Evidence from Crosslinking for Nearest-Neighbor Relationships among the Three Extrinsic Proteins of Spinach Photosystem II Complexes that are Associated with Oxygen Evolution

Treatment of oxygen-evolving Photosystem II complexes, whichlack light-harvesting chlorophyll a/b proteins, with a seriesof disuccinimidyl esters with different chain lengths yieldeda crosslinked product which consisted of one molecule each ofthe extrinsic 33 kDa and 23 kDa proteins. In addition, crosslinkingbetween the 33 kDa protein and the chlorophyll-carrying 47 kDaprotein and between the 23 kDa and 17 kDa proteins was confirmed.Thus, the three extrinsic proteins are closely associated witheach other to form a complex which is attached to the PS IIreaction center complexes. (Received December 1, 1989; Accepted May 2, 1990)     

Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice

BackgroundMutations in GJB2, which encodes connexin 26 (Cx26), a cochlear gap junction protein, represent a major cause of pre-lingual, non-syndromic deafness. The degeneration of the organ of Corti observed in Cx26 mutant—associated deafness is thought to be a secondary pathology of hearing loss. Here we focused on abnormal development of the organ of Corti followed by degeneration including outer hair cell (OHC) loss.MethodsWe investigated the crucial factors involved in late-onset degeneration and loss of OHC by ultrastructural observation, immunohistochemistry and protein analysis in our Cx26-deficient mice (Cx26^f/fP0Cre).ResultsIn ultrastructural observations of Cx26^f/fP0Cre mice, OHCs changed shape irregularly, and several folds or notches were observed in the plasma membrane. Furthermore, the mutant OHCs had a flat surface compared with the characteristic wavy surface structure of OHCs of normal mice. Protein analysis revealed an increased protein level of caveolin-2 (CAV2) in Cx26^f/fP0Cre mouse cochlea. In immunohistochemistry, a remarkable accumulation of CAV2 was observed in Cx26^f/fP0Cre mice. In particular, this accumulation of CAV2 was mainly observed around OHCs, and furthermore this accumulation was observed around the shrunken site of OHCs with an abnormal hourglass-like shape.ConclusionsThe deformation of OHCs and the accumulation of CAV2 in the organ of Corti may play a crucial role in the progression of, or secondary OHC loss in, GJB2-associated deafness. Investigation of these molecular pathways, including those involving CAV2, may contribute to the elucidation of a new pathogenic mechanism of GJB2-associated deafness and identify effective targets for new therapies.     

RNA-Seq Analysis of the Response of the Halophyte,Mesembryanthemum crystallinum (Ice Plant) to High Salinity

Understanding the molecular mechanisms that convey salt tolerance in plants is a crucial issue for increasing crop yield. The ice plant (Mesembryanthemum crystallinum) is a halophyte that is capable of growing under high salt conditions. For example, the roots of ice plant seedlings continue to grow in 140 mM NaCl, a salt concentration that completely inhibits Arabidopsis thaliana root growth. Identifying the molecular mechanisms responsible for this high level of salt tolerance in a halophyte has the potential of revealing tolerance mechanisms that have been evolutionarily successful. In the present study, deep sequencing (RNAseq) was used to examine gene expression in ice plant roots treated with various concentrations of NaCl. Sequencing resulted in the identification of 53,516 contigs, 10,818 of which were orthologs of Arabidopsis genes. In addition to the expression analysis, a web-based ice plant database was constructed that allows broad public access to the data. The results obtained from an analysis of the RNAseq data were confirmed by RT-qPCR. Novel patterns of gene expression in response to high salinity within 24 hours were identified in the ice plant when the RNAseq data from the ice plant was compared to gene expression data obtained from Arabidopsis plants exposed to high salt. Although ABA responsive genes and a sodium transporter protein (HKT1), are up-regulated and down-regulated respectively in both Arabidopsis and the ice plant; peroxidase genes exhibit opposite responses. The results of this study provide an important first step towards analyzing environmental tolerance mechanisms in a non-model organism and provide a useful dataset for predicting novel gene functions.     

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.     

Cell-free identification of novel N-myristoylated proteins from complementary DNA resources using bioorthogonal myristic acid analogues

To establish a non-radioactive, cell-free detection system for protein N-myristoylation, metabolic labeling in a cell-free protein synthesis system using bioorthogonal myristic acid analogues was performed. After Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) with a biotin tag, the tagged proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and blotted on a polyvinylidene fluoride (PVDF) membrane, and then protein N-myristoylation was detected by enhanced chemiluminescence (ECL) using horseradish peroxidase (HRP)-conjugated streptavidin. The results showed that metabolic labeling in an insect cell-free protein synthesis system using an azide analogue of myristic acid followed by CuAAC with alkynyl biotin was the most effective strategy for cell-free detection of protein N-myristoylation. To determine whether the newly developed detection method can be applied for the detection of novel N-myristoylated proteins from complementary DNA (cDNA) resources, four candidate cDNA clones were selected from a human cDNA resource and their susceptibility to protein N-myristoylation was evaluated using the newly developed strategy. As a result, the products of three cDNA clones were found to be novel N-myristoylated protein, and myristoylation-dependent specific intracellular localization was observed for two novel N-myristoylated proteins. Thus, the metabolic labeling in an insect cell-free protein synthesis system using bioorthogonal azide analogue of myristic acid was an effective strategy to identify novel N-myristoylated proteins from cDNA resources.     

Enhancement of phase II enzyme activity by purpurin resulting in the suppression of MeIQx-DNA-adduct formation in mice

We previously demonstrated using a bacterial system that the antigenotoxic activity of the anthraquinone compounds purpurin and alizarin was due to the suppression of microsomal enzyme activity involved in the activation of mutagens. In the present study we determined the effect of purpurin and alizarin on (i) MeIQx-DNA-adduct formation in mouse tissues and (ii) the activity of phases I and II enzymes in liver fractions, the liver being the target tissue of MeIQx. The amount of MeIQx-DNA adduct formed was determined using 32P-postlabeling methods. Methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD) enzyme activities, which reflect CYP 1A activity, were measured as markers for phase I enzymes, and UDP-glucuronyltransferase (UGT) and glutathione S-transferase (GST) activities were determined as markers for phase II enzymes. Mice fed with a diet containing 0.5% purpurin for 3 days prior to MeIQx administration had 70% fewer MeIQx-DNA adducts in the lung and kidney, and fewer DNA adducts (insignificant, statistically) in the liver compared with mice fed a diet lacking purpurin. MROD and EROD activities in the liver of these mice increased six- and eight-fold, respectively, and were higher than those determined for the control mice within 1 day following commencement of purpurin treatment. These elevated activities were maintained during treatment and declined immediately following removal of purpurin from the diet. GST and UGT activities gradually increased 2.5- and 3-fold, respectively, following purpurin treatment, and were maintained at significantly high levels even after purpurin administration ceased. Alizarin did not significantly affect DNA-adduct formation and enzyme activity, except in the case of UGT. Taken together, our results show that purpurin reduced MeIQx-DNA-adduct formation by maintaining elevated phase II enzyme activities, thereby facilitating accelerated excretion of MeIQx.     

Ovariectomy augments pressure overload-induced hypertrophy associated with changes in Akt and nitric oxide synthase signaling pathways in female rats

To elucidate the molecular mechanism underlying estrogen-mediated cardioprotection in left ventricular (LV) hypertrophy and remodeling, we analyzed myocardial hypertrophy as well as cardiac function and hypertrophy-related protein expression in ovariectomized, aortic-banded rats. Wistar rats subjected to bilateral ovariectomy (OVX) were further treated with abdominal aortic stenosis. Effects on LV morphology and function were assessed using echocardiography, and expression of protein levels was determined by Western blot analysis. The heart-to-body weight ratio was most significantly increased in the OVX-pressure overload (PO) group compared with the OVX group and in the PO group compared with sham. The LV weight-to-body weight ratio was also significantly increased in the OVX-PO group compared with the OVX group and in the PO group compared with sham. The most significant increases in LV end diastolic pressure, LV developed pressure, and +/-dp/dt(max) were observed in the OVX-PO group compared with the OVX group and represent compensatory phenotypes against hypertrophy. Both endothelial nitric oxide (eNOS) synthase expression and activity was markedly reduced in the OVX-PO group, and protein kinase B (Akt) activity was largely attenuated. Marked breakdown of dystrophin was also seen in hearts of OVX-PO groups. Finally, significantly increased mortality was observed in the OVX-PO group following chronic isoproterenol administration. Our results demonstrate that rats subjected to ovariectomy are unable to compensate for hypertrophy, showed deteriorated heart function, and demonstrated increased mortality. Simultaneous impairment of eNOS and Akt activities and reduced dystrophin by ovariectomy likely contribute to cardiac decompensation during PO-induced hypertrophy in ovariectomized rats.     

Ixodes philipi (Acari: Ixodidae): phylogenetic status inferred from mitochondrial cytochrome oxidase subunit I gene sequence comparison

Ixodes philipi ticks were collected from the nest burrows of streaked shearwaters, Calonectris luecomelas, on 3 different islands of Japan (Awashima: 38 degrees 45'N, 139 degrees 24'E; Mikurajima: 33 degrees 52'N, 139 degrees 36'E; and Omorijima: 36 degrees 8'N, 133 degrees 10'E). The mitochondrial cytochrome oxidase subunit I (COI) gene sequence was determined for each tick. The COI sequences of 9 other ixodid tick species also were determined, and they were used for taxonomic positioning of I. philipi. A metastriata tick, Amblyomma triguttatum, was used as an outgroup reference for the analysis. Phylogenetic examination indicated that the I. philipi ticks are on the branch with Ixodes turdus and Ixodes acutitarsus weakly, and the bootstrap value of this branching was low. Three different analyses, maximum parsimony, genetic distance, and maximum likelihood, support this conclusion. To further refine this analysis, 2761 base pairs (bp) of sequence, which included the genes for tRNA(Met), NADH dehydrogenase subunit 2 (ND2), tRNA(Trp), tRNA(Cys), tRNA(Tyr), and COI, were determined and compared for 6 I. philipi ticks from the 3 different collection sites. Although a base substitution (T to C in the ND2 gene for an Awashima tick) and 2 transitions (G to A in the COI gene for 1 Omorijima tick) have occurred, the overall sequences were highly conserved. Preserved mitochondrial sequences in the ticks from 3 widely separated locations suggest the possibility of gene flow, which was probably accomplished by migratory seabirds.