Abiotic stress-inducible receptor-like kinases negatively control ABA signaling in Arabidopsis

Membrane-anchored receptor-like protein kinases (RLKs) recognize extracellular signals at the cell surface and activate the downstream signaling pathway by phosphorylating specific target proteins. We analyzed a receptor-like cytosolic kinase (RLCK) gene, ARCK1, whose expression was induced by abiotic stress. ARCK1 belongs to the cysteine-rich repeat (CRR) RLK sub-family and encodes a cytosolic protein kinase. The arck1 mutant showed higher sensitivity than the wild-type to ABA and osmotic stress during the post-germinative growth phase. CRK36, an abiotic stress-inducible RLK belonging to the CRR RLK sub-family, was screened as a potential interacting factor with ARCK1 by co-expression analyses and a yeast two-hybrid system. CRK36 physically interacted with ARCK1 in plant cells, and the kinase domain of CRK36 phosphorylated ARCK1 in vitro. We generated CRK36 RNAi transgenic plants, and found that transgenic plants with suppressed CRK36 expression showed higher sensitivity than arck1-2 to ABA and osmotic stress during the post-germinative growth phase. Microarray analysis using CRK36 RNAi plants revealed that suppression of CRK36 up-regulates several ABA-responsive genes, such as LEA genes, oleosin, ABI4 and ABI5. These results suggest that CRK36 and ARCK1 form a complex and negatively control ABA and osmotic stress signal transduction.     

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.     

Near-infrared fluorescent labeled peptosome for application to cancer imaging

Nonionic amphiphilic copolypeptides, which were composed of hydrophilic poly(sarcosine) and hydrophobic poly(gamma-methyl L-glutamate) blocks, were synthesized with varying chain lengths of the blocks. The polypeptides having a suitable hydrophilic and hydrophobic balance were found to form vesicular assemblies of 100 nm size in buffer, which was evidenced by the TEM observation, the DLS analysis, and the encapsulation experiment. The genuine peptide vesicles, peptosomes, were labeled with a near-infrared fluorescence (NIRF) probe. In vivo retention in blood experiment showed long circulation of the peptosome in rat blood as stable as the PEGylated liposome. NIRF imaging of a small cancer on mouse by using the peptosome as a nanocarrier was successful due to the EPR effect of the peptosome. Peptosome is shown here as a novel excellent nanocarrier for molecular imaging.     

Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

Absorption of riboflavin is mediated by transporter(s). However, a mammalian riboflavin transporter has yet to be identified. In the present study, the novel human and rat riboflavin transporters hRFT1 and rRFT1 were identified on the basis of our rat kidney mRNA expression database (Horiba N, Masuda S, Takeuchi A, Saito H, Okuda M, Inui K. Kidney Int 66: 29-45, 2004). hRFT1 and rRFT1 cDNAs have an open reading frame encoding 448- and 450-amino acid proteins, respectively, that exhibit 81.1% identity and 96.4% similarity to one another. In addition, an inactive splice variant of hRFT1, hRFT1sv, was also cloned. The hRFT1sv cDNA, which encodes a 167-amino acid protein, retains an intron between exons 2 and 3 of hRFT1. Real-time PCR revealed that the sum of hRFT1 and hRFT1sv mRNAs was expressed strongly in the placenta and small intestine and was detected in all tissues examined. In addition, hRFT1 and hRFT1sv were expressed in human embryonic kidney (HEK)-293 and Caco-2 cells. HEK-293 cells transfected with green fluorescent protein-tagged hRFT1 and rRFT1 exhibited a fluorescent signal in the plasma membrane. Overexpression of hRFT1 and rRFT1, but not hRFT1sv, increased the cellular accumulation of [(3)H]riboflavin. The transfection of small interfering RNA targeting both hRFT1 and hRFT1sv significantly decreased the uptake of [(3)H]riboflavin by HEK-293 and Caco-2 cells. Riboflavin transport is Na(+), potential, and pH independent. Kinetic analyses demonstrated that the Michaelis-Menten constants for the uptake by HEK-293 and Caco-2 cells were 28.1 and 63.7 nM, respectively. We propose that hRFT1 and rRFT1 are novel mammalian riboflavin transporters, which belong to a new mammalian riboflavin transporter family.     

密穗型水稻品种籽粒垩白性状改良研究

采用籽粒长宽比较大、穗部着粒密的散穗型材料(EG23)改良粳稻密穗型品种的籽粒垩白性状.结果表明,经改良后得到的密穗型品系EA6,与原亲本浙粳20比较,其穗部长度缩短,每穗总粒数增加,着粒密度增大,而籽粒垩白特性得到明显的改善,表明在穗部长度和着粒结构未得到改良的情况下,调节籽粒长宽比对改善密穗型品种籽粒垩白性状具有可能性.穗部不同粒位籽粒垩白性状改良的效果不同,穗顶部和穗中部的改良效果明显优于穗基部.设计的4个不同杂交配组方式中,以反回交配组方式(浙粳20/ EG23//浙粳20)选育效果最好.EA6具有较好的农艺性状,既可作为优异种质资源利用,也可直接应用于生产.这一结果从育种实践上较好地协调了密穗型品种高产与优质的矛盾,对于培育既有密穗型的高产株型又有优良籽粒外观品质的水稻品种具有重要意义.     

Regulation of stress-activated protein kinase signaling pathways by protein phosphatases.

Stress-activated protein kinase (SAPK) signaling plays essential roles in eliciting adequate cellular responses to stresses and proinflammatory cytokines. SAPK pathways are composed of three successive protein kinase reactions. The phosphorylation of SAPK signaling components on Ser/Thr or Thr/Tyr residues suggests the involvement of various protein phosphatases in the negative regulation of these systems. Accumulating evidence indicates that three families of protein phosphatases, namely the Ser/Thr phosphatases, the Tyr phosphatases and the dual specificity Ser/Thr/Tyr phosphatases regulate these pathways, each mediating a distinct function. Differences in substrate specificities and regulatory mechanisms for these phosphatases form the molecular basis for the complex regulation of SAPK signaling. Here we describe the properties of the protein phosphatases responsible for the regulation of SAPK signaling pathways.     

Manipulation of a Large DNA Molecule using the Phase Transition

A conventional method of DNA sequencing can determine up to 1000 base pairs at one time. Therefore, long DNA should be cut into many short fragments that are suitable for DNA sequencing. Those fragments, however, lose their order information. If the fragments are prepared from the terminus of the long DNA, the reorganization process can be omitted. This process consists of following unit operations; manipulation of genomic DNA, fixation with a stretched form, cutting from the terminus, recovery and amplification. In these unit operations, manipulation and cutting of DNA are focused in this report. Globular transformation suppresses break down of long genome DNA and permits manipulation of large DNA. Because globular transition is reversible, the coiled DNA can be sequentially spun from the globular DNA like a spindle. Thespun DNA was successfully fixed on a glass surface in an arbitrary pattern. To prepare fragments from the stretched DNA molecule, a method to cut DNA moleculen was developed. Since most restriction enzyme requires magnesium ion for their activation, the restriction enzyme was successfully activated only when magnesium ion was electrochemically supplied.     

Deoxyribonucleoside-3',5'-cyclic silanediyl derivatives: formation and properties

Formation of 3',5'-O-(dialkylsilanediyl)deoxyribonucleosides was studied. Treatment of deoxythymidine in DMF with bifunctional silylating reagent such as di-t-butyldichlorosilane and diisopropyldichlorosilane in the presence of imidazole gave the expected silanediyl derivatives. The structure was confirmed mainly by NMR spectroscopy. The stability of these cyclic silyl derivatives toward hydrolysis is also described.