Differential requirement of Oryza sativa RAR1 in immune receptor-mediated resistance of rice to Magnaporthe oryzae

The required for Mla12 resistance (RAR1) protein is essential for the plant immune response. In rice, a model monocot species, the function of Oryza sativa RAR1 (OsRAR1) has been little explored. In our current study, we characterized the response of a rice osrar1 T-DNA insertion mutant to infection by Magnaporthe oryzae, the causal agent of rice blast disease. osrar1 mutants displayed reduced resistance compared with wild type rice when inoculated with the normally virulent M. oryzae isolate PO6-6, indicating that OsRAR1 is required for an immune response to this pathogen. We also investigated the function of OsRAR1 in the resistance mechanism mediated by the immune receptor genes Pib and Pi5 that encode nucleotide binding-leucine rich repeat (NB-LRR) proteins. We inoculated progeny from Pib/osrar1 and Pi5/osrar1 heterozygous plants with the avirulent M. oryzae isolates, race 007 and PO6-6, respectively. We found that only Pib-mediated resistance was compromised by the osrar1 mutation and that the introduction of the OsRAR1 cDNA into Pib/osrar1 rescued Pib-mediated resistance. These results indicate that OsRAR1 is required for Pib-mediated resistance but not Pi5-mediated resistance to M. oryzae.     

Verification of accuracy and validity of gait phase detection system using motion sensors for applying walking assistive FES

In this study, we have analysed heel strike (HS) and toe off (TO) of normal individuals and hemiplegic patients, taking advantage of output curves acquired from various sensors, and verified the validity of sensor detection methods and their effectiveness when they were used for hemiplegic gaits. Gait phase detections using three different motion sensors were valid, since they all had reliabilities more than 95%, when compared with foot velocity algorithm. Results showed that the tilt sensor and the gyrosensor could detect gait phase more accurately in normal individuals. Vertical acceleration could detect HS most accurately in hemiplegic patient group A. The gyrosensor could detect HS and TO most accurately in hemiplegic patient groups A and B. The detection of TO from all sensor signals was valid in both the patient groups A and B. However, the vertical acceleration detected HS validly in patient group A and the gyrosensor detected HS validly in patient group B.     

PTP1B inhibitory effects of tridepside and related metabolites isolated from the Antarctic lichen Umbilicaria antarctica

The selective inhibition of PTP1B has been widely recognized as a potential drug target for the treatment of type 2 diabetes and obesity. In the course of screening for PTP1B inhibitory natural products, the MeOH extract of the dried sample of the Antarctic lichen Umbilicaria antarctica was found to exhibit significant inhibitory effect, and the bioassay-guided fractionation and purification afforded three related lichen metabolites 1-3. Compounds 1-3 were identified as gyrophoric acid (1), lecanoric acid (2), and methyl orsellinate (3) mainly by analysis of NMR and MS data. These compounds inhibited PTP1B activity with 50% inhibitory concentration values of 3.6 ± 0.04 μM, 31 ± 2.7 μM, and 277 ± 8.6 μM, respectively. Furthermore, the kinetic analysis of PTP1B inhibition by compound 1 suggested that the compound inhibited PTP1B activity in a non-competitive manner.     

Delphinidin prevents hypoxia-induced mouse embryonic stem cell apoptosis through reduction of intracellular reactive oxygen species-mediated activation of JNK and NF-κB, and Akt inhibition

Delphinidin, gallic acid, betulinic acid, and ursolic acid, which are bio-active ingredients in a variety of fruits, vegetables, and herbs, have potent antioxidant activity and various biological activities. However, it is not clear whether these bio-active ingredients can significantly contribute to the protection of embryonic stem (ES) cells from hypoxia-induced apoptosis. In the present study, hypoxia-induced ES cells apoptosis with time, which were abrogated by pretreatment with all ingredients. Hypoxia-induced ROS generation was blocked by pretreatment with all ingredients in a dose-dependent manner, with the maximum ROS scavenging effect observed for delphinidin. Hypoxia increased phosphorylation of JNK and NF-κB were blocked by pretreatment of delphinidin as well as NAC. Hypoxia decreased phosphorylation of Akt^thr308 and ^ser473; these decreases were reversed by pretreatment with delphinidin or NAC. However, Akt inhibition did not affect NF-κB phosphorylation. Delphinidin attenuated the hypoxia-induced increase in Bax, cleaved caspase-9, cleaved caspase-3, and decrease in Bcl-2, which were diminished by pretreatment of Akt inhibitor. Hypoxia induced Bax translocation from the cytosol to mitochondria. Furthermore, hypoxia induced mitochondria membrane potential loss and cytochrome c release in cytosol, which were blocked by delphinidin pretreatment. Hypoxia induced cleavage of procaspase-9 and procaspase-3 which were blocked by delphinidin or SP600125, but Akt inhibitor abolished the protection effect of delphinidin. Moreover, inhibition of JNK and NF-κB abolished hypoxia-induced ES cell apoptosis and inhibition of Akt attenuated delphinidin-induced blockage of apoptosis. The results indicate that delphinidin can prevent hypoxia-induced apoptosis of ES cells through the inhibition of JNK and NF-κB phosphorylation, and restoration of Akt phosphorylation.     

Properties and Expression of Na+/K+-ATPase α-Subunit Isoforms in the Brain of the Swamp Eel,Monopterus albus,Which Has Unusually High Brain Ammonia Tolerance

The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l⁻¹) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g⁻¹). Na⁺/K⁺-ATPase (Nka) is an essential transporter in brain cells, and since NH₄ ⁺ can substitute for K⁺ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K⁺ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH₄ ⁺ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na⁺/K⁺-ATPase and Na⁺/NH₄ ⁺-ATPase activities over a range of K⁺/NH₄ ⁺ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l⁻¹ NH₄Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na⁺/NH₄ ⁺-ATPase activities were significantly lower than the Na⁺/K⁺-ATPase activities assayed at various NH₄ ⁺/K⁺ concentrations. Furthermore, the effectiveness of NH₄ ⁺ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K⁺ specificity of K⁺ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus.     

An Optimized Whole-Body Cortisol Quantification Method for Assessing Stress Levels in Larval Zebrafish

Glucocorticoids serve important regulatory functions for many physiological processes and are critical mediators of the stress response. The stress response is a set of bodily processes aimed at counteracting a state of threatened homeostasis. Proper stress response is critical for the survival of an animal, however prolonged or abnormal stress response can be detrimental and is implicated in a number of human diseases such as depression and metabolic diseases. To dissect the underlying mechanism of this complex and important response, the zebrafish, Danio rerio offer important advantages such as ease of genetic manipulations and high-throughput behavioral analyses. However, there is a paucity of suitable methods to measure stress level in larval zebrafish. Therefore, an efficient low-cost method to monitor stress hormone levels will greatly facilitate stress research in zebrafish larvae. In this study, we optimized sample collection as well as cortisol extraction methods and developed a home-made ELISA protocol for measuring whole-body cortisol level in zebrafish larvae. Further, using our customized protocols, we characterized the response of larval zebrafish to a variety of stressors. This assay, developed for efficient cortisol quantification, will be useful for systematic and large-scale stress analyses in larval zebrafish.     

Peripheral Afferent Mechanisms Underlying Acupuncture Inhibition of Cocaine Behavioral Effects in Rats

Administration of cocaine increases locomotor activity by enhancing dopamine transmission. To explore the peripheral mechanisms underlying acupuncture treatment for drug addiction, we developed a novel mechanical acupuncture instrument (MAI) for objective mechanical stimulation. The aim of this study was to evaluate whether acupuncture inhibition of cocaine-induced locomotor activity is mediated through specific peripheral nerves, the afferents from superficial or deep tissues, or specific groups of nerve fibers. Mechanical stimulation of acupuncture point HT7 with MAI suppressed cocaine-induced locomotor activity in a stimulus time-dependent manner, which was blocked by severing the ulnar nerve or by local anesthesia. Suppression of cocaine-induced locomotor activity was elicited after HT7 stimulation at frequencies of either 50 (for Meissner corpuscles) or 200 (for Pacinian corpuscles) Hz and was not affected by block of C/Aδ-fibers in the ulnar nerve with resiniferatoxin, nor generated by direct stimulation of C/Aδ-fiber afferents with capsaicin. These findings suggest that HT7 inhibition of cocaine-induced locomotor activity is mediated by A-fiber activation of ulnar nerve that originates in superficial and deep tissue.     

Crif1 Deficiency Reduces Adipose OXPHOS Capacity and Triggers Inflammation and Insulin Resistance in Mice

Impaired mitochondrial oxidative phosphorylation (OXPHOS) has been proposed as an etiological mechanism underlying insulin resistance. However, the initiating organ of OXPHOS dysfunction during the development of systemic insulin resistance has yet to be identified. To determine whether adipose OXPHOS deficiency plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with OXPHOS–deficient adipose tissue was examined. Crif1 is a protein required for the intramitochondrial production of mtDNA–encoded OXPHOS subunits; therefore, Crif1 haploinsufficient deficiency in mice results in a mild, but specific, failure of OXPHOS capacity in vivo. Although adipose-specific Crif1-haploinsufficient mice showed normal growth and development, they became insulin-resistant. Crif1-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon stress kinases and antioxidant. Accordingly, examination of adipose tissue from Crif1-haploinsufficient mice revealed increased secretion of MCP1 and TNFα, as well as marked infiltration by macrophages. These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance.     

Structural alteration of Escherichia coli Hsp31 by thermal unfolding increases chaperone activity

Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more β-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.