forms (ER-HY343 and ER-HY343F) have been dissected to reveal: 1) that Epo-dependent mitogenesis, survival, and bcl-x gene expression via ER-HY343 depend upon the intactness of the Tyr-343 STAT5 binding site; 2) that ER-HY343-dependent bcl-x L gene transcription is enhanced markedly via c-Kit; 3) that socs-3, plfap, dpp-1, and cacy-bp gene transcription is induced via ER-HY343, whereas dpp-1 and cacy-bp gene expression is also supported by ER-HY343F; 4) that ectopically expressed SOCS-3 suppresses proliferative signaling by not only ER-HY343 but also c-Kit; and 5) that in FDCER and primary erythroid cells, c-Kit appears to provide the primary route to MAPK activation. Thus, integration circuits exist in only select downstream pathways within Epo and stem call factor receptor signaling. Epo,1 the prime hormonal regulator of red cell development, initiates its effects by binding to receptor dimers on the surface of erythroid burst-and colony-forming units and activating the tethered Janus family kinase (JAK) 2 (1, 2). JAK2 then mediates the phosphorylation of eight cytoplasmic tyrosine sites within the Epo receptor, and via these sites, a complex set of Src homology 2 domain-encoding effectors (and associated cofactors) are engaged. These include STAT 5A and B; Grb2/ mSOS/Raf/Ras; phosphatidylinositol 3-kinase, phospholipase-␥1; and SHIP; Lyn, Syk, and Tec; SHPTP-1 and 2; the nucleotide exchange factors Vav and C3G (via Cbl); Cis and SOCS-3; and the adaptors Shc, Gab1, Gab2, CrkL, APS, and IRS-2 (reviewed by Wojchowski et al. (Ref. 3)). Although many of these are proto-oncogenic growth regulators, others are negative effectors whose action in terminating Epo-stimulated events is likewise crucial to regulated erythropoiesis. These include Cis, which appears to compete with STAT5 for binding at Tyr-343 (4, 5); SHPTP-1, which acts to dephosphorylate JAK2 (6), and the suppressor of cytokine signaling, SOCS-3, which also binds and inhibits JAK2 (7). In SOCS-3 Ϫ/Ϫ mice, in fact, a fatal erythrocytosis is precipitated (8).Despite the complexity of this signaling network, studies of tyrosine-mutated and -truncated Epo receptors in cell lines (9, 10), murine fetal liver (11, 12), and adult murine marrow and spleen (13) 2)), definitive erythropoiesis fails and lethal embryonic anemias are engendered. In at least certain systems, however, Epo receptor forms that lack all cytoplasmic Tyr(P) sites (yet activate JAK2) also have been reported to retain significant bioactivity (4), and among STAT5 a Ϫ/Ϫ and b Ϫ/Ϫ mice those which survive embryonic stre...

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been shown to differ from enterobacterial LPS in structure and function; therefore, the Toll-like receptors (TLRs) and the intracellular inflammatory signaling pathways are accordingly different. To elucidate the signal transduction pathway of P. gingivalis, LPS-induced pro-inflammatory cytokine production in the human monocytic cell line THP-1 was measured by ELISA, and the TLRs were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors as well as Phospho-ELISA kits were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. In this study, P. gingivalis LPS showed the ability to induce cytokine production in THP-1 cells and its induction was significantly (P < 0.05) suppressed by anti-TLR2 antibody or JNK inhibitor, and the phosphorylation level of JNK was significantly increased (P < 0.05). These results indicate that TLR2-JNK is the main signaling pathway of P. gingivalis LPS-induced cytokine production, while the cytokine induction by E. coli LPS was mainly via TLR4-NF-kappaB and TLR4-p38MAPK. This suggests that P. gingivalis LPS differs from E. coli LPS in its signaling pathway in THP-1 cells, and that the TLR2-JNK pathway might play a significant role in P. gingivalis LPS-induced chronic inflammatory periodontal disease.

Little is known of how Toll-like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL-1 is processed in macrophages after recognition by TLR2. FSL-1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl-beta-cyclodextrin, which inhibit clathrin-dependent endocytosis, reduced FSL-1 uptake by RAW264.7 cells in a dose-dependent manner but nystatin, which inhibits caveolae- and lipid raft-dependent endocytosis, did not. FSL-1 was co-localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL-1 is clathrin dependent. In addition, FSL-1 was internalized by peritoneal macrophages from TLR2-deficient mice. FSL-1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non-transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL-1 uptake. In this study, we suggest that (i) FSL-1 is internalized into macrophages via a clathrin-dependent endocytic pathway, (ii) the FSL-1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL-1.

The adult erythron is maintained via dynamic modulation of erythroblast survival potentials. Toward identifying novel regulators of this process, murine splenic erythroblasts at 3 developmental stages were prepared, purified and profiled. Stage-to-stage modulated genes were then functionally categorized, with a focus on apoptotic factors. In parallel with BCL-X and NIX, death-associated protein kinase-2 (DAPK2) was substantially upmodulated during late erythropoiesis. Among hematopoietic lineages, DAPK2 was expressed predominantly in erythroid cells. In a Gata1-IE3.9int-DAPK2 transgenic mouse model, effects on steady-state reticulocyte and red blood cell (RBC) levels were limited. During hemolytic anemia, however, erythropoiesis was markedly deficient. Ex vivo analyses revealed heightened apoptosis due to DAPK2 at a Kit ؊ CD71 high Ter119 ؊ stage, together with a subsequent multifold defect in late-stage Kit ؊ CD71 high Ter119 ؉ cell formation. In UT7epo cells, siRNA knockdown of DAPK2 enhanced survival due to cytokine withdrawal, and DAPK2's phosphorylation and kinase activity also were erythropoietin (EPO)-modulated. DAPK2 therefore comprises a new candidate attenuator of stress erythropoiesis. (Blood. 2008;112:886-890) IntroductionErythroid progenitor cell survival is regulated by unique networked mechanisms. As cell-extrinsic factors, FASL and TRAIL can induce apoptosis, while erythropoietin (EPO) provides essential survival signals via JAK2/STAT5, PI3K/AKT, and RAS/MEK/ ERK1,2 routes. 1 Cell-intrinsic regulators include BCL-X and NIX, plus GATA1 as a caspase-3 target. [2][3][4] To define new potential key survival-regulating factors, we presently profiled differentially staged primary murine splenic erythroblasts. One discovered erythroid-predominant factor was death-associated protein kinase-2 (DAPK2). Among 3 DAPK serine/threonine kinases, 5,6 DAPK1 first was identified as an IFN-␥-induced factor which facilitates cell death initiated by IFN-␥, TNF-␣, FAS, or oncogene expression. 7 DAPK1 possesses a DAP kinase upper-lobe signature, CaM regulatory domain, ankyrin repeats, and a C-terminal death domain. 5,6 ZIPK (zipper-interacting protein kinase/DAPK3/DLK) lacks a CaM domain, but possesses leucine zipper and nuclear localization motifs. 5,6,8 DAPK2 retains a related CaM domain, but possesses a unique C-terminal region. 5,6,9 Such structural differences further suggest that DAPK1, ZIP-K and DAPK2 likely play unique biologic roles.DAPK1 can exert proapoptotic effects, potentially via caspaseindependent type-II mechanisms. [5][6][7] Gene disruption experiments further place DAPK1 upstream of p53, 9 and decreased DAPK1 expression is linked to multiple myeloma, ALL, and colon, breast and lung cancers. 10 For DAPK2, overexpression studies in cell lines also point to potential proapoptotic effects, 11,12 but to date this has not been investigated in primary cells which normally express, and regulate endogenous DAPK2. Via transcriptome analyses, transgenic mouse experiments, and analyses in EPO-dependent erythroid...

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