Olig2-Induced Neural Stem Cell Differentiation Involves Downregulation of Wnt Signaling and Induction of Dickkopf-1 Expression

Understanding stem cell-differentiation at the molecular level is important for clinical applications of stem cells and for finding new therapeutic approaches in the context of cancer stem cells. To investigate genome-wide changes involved in differentiation, we have used immortalized neural stem cell (NSC) line (HB1.F3) and Olig2-induced NSC differentiation model (F3.Olig2). Using microarray analysis, we revealed that Olig2-induced NSC differentiation involves downregulation of Wnt pathway, which was further confirmed by TOPflash/FOPflash reporter assay, RT-PCR analysis, immunoblots, and immunocytochemistry. Furthermore, we found that Olig2-induced differentiation induces the expression of Dickkopf-1(Dkk1), a potent antagonist of Wnt signaling. Dkk1 treatment blocked Wnt signaling in HB1.F3 in a dosage-dependent manner, and induced differentiation into astrocytes, oligodendrocytes, and neurons. Our results support cancer stem cell hypothesis which implies that signaling pathway for self-renewal and proliferation of stem cells is maintained till the late stage of differentiation. In our proposed model, Dkk1 may play an important role in downregulating self-renewal and proliferation pathway of stem cells at the late stage of differentiation, and its failure may lead to carcinogenesis.     

Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by Src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr(9) antibodies showed that the level of Tyr(9) phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr(9), distinct from Tyr(373/376), is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr(9) phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.     

Production of cyclodextrin by poly-lysine fused Bacillus macerans cyclodextrin glycosyltransferase immobilized on cation exchanger

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at its C-terminus (CGTK10ase) was immobilized onto a cation exchanger by ionic interaction and used to produce -cyclodextrin (CD) from soluble starch. Poly-lysine fused immobilization increased the V_m of the immobilized CGTase by 40% without a change in K_m. The activation energies of thermal deactivation (E_a) were 41.4, 28.1, and 25.9 kcal mol⁻¹, respectively, for soluble wild-type (WT) CGTase, soluble CGTK10ase, and immobilized CGTK10ase, suggesting destabilization of CGTase by poly-lysine fusion and immobilization onto a cation exchanger. Maximum -CD productivity of 539.4 g l⁻¹ h⁻¹ was obtained with 2% soluble starch solution which was constantly fed at a flow rate of 4.0 ml min⁻¹ (D = 240 h⁻¹) in a continuous operation mode of a packed-bed reactor. The operational half-life of the packed-bed enzyme reactor was estimated 12 days at 25 °C and pH 6.0.     

Inhibitory effect of Eucommia ulmoides Oliver on adipogenic differentiation through proteome analysis

Eucommia ulmoides Oliver was tested to control the differentiation of cultured human mesenchymal stem cell into adipocyte. The extent of the inhibitory effect on the conversion of adipose was measured using Oil red O staining, which stains accumulated lipid droplets in the cytoplasm of adipocyte. The adipocyte differentiation was inhibited by an extract from E. ulmoides Oliver in the concentration range 0.01–50.0 g/l. After 2D PAGE, protein spots of E. ulmoides Oliver extract treated stem cells were analyzed. Several protein spots were strongly up-regulated in E. ulmoides Oliver extract treated cells. Among them, tropomyosin was identified by MALDI-TOF. These results suggest that E. ulmoides Oliver has an inhibitory activity toward the adipose conversion, which is a major cause of obesity.     

Activation of Lck is critically required for sphingosine-induced conformational activation of Bak and mitochondrial cell death

Despite extensive investigation, the molecular mechanism of anticancer activity of sphingolipid metabolites remains to be clarified. Here we demonstrate that sphingosine induces mitochondrial cell death via Lck-mediated conformational activation of Bak in Jurkat T cell lymphoma. Treatment of cells with sphingosine rapidly induced mitochondrial membrane potential loss, cytochrome c release from mitochondria, and apoptotic cell death. Sphingosine also induced conformational activation of Bak, but not Bax. siRNA targeting of Bak effectively attenuated sphingosine-induced mitochondrial cell death, indicating that Bak is involved in sphingosine-induced mitochondrial cell death. Sphingosine also induced activation of tyrosine kinase Lck. Inhibition of Lck by treatment of PP2, a Lck inhibitor or siRNA targeting of Lck suppressed sphingosine-induced conformational activation and oligomerization of Bak, mitochondrial membrane potential loss, and apoptotic cell death, implying that activation of Lck is critically required for sphingosine-induced conformational activation of Bak and mitochondrial cell death. The results elucidated in this study provide a novel cellular mechanism for the anticancer activity of sphingolipid metabolites.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

This review focuses on the mechanisms of stress response in the synovial tissue of rheumatoid arthritis. The major stress factors, such as heat stress, shear stress, proinflammatory cytokines and oxidative stress, are discussed and reviewed, focusing on their potential to induce a stress response in the synovial tissue. Several pathways of stress signalling molecules are found to be activated in the synovial membrane of rheumatoid arthritis; of these the most important examples are heat shock proteins, mitogen-activated protein kinases, stress-activated protein kinases and molecules involved in the oxidative stress pathways. The expression of these pathways in vitro and in vivo as well as the consequences of stress signalling in the rheumatoid synovium are discussed. Stress signalling is part of a cellular response to potentially harmful stimuli and thus is essentially involved in the process of synovitis. Stress signalling pathways are therefore new and promising targets of future anti-rheumatic therapies.     

Genus delimitation,biogeography and diversification of Choristoneura Lederer (Lepidoptera: Tortricidae) based on molecular evidence

Widely known for pest species that include major modulators of temperate forests, the genus Choristoneura is part of the species‐rich tribe Archipini of leafroller moths (Tortricidae). Delimitation of the genus has remained unresolved because no phylogeny has included species endemic to Africa and studies have often omitted the type species of the genus. Further taxonomic confusion has been generated by the transfer of Archips occidentalis (Walsingham) to Choristoneura, creating a homonym with Choristoneura occidentalis Freeman, an important defoliator of North American forests. To define the limits of the genus, we reconstructed a phylogeny using DNA sequences for mitochondrial cytochrome oxidase subunit I and nuclear ribosomal 28S genes. Our ingroup included 23 Choristoneura species‐level taxa, complemented by a large sample of outgroups comprising 82 species of Archipini and other Tortricidae. We generated a time‐calibrated tree using fossil and secondary calibrations and we inferred biogeographic and diversification processes in Choristoneura. Our analysis recovered the genus as polyphyletic, with Archips occidentalis, Choristoneura simonyi and Choristoneura evanidana excluded from the main clade. Based on the recovered phylogenies and a redefinition, we restrict Choristoneura primarily to species with a northern hemisphere distribution. Our analysis supports A. occidentalis as the sister group of Cacoecimorpha pronubana, C. simonyi as the sister of ‘Xenotemna’ pallorana, and C. evanidana as the sister of Archips purpurana. A new combination is proposed: Archips evanidana comb.n. ; the availability of ‘Xenotemna’ as a valid name is discussed and A. occidentalis is considered as an orphaned name within the Archipini. We found support for a Holarctic origin of Choristoneura about 23 Ma, followed by early divergence in the Palearctic region. The main divergence occurred at 16 Ma, with one clade in the Nearctic and another in the Palearctic. Subsequent cladogenetic events were synchronous and related to herbivorous specialization, with each clade divided into coniferophagous and polyphagous lineages. Their specialization as conifer feeders temporally matched the expansion of boreal forest during the Miocene.     

Heterogeneity of Regional Brain Atrophy Patterns Associated with Distinct Progression Rates in Alzheimer’s Disease

We aimed to identify and characterize subtypes of Alzheimer’s disease (AD) exhibiting different patterns of regional brain atrophy on MRI using age- and gender-specific norms of regional brain volumes. AD subjects included in the Alzheimer''s Disease Neuroimaging Initiative study were classified into subtypes based on standardized values (Z-scores) of hippocampal and regional cortical volumes on MRI with reference to age- and gender-specific norms obtained from 222 cognitively normal (CN) subjects. Baseline and longitudinal changes of clinical characteristics over 2 years were compared across subtypes. Whole-brain-level gray matter (GM) atrophy pattern using voxel-based morphometry (VBM) and cerebrospinal fluid (CSF) biomarkers of the subtypes were also investigated. Of 163 AD subjects, 58.9% were classified as the “both impaired” subtype with the typical hippocampal and cortical atrophy pattern, whereas 41.1% were classified as the subtypes with atypical atrophy patterns: “hippocampal atrophy only” (19.0%), “cortical atrophy only” (11.7%), and “both spared” (10.4%). Voxel-based morphometric analysis demonstrated whole-brain-level differences in overall GM atrophy across the subtypes. These subtypes showed different progression rates over 2 years; and all subtypes had significantly lower CSF amyloid-β_1–42 levels compared to CN. In conclusion, we identified four AD subtypes exhibiting heterogeneous atrophy patterns on MRI with different progression rates after controlling the effects of aging and gender on atrophy with normative information. CSF biomarker analysis suggests the presence of Aβ neuropathology irrespective of subtypes. Such heterogeneity of MRI-based neuronal injury biomarker and related heterogeneous progression patterns should be considered in clinical trials and practice with AD patients.     

Plant gene responses to frequency-specific sound signals

We identified a set of sound-responsive genes in plants using a sound-treated subtractive library and demonstrated sound regulation through mRNA expression analyses. Under both light and dark conditions, sound up-regulated expression of rbcS and ald. These are also light-responsive genes and these results suggest that sound could represent an alternative to light as a gene regulator. Ald mRNA expression increased significantly with treatment at 125 and 250 Hz, whereas levels decreased significantly with treatment at 50 Hz, indicating a frequency-specific response. To investigate whether the ald promoter responds to sound, we generated transgenic rice plants harboring a chimeric gene comprising a fusion of the ald promoter and GUS reporter. In three independent transgenic lines treated with 50 or 250 Hz for 4 h, GUS mRNA expression was up-regulated at 250 Hz, but down-regulated at 50 Hz. Thus, the sound-responsive mRNA expression pattern observed for the ald promoter correlated closely with that of ald, suggesting that the 1,506 bp ald promoter is sound-responsive. Therefore, we propose that in transgenic plants, specific frequencies of sound treatment could be used to regulate the expression of any gene fused to the ald promoter.