Identification of Surface-Exposed Parts of Red-Light- and Far-Red-Light- Absorbing Forms of Native Pea Phytochrome by Limited Proteolysis

Peptide fragments were obtained by limited proteolysis withtrypsin and Staphylococcus aureus V 8 protease from either theP_R or the P_FR form of 121-kDa phytochrome purified from etiolatedpea (Pisum sativum L.) shoots. Patterns of bands after polyacrylamidegel electrophoresis in the presence of SDS of the digests weredifferent, with some bands appearing preferentially when thedigestions were carried out with the P_R or the P_FR form. Amino-terminalsequences of the fragments were analyzed to determine the exactlocations of the amino-termini of the fragments within the aminoacid sequence of the apoprotein of pea phytochrome. The aminoacid compositions of some of the sequenced fragments were determinedin order to confirm the carboxy-terminal amino acids. Threecleavage regions were identified as kinetically favored sitesof cleavage of P_FR (Arg-746 to Lys-752, around Glu-877 and aroundArg-1010), whereas only one was identified for P_R (Glu-38 toArg-62). Regions of Glu-255, Arg-383, Arg-583 to Glu-620 andLys-1093 to Glu-1115 were also identified as potential sitesof proteolytic cleavage in both forms of the phytochrome. Othercleavage sites, the specificities of which have not yet beendetermined, are Glu-404, Glu-695 and Lys-1045. Surface-exposed parts of phytochrome in the P_R and P_FR formsare discussed. (Received June 13, 1992; Accepted October 27, 1992)     

Cellular level observation of water loss and the refilling of tracheids in the xylem of Cryptomeria japonica during heartwood formation

The mechanism of heartwood formation in Cryptomeria japonica D. Don has long been studied since heartwood formation is a fundamental physiological feature of trees. In this study, the water distribution in the xylem of C. japonica was investigated at the cellular level to reveal the role of water distribution in the xylem during heartwood formation. Samples were taken from different heights of each trunk, in which the phases of heartwood formation differed. These were designated as SIH, which consisted of sapwood, intermediate wood, and heartwood; SI, which consisted of sapwood and intermediate wood but no heartwood; and S-all, which consisted entirely of sapwood. Cryo-scanning electron microscopic observations of the heartwood-formed (SIH) and non-heartwood-formed (SI and S-all) xylem revealed different patterns of water distribution changes in tracheids between the latewood and earlywood. In the latewood, almost all tracheids were filled with water in all areas from the sapwood to the heartwood (98–100% of tracheids had water in their lumina). In the earlywood, however, the water distribution differed between the sapwood (95–99%), intermediate wood (7–12%), and heartwood (4–100%). Many of the tracheids in the xylem, where the sapwood changed to intermediate wood lost water. In the heartwood, some tracheids remained empty, while others were refilled with water. These results suggest that the water distribution changes in individual tracheids are closely related to heartwood formation. Water loss from tracheids may be an important factor inducing heartwood formation in the xylem of C. japonica.     

SNP-SNP Interactions Discovered by Logic Regression Explain Crohn's Disease Genetics

In genome-wide association studies (GWAS), the association between each single nucleotide polymorphism (SNP) and a phenotype is assessed statistically. To further explore genetic associations in GWAS, we considered two specific forms of biologically plausible SNP-SNP interactions, ‘SNP intersection’ and ‘SNP union,’ and analyzed the Crohn''s Disease (CD) GWAS data of the Wellcome Trust Case Control Consortium for these interactions using a limited form of logic regression. We found strong evidence of CD-association for 195 genes, identifying novel susceptibility genes (e.g., ISX, SLCO6A1, TMEM183A) as well as confirming many previously identified susceptibility genes in CD GWAS (e.g., IL23R, NOD2, CYLD, NKX2-3, IL12RB2, ATG16L1). Notably, 37 of the 59 chromosomal locations indicated for CD-association by a meta-analysis of CD GWAS, involving over 22,000 cases and 29,000 controls, were represented in the 195 genes, as well as some chromosomal locations previously indicated only in linkage studies, but not in GWAS. We repeated the analysis with two smaller GWASs from the Database of Genotype and Phenotype (dbGaP): in spite of differences of populations and study power across the three datasets, we observed some consistencies across the three datasets. Notable examples included TMEM183A and SLCO6A1 which exhibited strong evidence consistently in our WTCCC and both of the dbGaP SNP-SNP interaction analyses. Examining these specific forms of SNP interactions could identify additional genetic associations from GWAS. R codes, data examples, and a ReadMe file are available for download from our website: http://www.ualberta.ca/~yyasui/homepage.html.     

Filamentous bacteriophages of vibrios are integrated into the dif-like site of the host chromosome

The dif site is located in the replication terminus region of bacterial chromosomes, having a function of resolving dimeric chromosomes formed during replication. We demonstrate that filamentous bacteriophages of vibrios, such as f237 (Vibrio parahaemolyticus) and CTXphi (V. cholerae), are integrated into the dif-like site of host chromosome.     

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.     

siRNA-mediated inhibition of endogenous Huntington disease gene expression induces an aberrant configuration of the ER network in vitro

Huntingtin is a ubiquitously expressed cytoplasmic protein encoded by the Huntington disease (HD) gene, in which a CAG expansion induces an autosomal dominant progressive neurodegenerative disorder; however, its biological function has not been completely elucidated. Here, we report for the first time that short interfering RNA (siRNA)-mediated inhibition of endogenous Hdh (a mouse homologue of huntingtin) gene expression induced an aberrant configuration of the endoplasmic reticulum (ER) network in vitro. Studies using immunofluorescence microscopy with several ER markers revealed that the ER network appeared to be congregated in various types of cell lines transfected with siRNA directed against Hdh, but not with other siRNAs so far tested. Other subcellular organelles and structures, including the nucleus, Golgi apparatus, mitochondria, lysosomes, microtubules, actin cytoskeletons, cytoplasm, lipid rafts, and plasma membrane, exhibited normal configurations. Western blot analysis of cellular prion protein (PrP(C)) revealed normal glycosylation, which is a simple marker of post-translational modification in the ER and Golgi compartments, and immunofluorescence microscopy detected no altered subcellular distribution of PrP(C) in the post-ER compartments. Further investigation is required to determine whether the distorted ER network, i.e., loss of the huntingtin function, participates in the development of HD.     

Contactin-associated protein (Caspr) 2 interacts with carboxypeptidase E in the CNS

To identify proteins interacting with the intracellular domain of the neural cell adhesion molecule contactin-associated protein 2 (Caspr2), yeast two-hybrid screening was performed. We identified carboxypeptidase E (CPE) as a Caspr2-interacting candidate protein. Glutathione S -transferase pull-down and immunoprecipitation analyses indicated that Caspr2 was associated with CPE in vitro and in vivo . Both Caspr2 and CPE were expressed predominantly in the CNS. Immunohistochemical analyses revealed that both Caspr2- and CPE-like immunoreactivities were found to co-localize in the apical dendrites and cell bodies of rat cortical neurons. In subcellular localization analysis, Caspr2- and CPE-like immunoreactivities were co-migrated in the fractions of Golgi/ER. Additionally, in COS-7 cells co-transfected with CPE and Caspr2 cDNAs, Caspr2- and CPE-immunoreactivities were co-localized in both Golgi and membrane, whereas it was only observed in Golgi of either COS-7 cell transfected with CPE or Caspr2 cDNA alone. It is known that the membrane-bound form of CPE functions as a sorting receptor of prohormones in the trans -Golgi network. Taken together, our data suggest that CPE may be a key molecule to regulate Caspr2 trafficking to the cell membrane.