Suppression of the rice heterotrimeric G protein β-subunit gene, RGB1, causes dwarfism and browning of internodes and lamina joint regions

In the present study, we investigated the function of the heterotrimeric G protein β-subunit (Gβ) gene (RGB1) in rice. RGB1 knock-down lines were generated in the wild type and d1-5, a mutant deficient for the heterotrimeric G protein α-subunit (Gα) gene (RGA1). Both transgenic lines showed browning of the lamina joint regions and nodes that could be attributed to a reduction of RGB1 function, as the abnormality was not observed in d1-5. The RGB1 knock-down lines generated in d1-5 were shorter, suggesting RGB1 to be a positive regulator of cellular proliferation, in addition to RGA1. The number of sterile seeds also increased in both RGB1 knock-down lines. These results suggest that Gβγ and Gα cooperatively function in cellular proliferation and seed fertility. We discuss the potential predominant role of RGB1 in G protein signaling in rice.     

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.     

Partial hepatectomy aggravates cyclosporin A-induced neurotoxicity by lowering the function of the blood-brain barrier in mice

AimsCyclosporin A, a calcineurin inhibitor, produces neurotoxicity with relatively high frequency in organ-transplanted patients. The aim of the present study was to clarify whether acute liver failure (ALF) simulated to the transient liver dysfunction at an early phase after liver transplantation increases the susceptibility to cyclosporin A-induced neurotoxicity through the blood–brain barrier (BBB) dysfunction.Main methodsThe right internal, left lateral and left internal lobes in male ddy mice were surgically excised under sodium pentobarbital anesthesia. Effect of cyclosporin A on harmine-induced tremors was examined and BBB permeability to ³[H]cyclosporin A was assessed in partially (70%) hepatectomized mice at postoperative days 1, 3 and 7.Key findingsPatrial hepatectomy aggravated harmine-induced tremors. Cyclosporin A (50 mg/kg, i.p.) markedly augmented harmine-induced tremors in partially hepatectomized mice at postoperative day 1. Consistent with these behavioral findings, the brain uptake of ³[H]cyclosporin A in mice injected with ³[H]cyclosporin A into the jugular vein at postoperative day 1 was significantly increased by partial hepatectomy compared with sham operation.SignificanceOur results indicate that ALF increases BBB permeability to cyclosporin A by lowering the function of P-glycoprotein and tight junctions, consequently leading to augmentation of cyclosporin A-induced neurotoxicity. The possibility that cyclosporin A increases the risk of neurotoxicity including tremors at an early phase of liver transplantation must be considered.     

Increase in expression level of alpha-tubulin gene in Arabidopsis seedlings under hypergravity conditions.

Under hypergravity conditions, elongation growth of plant shoots is suppressed. The analysis of the changes in gene expression by hypergravity treatment in Arabidopsis hypocotyls by the differential display method showed that a gene encoding alpha-tubulin, which is a component of microtubules, was up-regulated by hypergravity. In Arabidopsis six genes encoding alpha-tubulin (TUA1-TUA6) have been identified. In the present study, we examined the dose-response and the time course relations of the changes in the expression of all six alpha-tubulin genes in Arabidopsis hypocotyls grown under hypergravity conditions. The expression levels of all six alpha-tubulin genes, TUA1-TUA6, were increased by increasing gravity, although the extent was variable among genes. The increase in expression of all alpha-tubulin genes was detected within a few hours, when the seedlings grown at 1 g were transferred to 300 g condition. These results suggest that Arabidopsis hypocotyls regulate the expression level of six alpha-tubulin genes promptly in response to gravity stimuli. The increase in the amount of microtubules due to the activation of tubulin gene expression may be involved in the regulation by gravity signal of shoot growth.     

A Fas-associated death domain protein/caspase-8-signaling axis promotes S-phase entry and maintains S6 kinase activity in T cells responding to IL-2

Fas-associated death domain protein (FADD) constitutes an essential component of TNFR-induced apoptotic signaling. Paradoxically, FADD has also been shown to be crucial for lymphocyte development and activation. In this study, we report that FADD is necessary for long-term maintenance of S6 kinase (S6K) activity. S6 phosphorylation at serines 240 and 244 was only observed after long-term stimulation of wild-type cells, roughly corresponding to the time before S-phase entry, and was poorly induced in T cells expressing a dominantly interfering form of FADD (FADDdd), viral FLIP, or possessing a deficiency in caspase-8. Defects in S6K1 phosphorylation were also observed. However, defective S6K1 phosphorylation was not a consequence of a wholesale defect in mammalian target of rapamycin function, because 4E-BP1 phosphorylation following T cell activation was unaffected by FADDdd expression. Although cyclin D3 up-regulation and retinoblastoma hypophosphorylation occurred normally in FADDdd T cells, cyclin E expression and cyclin-dependent kinase 2 activation were markedly impaired in FADDdd T cells. These results demonstrate that a FADD/caspase-8-signaling axis promotes T cell cycle progression and sustained S6K activity.     

Resistance to experimental autoimmune encephalomyelitis and impaired T cell priming by dendritic cells in Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 mutant mice

Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is expressed on the surface of CD11c(+) dendritic cells (DCs) and macrophages. In this study, we show that mice that express a mutant form of SHPS-1 lacking most of the cytoplasmic region are resistant to experimental autoimmune encephalomyelitis (EAE) in response to immunization with a peptide derived from myelin oligodendrocyte glycoprotein (MOG (35-55)). The MOG (35-55)-induced proliferation of, and production of IFN-gamma, IL-2, and IL-17, by T cells from immunized SHPS-1 mutant mice were reduced compared with those apparent for wild-type cells. The abilities of splenic DCs from mutant mice to stimulate an allogenic MLR and to prime Ag-specific T cells were reduced. Both IL-12-stimulated and TLR-dependent cytokine production by DCs of mutant mice were also impaired. Finally, SHPS-1 mutant mice were resistant to induction of EAE by adoptive transfer of MOG (35-55)-specific T cells. These results show that SHPS-1 on DCs is essential for priming of naive T cells and the development of EAE. SHPS-1 is thus a potential therapeutic target in inflammatory disorders of the CNS and other autoimmune diseases.     

Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is primarily characterized by the degeneration of dopaminergic neurons in the nigrostriatal pathway. Previous studies have demonstrated that chronic systemic exposure of Lewis rats to rotenone produced many features of PD, and cerebral tauopathy was also detected in the case of severe weight loss. The present study was designed to assess the neurotoxicity of rotenone after daily oral administration for 28 days at several doses in C57BL/6 mice. In addition, we examined the protective effects of 4-phenylbutyrate (4-PBA) on nigral dopamine (DA) neurons in rotenone-treated mice. 4-PBA was injected intraperitoneally daily 30 min before each oral administration of rotenone. Chronic oral administration of rotenone at high doses induced specific nigrostriatal DA neurodegeneration, motor deficits and the up-regulation of alpha-synuclein in the surviving DA neurons. In contrast to the Lewis rat model, cerebral tauopathy was not detected in this mouse model. 4-PBA inhibited rotenone-induced neuronal death and decreased the protein level of alpha-synuclein. These results suggest that this rotenone mouse model may be useful for understanding the mechanism of DA neurodegeneration in PD, and that 4-PBA has a neuroprotective effect in the treatment of PD.     

Identification of a ribonucleoprotein intermediate of tomato mosaic virus RNA replication complex formation

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the "pre-membrane-targeting complex." Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.