A simple and high-sensitivity method for analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis

<p>Abstract</p> <p>Background</p> <p>Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for <it>in vitro </it>analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection.</p> <p>Results</p> <p>Using wheat cell-free synthesis, 11 E3 proteins from Arabidopsis full-length cDNA templates were produced. These proteins were analyzed either in the translation mixture or purified recombinant protein from the translation mixture. In our luminescent method using FLAG- or His-tagged and biotinylated ubiquitins, the polyubiquitin chain on AtUBC22, UPL5 and UPL7 (HECT) and CIP8 (RING) was detected. Also, binding of ubiquitin to these proteins was detected using biotinylated ubiquitin and FLAG-tagged recombinant protein. Furthermore, screening of the RING 6 subgroup demonstrated that At1g55530 was capable of polyubiquitin chain formation like CIP8. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. The amount of polyubiquitinated proteins in the crude translation reaction mixture was unaffected by treatment with MG132, suggesting that our system does not contain 26S proteasome-dependent protein degradation activity.</p> <p>Conclusion</p> <p>In this study, we developed a simple wheat cell-free based luminescence method that could be a powerful tool for comprehensive ubiquitination analysis.</p

The Relationship between Inferior Vena Cava Distensibility and Arterial Blood Pressure

The purpose of this study was to clarify the relationship between the inferior vena cava distensibility and blood pressure. Six Japanese healthy males volunteered to participate in this study. We measured the cross-sectional area of inferior vena cava (CSAivc) and blood pressure at rest and during passive leg raising in supine position. We calculated the change rates of each parameter, based on the value at rest in supine position. We observed a negative correlation between the change rate of CSAivc and systolic blood pressure (P<0.05). These results suggest that the inferior vena cava distensibility affects partially systolic blood pressure

Improved Path Planning of the Blade for an Automatic Spinach Harvester

ArticleIFAC-PapersOnLine. 53(2): 15816-15823. (2020)journal articl

Motivational orientation of college students who self-select to study English

This study examined the motivational orientation of Japanese college students who self-selected to study English. Self-determination theory (SDT) was used as the theoretical base. Semi-structured retrospective interviews were conducted with 35 EFL college students in order to identify their motivation at the beginning of their college English courses and motivation changes that occurred over the semester. A qualitative analysis of audio-recorded interviews demonstrated that a combination of intrinsic and extrinsic motivation is important when starting college English courses. In some cases, however, a strong single motivation type also played a prominent role. For continuing foreign language learning, maintaining the combination of self-determined motivation (both extrinsic and intrinsic) is crucial, but the effect of a strong single type of motivation disappears. The analysis also suggested the possibility that intrinsic motivation can be enhanced by course contents and teaching approach; however, no clear method for enhancing the "internalization" of extrinsic regulation was found. Moreover, our findings pose the possibility of the developmental nature of motivation, which SDT does not support

Day-to-Day Variation in the Hoffmann Reflex in Females

The present study aimed to investigate changes in the Hoffmann reflex (H-reflex) at the same time on different days and to compare the findings between genders. Four males and five females in healthy subjects agreed to participate in the study and provided informed consent. All subjects were nonsmokers, and none of the females were consuming any oral contraceptives. The H-reflex was elicited in the right soleus muscle by constant current stimulation of the tibial nerve in the popliteal fossa with the subjects in the prone position. As a reference, the maximal amplitude of the H-wave relative to that of the M-wave (Hmax/Mmax) was measured to determine changes in α-motoneuron excitability. Hmax/Mmax of 6 to 9 trials and was represented by the coefficient of variation (CV). The CV of Hmax/Mmax in females was significantly higher than that in males. The level of Hmax/Mmax for males was highly reproducible, suggesting that fewer factors alter α-motoneuron excitability in males compared with those in females. Several female subjects experienced symptoms of depression and suffered from body and menstrual pain. Variations in Hmax/Mmax values were considerably greater for females than for males, suggesting that the α-motoneuron might be influenced by the menstrual cycle phase as well as by individual differences

CAUSES OF FUNCTIONAL DECLINE IN ELDERLY HOSPITALIZED PATIENTS RECEIVING EITHER INDIVIDUAL OR EXCLUSIVE REHABILITATION THERAPY: A COHORT STUDY

Background: Recently, exclusive rehabilitation therapy was introduced to prevent functional decline due to hospital-associated deconditioning by managing older inpatients’ activities of daily living in Japan. However, this type of therapy does not provide one-on-one exercises similar to individual rehabilitation therapy. This study aimed to report the present ward conditions and the causes of the functional decline in elderly patients receiving individual or exclusive rehabilitation therapy. Methods: A total of 1,636 inpatients, aged 65 years or older, were included in the study. Barthel Index at admission and discharge was assessed prospectively to analyze functional decline. We further analyzed the causes of functional decline by investigating the inpatient’s medical records. Results: Forty-three inpatients (2.6%) had functional decline during hospitalization. There were no significant differences in age, Barthel Index at the time of admission, and the type of clinical department between inpatients with and without functional decline. The functional decline rate in individual rehabilitation therapy was 8.2%, which was significantly higher compared to exclusive rehabilitation therapy (0.8%). The most common causes of functional decline were a pain, low postoperative physical fitness, malignant neoplasm, and new-onset cerebral stroke. Conclusion: We report the present ward conditions in elderly patients receiving either individual or exclusive rehabilitation therapies. Functional decline was correlated to the inpatients’ disease and conditions. The causes of the functional decline can be classified based on whether rehabilitation was effective or ineffective. If the functional decline was caused by hospital-associated deconditioning, we should address the functional decline by providing appropriate rehabilitation methods

The long noncoding RNA SPRIGHTLY acts as an intranuclear organizing hub for pre-mRNA molecules

Molecular mechanisms by which long noncoding RNA (lncRNA) molecules may influence cancerous condition are poorly understood. The aberrant expression of SPRIGHTLY lncRNA, encoded within the drosophila gene homolog Sprouty-4 intron, is correlated with a variety of cancers, including human melanomas. We demonstrate by SHAPE-seq and dChIRP that SPRIGHTLY RNA secondary structure has a core pseudoknotted domain. This lncRNA interacts with the intronic regions of six pre-mRNAs: SOX5, SMYD3, SND1, MEOX2, DCTN6, and RASAL2, all of which have cancer-related functions. Hemizygous knockout of SPRIGHTLY by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 in melanoma cells significantly decreases SPRIGHTLY lncRNA levels, simultaneously decreases the levels of its interacting pre-mRNA molecules, and decreases anchorage-independent growth rate of cells and the rate of in vivo tumor growth in mouse xenografts. These results provide the first demonstration of an lncRNA’s three-dimensional coordinating role in facilitating cancer-related gene expression in human melanomas

Definition of the profile gain factor and its application for internal transport barrier analysis in torus plasmas

In this paper, a new criterion for the internal transport barrier (ITB) formation is proposed by defining a unique scalar parameter, the profile gain factor. The profile gain factor shows degree of the confinement improvement with respect to an arbitrary reference temperature profile in the L-mode. As the reference L-mode profile for the large helical device (LHD), the edge ion temperature profile data is extrapolated to the core by the L-mode profile function, which is characterized by the thermal diffusion coefficient being proportional to the local ion temperature. The profile gain factor is defined as the ratio of the ion stored energy experimentally measured to that evaluated from the reference L-mode profile. The proposed method is applied to the LHD experimental data, and its capability for quantification of the ITB strength is demonstrated