Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling

The ephrin/Eph system plays a central role in neuronal circuit formation; however, its downstream effectors are poorly understood. Here we show that alpha-chimerin Rac GTPase-activating protein mediates ephrinB3/EphA4 forward signaling. We discovered a spontaneous mouse mutation, miffy (mfy), which results in a rabbit-like hopping gait, impaired corticospinal axon guidance, and abnormal spinal central pattern generators. Using positional cloning, transgene rescue, and gene targeting, we demonstrated that loss of alpha-chimerin leads to mfy phenotypes similar to those of EphA4(-/-) and ephrinB3(-/-) mice. alpha-chimerin interacts with EphA4 and, in response to ephrinB3/EphA4 signaling, inactivates Rac, which is a positive regulator of process outgrowth. Moreover, downregulation of alpha-chimerin suppresses ephrinB3-induced growth cone collapse in cultured neurons. Our findings indicate that ephrinB3/EphA4 signaling prevents growth cone extension in motor circuit formation via alpha-chimerin-induced inactivation of Rac. They also highlight the role of a Rho family GTPase-activating protein as a key mediator of ephrin/Eph signaling.     

Effective syntheses of 2′,4′-BNANC monomers bearing adenine,guanine, thymine,and 5-methylcytosine,and the properties of oligonucleotides fully modified with 2′,4′-BNANC

We efficiently synthesized 2′-O,4′-C-aminomethylene-bridged nucleic acid (2′,4′-BNA^NC) monomers bearing the four nucleobases, guanine, adenine, thymine, and 5-methylcytosine and incorporated these monomers into oligonucleotides. Initially, we carried out the transglycosylation reaction on several 2′-O-substituted 5-methyluridines to evaluate the effects of 2′-substitutions on this reaction. Under the optimized conditions, purine nucleobases were successfully introduced, and 2′,4′-BNA^NC monomers bearing adenine or guanine were obtained over several steps. In addition, the improved synthesis of the 2′,4′-BNA^NC monomers bearing thymine or 5-methylcytosine was also achieved. The obtained 2′,4′-BNA^NC monomers were subsequently incorporated into oligonucleotides and the duplex-forming abilities of the modified oligonucleotides were investigated. Duplexes containing 2′,4′-BNA^NC monomers in both or either strands were found to possess excellent thermal stabilities.     

Factors Affecting Cerebral Oxygenation in Hemodialysis Patients: Cerebral Oxygenation Associates with pH,Hemodialysis Duration,Serum Albumin Concentration,and Diabetes Mellitus

BackgroundPatients undergoing hemodialysis (HD) often develop cerebral disease complications. Furthermore, cerebral regional saturation of oxygen (rSO₂) was previously reported to be significantly lower in HD patients than in healthy subjects. We aimed to identify the factors affecting the cerebral rSO₂ in HD patients.MethodsFifty-four HD patients (38 men and 16 women; mean age, 67.7 ± 1.2 years, HD duration, 6.5 ± 1.9 years) were recruited. Cerebral rSO₂ was monitored at the forehead before HD using an INVOS 5100C (Covidien Japan, Tokyo, Japan).ResultsThe rSO₂ levels were significantly lower in HD patients compared with healthy controls (49.5 ± 1.7% vs. 68.9 ± 1.6%, p <0.001). Multiple regression analysis showed that cerebral rSO₂ independently associated with pH (standardized coefficient: -0.35), HD duration (standardized coefficient: -0.33), and serum albumin concentration (standardized coefficient: 0.28). Furthermore, the rSO₂ was significantly lower in HD patients with diabetes mellitus (DM), compared with patients without DM (46.8 ± 1.7% vs. 52.1 ± 1.8%, p <0.05).ConclusionsIn HD patients, cerebral rSO₂ was affected by multiple factors, including pH, HD duration, and serum albumin concentration. Furthermore, this is the first report describing significantly lower levels of rSO₂ in HD patients with DM than in those without DM.     

Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.     

Preparation of an antibody that recognizes and neutralizes Dictyostelium differentiation-inducing factor-1

In the development of the cellular slime mold Dictyostelium discoideum, the differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) plays an important role in the regulation of cell differentiation and chemotaxis; however, the cellular signaling systems involving DIF-1 remain to be elucidated. To obtain a probe for DIF-1, we synthesized a DIF derivative (DIF-1-NH₂; 6-amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), and prepared an anti-DIF-1 antibody using a DIF-1-NH₂-conjugated macromolecule as the immunogen. A 100-fold dilution of the antibody bound to DIF-1-NH₂-conjugated resin, and this binding was inhibited by co-addition of 20 μM DIF-1 or DIF-1-NH₂. In a monolayer culture of HM44 cells, a DIF-deficient D. discoideum strain, 0.5 nM exogenous DIF-1 induced stalk cell formation in ∼60% of the cells; this induction was dose-dependently inhibited by the antibody (diluted 12.5- or 25-fold). Furthermore, this inhibition by the antibody was recovered by co-addition of 2.5 or10 nM DIF-1. The results indicate that the anti-DIF-1 antibody recognizes DIF-1 and neutralizes its function.     

Anti-leukemic activities of Dictyostelium secondary metabolites: a novel aromatic metabolite, 4-methyl-5-n-pentylbenzene-1,3-diol, isolated from Dictyostelium mucoroides suppresses cell growth in human leukemia K562 and HL-60 cells

It has previously been shown that DIF-1, a differentiation-inducing factor of the cellular slime mold Dictyostelium discoideum, possesses antitumor activities in mammalian tumor cells and that neuronal differentiation of PC12 cells can be induced with furanodictines (FDs), aminosugar analogs found in D. discoideum, or dictyoglucosamines (DGs), N-acetyl glucosamine derivatives (DG-A from D. purpureum and DG-B from D. discoideum). Thus, cellular slime molds are attractive natural resources that may provide valuable lead compounds to be utilized in the field of pharmacology and medicine. In this study, we have isolated a novel aromatic compound, 4-methyl-5-n-pentylbenzene-1,3-diol (MPBD), from fruiting bodies of the cellular slime mold D. mucoroides and assessed the in vitro antiproliferative activities of MPBD, FDs, and DGs in human leukemia K562 and HL-60 cells. MPBD at 20-80 microM dose-dependently suppressed cell growth in both K562 and HL-60 cells. While FDs at 10-80 microM did not affect cell growth, DGs at 10-40 microM dose-dependently suppressed cell growth in the cells. Although we failed to find the roles of FDs and DGs in the original organisms, MPBD at 5-20 microM was found to promote stalk cell formation in D. discoideum. The present results indicate that MPBD, DGs or their derivatives may have therapeutic potential in the treatment of cancer and confirm our expectations regarding cellular slime molds as drug resources.     

NCI-H295R,a Human Adrenal Cortex-Derived Cell Line,Expresses Purinergic Receptors Linked to Ca2+-Mobilization/Influx and Cortisol Secretion

Purinergic receptor expression and involvement in steroidogenesis were examined in NCI-H295R (H295R), a human adrenal cortex cell line which expresses all the key enzymes necessary for steroidogenesis. mRNA/protein for multiple P1 (A_2A and A_2B), P2X (P2X₅ and P2X₇), and P2Y (P2Y₁, P2Y₂, P2Y₆, P2Y₁₂, P2Y₁₃, and P2Y₁₄) purinergic receptors were detected in H295R. 2MeS-ATP (10–1000 µM), a P2Y₁ agonist, induced glucocorticoid (GC) secretion in a dose-dependent manner, while other extracellular purine/pyrimidine agonists (1–1000 µM) had no distinct effect on GC secretion. Extracellular purines, even non-steroidogenic ones, induced Ca²⁺-mobilization in the cells, independently of the extracellular Ca²⁺ concentration. Increases in intracellular Ca²⁺ concentration induced by extracellular purine agonists were transient, except when induced by ATP or 2MeS-ATP. Angiotensin II (AngII: 100 nM) and dibutyryl-cyclic AMP (db-cAMP: 500 µM) induced both GC secretion and Ca²⁺-mobilization in the presence of extracellular Ca²⁺ (1.2 mM). GC secretion by AngII was reduced by nifedipine (10–100 µM); whereas the Ca²⁺ channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca²⁺ exposure induced Ca²⁺-influx in H295R, and the cells expressed mRNA/protein of the component molecules for store-operated calcium entry (SOCE): transient receptor C (TRPC) channels, calcium release-activated calcium channel protein 1 (Orai-1), and the stromal interaction molecule 1 (STIM1). In P2Y₁-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y₁ purinergic receptor for intracellular Ca²⁺-mobilization, and that P2Y₁ is linked to SOCE-activation, leading to Ca²⁺-influx which might be necessary for glucocorticoid secretion.     

Modeling Alzheimer’s Disease with iPSCs Reveals Stress Phenotypes Associated with Intracellular Aβ and Differential Drug Responsiveness

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Highlights? Familial and sporadic Alzheimer’s patient iPSC-derived neural cells were analyzed ? Intracellular Aβ oligomers accumulate in lines from some patients ? Aβ oligomer accumulation is associated with ER and oxidative stress ? DHA-alleviated ER and oxidative stresses improve cell viability