A non-canonical Puf3p-binding sequence regulates CAT5/COQ7 mRNA under both fermentable and respiratory conditions in budding yeast.

The Saccharomyces cerevisiae uses a highly glycolytic metabolism, if glucose is available, through appropriately suppressing mitochondrial functions except for some of them such as Fe/S cluster biogenesis. Puf3p, a Pumillio family protein, plays a pivotal role in modulating mitochondrial activity, especially during fermentation, by destabilizing its target mRNAs and/or by repressing their translation. Puf3p preferentially binds to 8-nt conserved binding sequences in the 3'-UTR of nuclear-encoded mitochondrial (nc-mitochondrial) mRNAs, leading to broad effects on gene expression under fermentable conditions. To further explore how Puf3p post-transcriptionally regulates nc-mitochondrial mRNAs in response to cell growth conditions, we initially focused on nc-mitochondrial mRNAs known to be enriched in monosomes in a glucose-rich environment. We unexpectedly found that one of the monosome-enriched mRNAs, CAT5/COQ7 mRNA, directly interacts with Puf3p through its non-canonical Puf3p binding sequence, which is generally less considered as a Puf3p binding site. Western blot analysis showed that Puf3p represses translation of Cat5p, regardless of culture in fermentable or respiratory medium. In vitro binding assay confirmed Puf3p's direct interaction with CAT5 mRNA via this non-canonical Puf3p-binding site. Although cat5 mutants of the non-canonical Puf3p-binding site grow normally, Cat5p expression is altered, indicating that CAT5 mRNA is a bona fide Puf3p target with additional regulatory factors acting through this sequence. Unlike other yeast PUF proteins, Puf3p uniquely regulates Cat5p by destabilizing mRNA and repressing translation, shedding new light on an unknown part of the Puf3p regulatory network. Given that pathological variants of human COQ7 lead to CoQ10 deficiency and yeast cat5Δ can be complemented by hCOQ7, our findings may also offer some insights into clinical aspects of COQ7-related disorders.

Six identical tRNATrpCCA genes express a similar amount of mature tRNATrpCCA but unequally contribute to yeast cell growth.

Saccharomyces cerevisiae has 6 synonymous tRNATrpCCA genes encoding the identical sequence, including their intronic region. They are supposed to express tRNATrpCCA in the same quality and quantity. Here, we generated single to quintuple deletion strains with all the possible combinations of the synonymous tRNATrpCCA genes to analyze whether those individual genes equally contribute cell viability and tRNA production. The quintuple deletion strains that only harbor tW(CCA)J, tW(CCA)M, or tW(CCA)P were viable but almost lethal while the other quintuple deletions showed moderately impaired growth. These growth differences were not obvious among the quadruple deletion strains, which expressed almost one third of mature tRNATrpCCA in the wild type. Therefore, no dosage compensation operates for tRNATrpCCA amount, and growth variations among the quintuple deletion strains may not simply reflect differences in tRNATrpCCA shortage. Yeast may retain the redundancy of tRNATrpCCA genes for a noncanonical function(s) beyond the supply of the tRNA to translation.

Impact of intron removal from tRNA genes on Saccharomyces cerevisiae.

In eukaryotes and archaea, tRNA genes frequently contain introns, which are removed during maturation. However, biological roles of tRNA introns remain elusive. Here, we constructed a complete set of Saccharomyces cerevisiae strains in which the introns were removed from all the synonymous genes encoding 10 different tRNA species. All the intronless strains were viable, but the tRNAPheGAA and tRNATyrGUA intronless strains displayed slow growth, cold sensitivity and defective growth under respiratory conditions, indicating physiological importance of certain tRNA introns. Northern analyses revealed that removal of the introns from genes encoding three tRNAs reduced the amounts of the corresponding mature tRNAs, while it did not affect aminoacylation. Unexpectedly, the tRNALeuCAA intronless strain showed reduced 5.8S rRNA levels and abnormal nucleolar morphology. Because pseudouridine (Ψ) occurs at position 34 of the tRNAIleUAU anticodon in an intron-dependent manner, tRNAIleUAU in the intronless strain lost Ψ34. However, in a portion of tRNAIleUAU population, position 34 was converted into 5-carbamoylmethyluridine (ncm5U), which could reduce decoding fidelity. In summary, our results demonstrate that, while introns are dispensable for cell viability, some introns have diverse roles, such as ensuring proper growth under various conditions and controlling the appropriate anticodon modifications for accurate pairing with the codon.

Thoracic Endovascular Aortic Repair Including Total Debranching on Femoral Artery for Arch Rupture.

A 94-year-old woman with rupture of a thoracic aortic aneurysm (rTAA) was referred to us. She previously underwent thoracic endovascular aortic repair and was considered to be at high risk for a conventional open operation. Therefore an endovascular procedure was planned. The proximal landing zone needed to be placed at the ascending aorta to seal a type 1a endoleak. A hybrid operation consisting of supraaortic total debranching on the common femoral artery and endovascular repair was performed. All debranched bypasses were patent and the aneurysm was excluded. The patient regained sufficient ambulatory strength and showed no symptoms of syncope.

Saccharomyces cerevisiae MSA1 mRNA has a sequence for localization at the bud tip.

MSA1 mRNA encodes Msa1p, a protein associated with the SCB-binding factor (SBF) and MCB-binding factor (MBF) complex. Msa1p promotes the transcription of G1 phase-specific genes, and is subjected to cell cycle-dependent regulation for its abundance and subcellular localization. MSA1 mRNA and Msa1p levels oscillate in the cell cycle with peaks at the late M/early G1 phase and early G1 phase, respectively. Phosphorylation by CDK1 negatively regulates the nuclear localization of Msa1p. In the present study, we identified MSA1 mRNA as a bud tip-localized mRNA in screening using a Tag-GFP system. A fragmentation analysis revealed a sequence of ~145 bases for the bud tip localization. Endogenous MSA1 mRNA localized at the bud tip in a manner that depended on SHE2. Msa1p levels were also affected by SHE2 in cells constitutively expressing MSA1 mRNA. These results suggest the existence of a regulatory mechanism for Msa1p through the localized control of MSA1 mRNA.

Total thoracoscopic left ventricular lead implantation for hybrid cardiac resynchronization therapy in pacemaker-mediated cardiomyopathy.

Cardiac resynchronization therapy (CRT) has been increasingly performed in patients having heart failure with dyssynchrony. We report a successful case of total thoracoscopic left ventricular (LV) lead implantation in CRT. A 77-year-old man with marked dyssynchrony of the LV wall motion and a low ejection fraction (EF17%) due to pacemaker-mediated cardiomyopathy was referred to us. CRT was planned, but percutaneous LV lead implantation proved difficult owing to anatomical variations. The LV lead was placed in the post-lateral wall of the LV base using a total thoracoscopic procedure. Preoperative dyspnea and dyssynchrony were clearly improved. In CRT, the LV wall stimulation site is important. The LV lead should be implanted in the latest activation area, which can be detected using speckle tracking echocardiography. Surgical lead implantation can be performed in the ideal area, and this procedure may play a new role as a hybrid CRT.

Surgical Strategy for Thoracic Aortic Pseudoaneurysm with Sternal Adherence.

A thoracic aortic pseudoaneurysm is a life-threatening complication following thoracic aortic surgery. We describe a surgical strategy for this pseudoaneurysm with a high risk for rupture during median sternotomy. The pseudoaneurysm was distended and widely adherent to the posterior sternum. Elective cardiopulmonary bypass and moderate hypothermia were established, and sternotomy was performed without left ventricle distention or brain ischemia. Total arch replacement was successful and the patient was discharged on post operative day (POD) 18. A key surgical strategy was to avoid ventricular fibrillation before sternotomy. Appropriate sternotomy timing and perfusion strategy are crucial for successful treatment.

Peripartum Type A Aortic Dissection Repair Using Frozen Elephant Trunk Technique.

A 43-year-old woman with abdominal and back pain during childbirth consulted us 1 day postdelivery. Contrast-enhanced computed tomography (CT) revealed partially thrombosed type A aortic dissection with intimal tear in the proximal descending thoracic aorta. Conservative antihypertensive treatment was started. However, her abdominal pain progressively deteriorated. Repeat CT revealed narrowing of the descending aorta true lumen and progressive bowel malperfusion. Total arch replacement was urgently performed using the frozen elephant trunk technique. Postoperative CT showed true lumen widening and symptom disappearance. Follow-up CT demonstrated excellent aortic remodeling.

A simplified vector system for visualization of localized RNAs in Schizosaccharomyces pombe.

RNA localization is an important event that is essential for the polarization and differentiation of a cell. Although several methods are currently used to detect localized RNAs, a simplified detection system has not yet been developed for Schizosaccharomyces pombe. In the present study, we describe a new vector system for the visualization of localized RNAs in S. pombe using a U1A-tag-GFP system. A pREP1-U1A-tag vector plasmid to express U1A-tagged RNA and a pREP2-U1AGFP plasmid to produce a U1A-GFP fusion protein were constructed for this system. Since the U1A-GFP protein binds U1A-tagged RNA, fluorescence is observed at the location of U1A-tagged RNA in cells expressing both of these. The nucleolar localization of U3 snoRNA was successfully detected using this system, and a novel RNA localized at the DNA region of the nucleus was found by screening localized RNAs. This system will accelerate the study of localized RNAs in S. pombe.

[Rapid Determination of Seven Fungicides in Citrus Fruits].

A rapid and simple determination method of seven fungicides, thiabendazole (TBZ), pyrimethanil (PYR), o-phenylphenol (OPP), fludioxonil (FLD), azoxystrobin (AZX), imazalil (IMZ) and diphenyl (DP) in citrus fruits by LC-MS and HPLC-FL was developed. The seven fungicides were extracted with acetonitrile from citrus fruits and cleaned up with Z-Sep/C18 cartridges. The LC separation was performed on a phenyl-hexyl column with methanol-acetonitrile-10 mmol/L ammonium formate (10 : 35 : 55) as a mobile phase. The recoveries from citrus fruits fortified with the compounds at the MRLs and at 0.1 µg/g ranged from 85.4 to 106.3% and from 75.8 to 99.7%, respectively. The quantitation limits (S/N=10) were 0.03-0.07 µg/g.

Repair of Acute Type B Aortic Dissection Complicated by Aortic Rupture with Debranching Thoracic Endovascular Aortic Repair and Left Subclavian Artery Occlusion Using Amplatzer Vascular Plug II.

An 88-year-old man with severe chest pain and syncope was admitted to our hospital. Contrast-enhanced computed tomography (CT) revealed acute type B aortic dissection with rupture. Considering age and operative risk, we performed emergency thoracic aortic endovascular repair with two-debranching of the left common carotid and left subclavian arteries. To prevent type II endoleak, we used Amplatzer Vascular Plug (AVP) II for left subclavian artery embolization. Postoperative contrast-enhanced CT showed no type II endoleak and rupture site exclusion. As postoperative persistent blood flow to the primary entry or rupture site causes re-rupture, AVP II was crucial in preventing type II endoleak.

Postoperative usage of tolvaptan in a patient with aortic valve stenosis complicated by Child-Pugh classification B liver cirrhosis and hepatic edema.

We report the case of an 80-year-old woman with postoperative congestive heart failure (CHF) complicated by Child-Pugh classification B liver cirrhosis and hepatic edema successfully treated with tolvaptan. The patient suffered from liver cirrhosis and underwent partial hepatectomy for a hepatocellular carcinoma diagnosed together with a severe aortic valve stenosis. Aortic valve replacement was performed under cardiopulmonary bypass. The postoperative course was uneventful until CHF and hepatic edema symptoms appeared on postoperative day (POD) 2. The symptoms were treated with intravenous human atrial natriuretic peptide and oral diuretics. As the condition showed no improvement, oral tolvaptan was administered on POD 11 and thereafter, which markedly improved the symptoms. This is apparently the first report describing the effectiveness of tolvaptan for the postoperative management of fluid balance in a patient with cardiac and liver dysfunction.

Release factor eRF3 mediates premature translation termination on polylysine-stalled ribosomes in Saccharomyces cerevisiae.

Ribosome stalling is an important incident enabling the cellular quality control machinery to detect aberrant mRNA. Saccharomyces cerevisiae Hbs1-Dom34 and Ski7 are homologs of the canonical release factor eRF3-eRF1, which recognize stalled ribosomes, promote ribosome release, and induce the decay of aberrant mRNA. Polyadenylated nonstop mRNA encodes aberrant proteins containing C-terminal polylysine segments which cause ribosome stalling due to electrostatic interaction with the ribosomal exit tunnel. Here we describe a novel mechanism, termed premature translation termination, which releases C-terminally truncated translation products from ribosomes stalled on polylysine segments. Premature termination during polylysine synthesis was abolished when ribosome stalling was prevented due to the absence of the ribosomal protein Asc1. In contrast, premature termination was enhanced, when the general rate of translation elongation was lowered. The unconventional termination event was independent of Hbs1-Dom34 and Ski7, but it was dependent on eRF3. Moreover, premature termination during polylysine synthesis was strongly increased in the absence of the ribosome-bound chaperones ribosome-associated complex (RAC) and Ssb (Ssb1 and Ssb2). On the basis of the data, we suggest a model in which eRF3-eRF1 can catalyze the release of nascent polypeptides even though the ribosomal A-site contains a sense codon when the rate of translation is abnormally low.

Sinodielide A exerts thermosensitizing effects and induces apoptosis and G2/M cell cycle arrest in DU145 human prostate cancer cells via the Ras/Raf/MAPK and PI3K/Akt signaling pathways.

Sinodielide A (SA) is a naturally occurring guaianolide, which is isolated from the root of Sinodielsia yunnanensis. This root, commonly found in Yunnan province, is used in traditional Chinese medicine as an antipyretic, analgesic and diaphoretic agent. A number of studies have reported that agents isolated from a species of Umbelliferae (Apiaceae) have antitumor activities. We previously reported, using combined treatments with this medicinal herb and hyperthermia at various temperatures, an enhanced cytotoxicity in the human prostate cancer androgen­independent cell lines, PC3 and DU145, and analyzed the related mechanisms. In the present study, we investigated the effects of treatment with SA prior to hyperthermia on the thermosensitivity of DU145 cells, and the mechanisms related to the induction of apoptosis and G(2)/M cell cycle arrest via the activation of extracellular-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling pathways, as well as the phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. Cells were exposed to hyperthermia alone (40-44˚C) or hyperthermia in combination with SA. Lethal damage to cells treated with mild hyperthermia (40 or 42˚C) for up to 6 h was slight; however, hyperthermia in combination with SA synergistically enhanced thermosensivity. Lethal damage to cells treated with acute hyperthermia (43 or 44˚C) was more severe, but these effects were also enhanced and were more significant by the combined treatment with SA. The kinetics of apoptosis induction and cell cycle distribution were analyzed by flow cytometry. In addition, the levels of ERK1/2, JNK and Akt were determined by western blot analysis. The incidence of apoptotic cells after treatment with SA (20.0 µM) at 37˚C for 4 h, hyperthermia (44˚C) alone for 30 min, and the combination in sequence were examined. The sub-G1 division (%) in the diagram obtained by flow cytometry was applied to that assay. The percentage of apoptotic cells (10.53±5.02%) was higher at 48 h as compared to 0, 12 and 24 h after treatment. The distribution of DU145 cells in the G2/M cell cycle phase was markedly increased after 24 h of heating at 44˚C and after the combined treatment with heating and SA. The phosphorylation of ERK1/2 was reduced following treatment with heating and SA, while the levels of phosphorylated JNK (p-JNK) were markedly increased immediately after heating at 44˚C and when heating was combined with SA. By contrast, the levels of phosphorylated Akt (p-Akt) were immediately increased only after heating at 44˚C. Thus, we concluded that SA exerts its thermosensitizing effects on DU145 cells by inhibiting the activation of the MAPK/ERK1/2 and PI3K/Akt signaling pathways.

Five reasons for the lack of nursing students' motivation to learn public health.

Prevention is better than cure. Public health plays an important role in promoting prevent medicine. To obtain the abilities to provide appropriate nursing services, learning public health is necessary for students who want to become registered nurses. When teachers teach public health to nursing students, it is important to motivate them to learn it. Therefore, we investigated the reasons for the lack of motivation to learn public health by conducting a questionnaire survey. The subjects were female nursing students in 29 vocational schools in Kanagawa and Chiba prefectures of Japan that allow graduation after a 3-year study period. We asked the students whether or not they had completed the subject of public health and analyzed those students who answered affirmatively. We analyzed 1,553 respondents whose average age was 22.6 ± 5.2 years (range, 18 to 45). Using factor analysis, we discovered the 5 reasons that lead to the lack of nursing students' motivation to learn public health: "Difficulties acquiring knowledge of public health," "Inappropriate attitudes of public health teachers," "Thinking lightly about the national examination in the field of public health," "Lack of understanding the importance of learning public health," and "Future plans that do not specialize in public health." Using multiple linear regression analysis, these 5 reasons were significant predictors for the lack of students' motivation. Older students also had significantly less motivation to learn public health than did younger students. When teachers instruct their students, they should teach public health better with the present knowledge.

Nursing students' learning motivation toward technical knowledge and their ethics regarding patients' rights.

Nursing students must develop their abilities to provide appropriate nursing services. They need to acquire the level of nursing knowledge to pass the national examination according to Japanese law. Moreover, even if the awareness of the rights of people who receive nursing services increases, students must not have a sense of resistance toward those rights. Therefore, we investigated the factors associated with students' motivation to pass their examination and such a sense of resistance. We produced items related to reasons students wanted to become registered nurses with reference to job satisfaction and their learning environment (e.g., teachers' manners and school events unrelated to the examination). There were 3,417 female nursing students analyzed in 29 vocational schools that allow graduation after a 3-year study period (average age, 21.93 years [standard deviation, 5.44]). Older and third-year students had a stronger motivation to pass the examination and a weaker sense of resistance to people's rights compared with younger and first- to second-year students. Students who answered a "Lack of enthusiasm for becoming a registered nurse" had a weakened motivation and a strengthened sense of resistance. Factors enhancing students' motivation to pass their examination were "Professional commitment," "Desire for companionship," and "School events unrelated to the national examination." Factors strengthening students' sense of resistance to people's rights were "Living stability" and "Social appraisal." Teachers must develop methods to teach ethics so that their students respect the rights of people who receive nursing services and to ensure that they acquire the necessary nursing knowledge.

Role of the RNA-binding protein Nrd1 in stress granule formation and its implication in the stress response in fission yeast.

We have previously identified the RNA recognition motif (RRM)-type RNA-binding protein Nrd1 as an important regulator of the posttranscriptional expression of myosin in fission yeast. Pmk1 MAPK-dependent phosphorylation negatively regulates the RNA-binding activity of Nrd1. Here, we report the role of Nrd1 in stress-induced RNA granules. Nrd1 can localize to poly(A)-binding protein (Pabp)-positive RNA granules in response to various stress stimuli, including heat shock, arsenite treatment, and oxidative stress. Interestingly, compared with the unphosphorylatable Nrd1, Nrd1(DD) (phosphorylation-mimic version of Nrd1) translocates more quickly from the cytoplasm to the stress granules in response to various stimuli; this suggests that the phosphorylation of Nrd1 by MAPK enhances its localization to stress-induced cytoplasmic granules. Nrd1 binds to Cpc2 (fission yeast RACK) in a phosphorylation-dependent manner and deletion of Cpc2 affects the formation of Nrd1-positive granules upon arsenite treatment. Moreover, the depletion of Nrd1 leads to a delay in Pabp-positive RNA granule formation, and overexpression of Nrd1 results in an increased size and number of Pabp-positive granules. Interestingly, Nrd1 deletion induced resistance to sustained stresses and enhanced sensitivity to transient stresses. In conclusion, our results indicate that Nrd1 plays a role in stress-induced granule formation, which affects stress resistance in fission yeast.

Thermosensitization and induction of apoptosis or cell-cycle arrest via the MAPK cascade by parthenolide, an NF-κB inhibitor, in human prostate cancer androgen-independent cell lines.

Parthenolide (PTL), a nuclear factor-κB (NF-κB) inhibitor, has a significant thermo-enhancement effect. Modification of thermosensitivity by treatment with PTL prior to hyperthermia was investigated in the human prostate cancer androgen-independent cell lines PC3 and DU145. In addition, we analyzed the mechanisms related to induction of apoptosis or G2/M cell-cycle arrest via the effects of ERK1/2, p38 and SAPK/JNK signaling on mitogen-activated protein kinase (MAPK). Lethal damage caused by mild hyperthermia at 41.0˚C or 42.0˚C in both cell lines resulted in a low level of thermosensitivity, while sequential combination with PTL showed significant thermosensitization. Step-up hyperthermia (SUH) (42˚C for 30 min, 43.0˚C or 43.5˚C for various periods) reduced the thermosensitivity of the cells to second heating. However, PTL given as pre-treatment prior to SUH prevented SUH-induced thermal tolerance and resulted in significant thermosensitization. Induction of apoptosis by the combination of PTL and hyperthermia at 44.0˚C was determined by the ratio of sub-G1 division cells using flow cytometry, which was increased significantly in comparison with single treatment, and was more effective in PC3 than DU145 cells. The behavior of ERK1/2, p38, and SAPK/JNK signaling in the MAPK cascade by treatment with PTL and hyperthermia were examined by Western blotting. As for PC3 cells, ras-downstream p-ERK1/2 was activated and p-p38 slightly activated by combined treatment with PTL and hyperthermia in comparison with each alone. As for DU145 cells, ERK1/2 was not changed, while p38 and SAPK/JNK were slightly activated by combination treatment. These results were related to increases in the induction of apoptosis, G2/M cell cycle arrest, and lethal damage of cells via the MAPK cascade. Together, our findings demonstrate that PTL is an effective thermosensitizing agent for multidisciplinary therapy for human prostate cancer.

EGD1 (ß-NAC) mRNA is localized in a novel cytoplasmic structure in Saccharomyces cerevisiae.

RNA localization is a common mechanism for recruiting proteins to specific regions of a cell, which causes cell polarization and sometimes asymmetric division. We found that EGD1 mRNA accumulates dose-dependently as a cytoplasmic granule in Saccharomyces cerevisiae. EGD1 encodes a ß-subunit of the nascent polypeptide-associated complex (NAC). NAC is a heterodimer consisting of α- and ß-subunits, associated with ribosomes and thought to be involved in the folding of nascent polypeptide chains. Analysis of deletion constructs showed that the localization of EGD1 mRNA requires both an upstream region and an ORF of EGD1, suggesting that the translation of Egd1p is important for localization. We also showed that Egd1p and P-body components are co-localized with EGD1 mRNA. This granule, named the EGD1 granule, has features similar to cellular inclusions containing aggregated proteins. Disruption of microtubules by treatment with a drug, benomyl, resulted in loss of the EGD1 granule. When the expression level of EGD2 encoding the αNAC increased, the percentage of cells showing the EGD1 granule decreased, suggesting that the granular distribution of EGD1 depends on the quantitative balance between α- and ß-subunits of NAC. Taken together, we propose a novel microtubule-dependent mechanism for controlling NAC through RNA localization.