Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

Akhirin regulates the proliferation and differentiation of neural stem cells in intact and injured mouse spinal cord

Although the central nervous system is considered a comparatively static tissue with limited cell turnover, cells with stem cell properties have been isolated from most neural tissues. The spinal cord ependymal cells show neural stem cell potential in vitro and in vivo in injured spinal cord. However, very little is known regarding the ependymal niche in the mouse spinal cord. We previously reported that a secreted factor, chick Akhirin, is expressed in the ciliary marginal zone of the eye, where it works as a heterophilic cell‐adhesion molecule. Here, we describe a new crucial function for mouse Akhirin (M‐AKH) in regulating the proliferation and differentiation of progenitors in the mouse spinal cord. During embryonic spinal cord development, M‐AKH is transiently expressed in the central canal ependymal cells, which possess latent neural stem cell properties. Targeted inactivation of the AKH gene in mice causes a reduction in the size of the spinal cord and decreases BrdU incorporation in the spinal cord. Remarkably, the expression patterns of ependymal niche molecules in AKH knockout (AKH?/?) mice are different from those of AKH+/+, both in vitro and in vivo. Furthermore, we provide evidence that AKH expression in the central canal is rapidly upregulated in the injured spinal cord. Taken together, these results indicate that M‐AKH plays a crucial role in mouse spinal cord formation by regulating the ependymal niche in the central canal. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 494–504, 2015     

Comparison of the Japanese Orthopaedic Association (JOA) Score and Modified JOA (mJOA) Score for the Assessment of Cervical Myelopathy: A Multicenter Observational Study

Objectives

The Japanese Orthopaedic Association (JOA) score is widely used to assess the severity of clinical symptoms in patients with cervical compressive myelopathy, particularly in East Asian countries. In contrast, modified versions of the JOA score are currently accepted as the standard tool for assessment in Western countries. The objective of the present study is to compare these scales and clarify their differences and interchangeability and verify their validity by comparing them to other outcome measures.

Materials and Methods

Five institutions participated in this prospective multicenter observational study. The JOA and modified JOA (mJOA) proposed by Benzel were recorded preoperatively and at three months postoperatively in patients with cervical compressive myelopathy who underwent decompression surgery. Patient reported outcome (PRO) measures, including Japanese Orthopaedic Association Cervical Myelopathy Evaluation Questionnaire (JOACMEQ), the Short Form-12 (SF-12) and the Neck Disability Index (NDI), were also recorded. The preoperative JOA score and mJOA score were compared to each other and the PRO values. A Bland-Altman analysis was performed to investigate their limits of agreement.

Results

A total of ninety-two patients were included. The correlation coefficient (Spearman’s rho) between the JOA and mJOA was 0.87. In contrast, the correlations between JOA/mJOA and the other PRO values were moderate (|rho| = 0.03 – 0.51). The correlation coefficient of the recovery rate between the JOA and mJOA was 0.75. The Bland-Altman analyses showed that limits of agreement were 3.6 to -1.2 for the total score, and 55.1% to -68.8% for the recovery rates.

Conclusions

In the present study, the JOA score and the mJOA score showed good correlation with each other in terms of their total scores and recovery rates. Previous studies using the JOA can be interpreted based on the mJOA; however it is not ideal to use them interchangeably. The validity of both scores was demonstrated by comparing these values to the PRO values.     

Endo-β-N-acetylglucosamidases (ENGases) in the fungus Trichoderma atroviride: Possible involvement of the filamentous fungi-specific cytosolic ENGase in the ERAD process

N-Glycosylation is an important post-translational modification of proteins, which mainly occurs in the endoplasmic reticulum (ER). Glycoproteins that are unable to fold properly are exported to the cytosol for degradation by a cellular system called ER-associated degradation (ERAD). Once misfolded glycoproteins are exported to the cytosol, they are subjected to deglycosylation by peptide:N-glycanase (PNGase) to facilitate the efficient degradation of misfolded proteins by the proteasome. Interestingly, the ortholog of PNGase in some filamentous fungi was found to be an inactive deglycosylating enzyme. On the other hand, it has been shown that in filamentous fungi genomes, usually two different fungi-specific endo-β-N-acetylglucosamidases (ENGases) can be found; one is predicted to be localized in the cytosol and the other to have a signal sequence, while the functional importance of these enzymes remains to be clarified. In this study the ENGases of the filamentous fungus Trichoderma atroviride was characterized. By heterologous expression of the ENGases Eng18A and Eng18B in Saccharomyces cerevisiae, it was found that both ENGases are active deglycosylating enzymes. Interestingly, only Eng18B was able to enhance the efficient degradation of the RTL protein, a PNGase-dependent ERAD substrate, implying the involvement of this enzyme in the ERAD process. These results indicate that T. atroviride Eng18B may deglycosylate misfolded glycoproteins, substituting the function of the cytoplasmic PNGase in the ERAD process.     

Proteolytic processing regulates pathological accumulation in dentatorubral-pallidoluysian atrophy

Dentatorubral-pallidoluysian atrophy is caused by polyglutamine (polyQ) expansion in atrophin-1 (ATN1). Recent studies have shown that nuclear accumulation of ATN1 and cleaved fragments with expanded polyQ is the pathological process underlying neurodegeneration in dentatorubral-pallidoluysian atrophy. However, the mechanism underlying the proteolytic processing of ATN1 remains unclear. In the present study, we examined the proteolytic processing of ATN1 aiming to understand the mechanisms of ATN1 accumulation with polyQ expansion. Using COS-7 and Neuro2a cells that express the ATN1 gene, in which ATN1 was accumulated by increasing the number of polyQs, we identified a novel C-terminal fragment containing a polyQ tract. The mutant C-terminal fragment with expanded polyQ selectively accumulated in the cells, and this was also demonstrated in the brain tissues of patients with dentatorubral-pallidoluysian atrophy. Immunocytochemical and biochemical studies revealed that full-length ATN1 and C-terminal fragments displayed individual localization. The mutant C-terminal fragment was preferentially found in the cytoplasmic membrane/organelle and insoluble fractions. Accordingly, it is assumed that the proteolytic processing of ATN1 regulates the localization of C-terminal fragments. Accumulation of the C-terminal fragment was enhanced by inhibition of caspases in the cytoplasm of COS-7 cells. Collectively, these results suggest that the C-terminal fragment plays a principal role in the pathological accumulation of ATN1 in dentatorubral-pallidoluysian atrophy.     

Dopamine-selective potentiometric responses by new ditopic sensory elements based on a hexahomotrioxacalix[3]arene

New ditopic sensory elements 2 and 3 for catecholamines based on a hexahomotrioxacalix[3]arene, with a boronic acid substituent appended, were designed and synthesized. As an interesting mode of molecular recognition at membrane surfaces, the host, when incorporated into poly(vinyl chloride) (PVC) liquid membranes, displayed excellent potentiometric selectivity for dopamine over other catecholamines (noradrenaline and adrenaline) and inorganic cations (Na+, K+, and NH4+).     

Small interfering RNA-mediated silencing induces target-dependent assembly of GW/P bodies

Gene silencing using small interfering RNA (siRNA) is a valuable laboratory tool and a promising approach to therapeutics for a variety of human diseases. Recently, RNA interference (RNAi) has been linked to cytoplasmic GW bodies (GWB). However, the correlation between RNAi and the formation of GWB, also known as mammalian processing bodies, remains unclear. In this report, we show that transfection of functional siRNA induced larger and greater numbers of GWB. This siRNA-induced increase of GWB depended on the endogenous expression of the target mRNA. Knockdown of GW182 or Ago2 demonstrated that the siRNA-induced increase of GWB required these two proteins and correlated with RNAi. Furthermore, knockdown of rck/p54 or LSm1 did not prevent the reassembly of GWB that were induced by and correlated with siRNA-mediated RNA silencing. We propose that RNAi is a key regulatory mechanism for the assembly of GWB, and in some cases, GWB may serve as markers for RNAi in mammalian cells.     

Newly established in vitro system with fluorescent proteins shows that abnormal expression of downstream prion protein-like protein in mice is probably due to functional disconnection between splicing and 3' formation of prion protein pre-mRNA

We and others previously showed that, in some lines of prion protein (PrP)-knockout mice, the downstream PrP-like protein (PrPLP/Dpl) was abnormally expressed in brains partly due to impaired cleavage/polyadenylation of the residual PrP promoter-driven pre-mRNA despite the presence of a poly(A) signal. In this study, we newly established an in vitro transient transfection system in which abnormal expression of PrPLP/Dpl can be visualized by expression of the green fluorescence protein, EGFP, in cultured cells. No EGFP was detected in cells transfected by a vector carrying a PrP genomic fragment including the region targeted in the knockout mice intact upstream of the PrPLP/Dpl gene. In contrast, deletion of the targeted region from the vector caused expression of EGFP. By employing this system with other vectors carrying various deletions or point mutations in the targeted region, we identified that disruption of the splicing elements in the PrP terminal intron caused the expression of EGFP. Recent lines of evidence indicate that terminal intron splicing and cleavage/polyadenylation of pre-mRNA are functionally linked to each other. Taken together, our newly established system shows that the abnormal expression of PrPLP/Dpl in PrP-knockout mice caused by the impaired cleavage/polyadenylation of the PrP promoter-driven pre-mRNA is due to the functional dissociation between the pre-mRNA machineries, in particular those of cleavage/polyadenylation and splicing. Our newly established in vitro system, in which the functional dissociation between the pre-mRNA machineries can be visualized by EGFP green fluorescence, may be useful for studies of the functional connection of pre-mRNA machineries.     

A mobile agent model for fault-tolerant manipulation on distributed objects

In this paper, we discuss how to realize fault-tolerant applications on distributed objects. Servers supporting objects can be fault-tolerant by taking advantage of replication and checkpointing technologies. However, there is no discussion on how application programs being performed on clients are tolerant of clients faults. For example, servers might block in the two-phase commitment protocol due to the client fault. We newly discuss how to make application programs fault-tolerant by taking advantage of mobile agent technologies where a program can move from a computer to another computer in networks. An application program to be performed on a faulty computer can be performed on another operational computer by moving the program in the mobile agent model. In this paper, we discuss a transactional agent model where a reliable and efficient application for manipulating objects in multiple computers is realized in the mobile agent model. In the transactional agent model, only a small part of the application program named routing subagent moves around computers. A routing subagent autonomously finds a computer which to visit next. We discuss a hierarchical navigation map which computer should be visited price to another computer in a transactional agent. A routing subagent makes a decision on which computer visit for the hierarchical navigation map. Programs manipulating objects in a computer are loaded to the computer on arrival of the routing subagent in order to reduce the communication overhead. This part of the transactional agent is a manipulating subagent. The manipulation subagent still exists on the computer even after the routing subagent leaves the computer in order to hold objects until the commitment. We assume every computer may stop by fault while networks are reliable. There are kinds of faulty computers for a transactional agent; current, destination, and sibling computers where a transactional agent now exists, will move, and has visited, respectively. The types of faults are detected by neighbouring manipulation subagents by communicating with each other. If some of the manipulation subagents are faulty, the routing subagent has to be aborted. However, the routing subagent is still moving. We discuss how to efficiently deliver the abort message to the moving routing subagent. We evaluate the transactional agent model in terms of how long it takes to abort the routing subagent if some computer is faulty.