Sequence-specific cleavage of RNA by a hybrid ribonuclease H

AbstractSite-specific cleavage of the 22-, 132- and 534-base RNAs by the DNA/protein hybrid R Nase H were examined. The 22-base RNA was chemically synthesized, and 132- and 534-base RNAs were prepared by run-off transcription. The hybrid enzyme cleaves these RNAs, which contain a single target sequence, primarily at the unique phosphodiester bond within the target sequence. The hybrid enzyme performs multiple turnovers, and at a substrate/enzyme ratio of 10:1 the RNAs are almost completely cleaved by the hybrid enzyme at 37°C within 1 h. We propose that hybrid RNase H molecules with various oligodeoxyribonucleotides function as RNA restriction enzymes and are useful for structural and functional studies of RNA

New NTP analogs: the synthesis of 4′-thioUTP and 4′-thioCTP and their utility for SELEX

The synthesis of the triphosphates of 4′-thiouridine and 4′-thiocytidine, 4′-thioUTP (7; thioUTP) and 4′-thioCTP (8; thioCTP), and their utility for SELEX (systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate (NTP) analogs 7 and 8 were prepared from appropriately protected 4′-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [(1989) J. Org. Chem., 54, 631–635]. Because SELEX requires both in vitro transcription and reverse transcription, we examined the ability of 7 and 8 for SELEX by focusing on the two steps. Incorporation of 7 and 8 by T7 RNA polymerase to give 4′-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2′-NH(2)dUTP and 2′-NH(2)dCTP; 2′-FdUTP and 2′-FdCTP), when an adequate leader sequence of DNA template was selected. We revealed that a leader sequence of about +15 of DNA template is important for the effective incorporation of modified NTP analogs by T7 RNA polymerase. In addition, reverse transcription of the resulting thioRNA into the complementary DNA in the presence of 2′-deoxynucleoside triphosphates (dNTPs) also proceeded smoothly and precisely. The stability of the thioRNA toward RNase A was 50 times greater than that of the corresponding natural RNA. With these successful results in hand, we attempted the selection of thioRNA aptamers to human α-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (K(d) = 4.7 nM)

Investigation of oxidative stress and dietary habits in Mongolian people, compared to Japanese people

BACKGROUND: The average life span of Mongolians is 62 years for males and 69 years for females. This life span is about 16 years shorter than that of Japanese. Mongolian people generally eat meat, fat and diary products but less vegetables or fruit. Thus, we investigated the state of oxidative stress and dietary habits of Mongolians. METHODS: The investigation was performed in Murun city in the northwest area of Mongolia. A total of 164 healthy subjects (24–66 y) were enrolled. As a marker of reactive oxygen species, the levels of reactive oxygen metabolites (ROM) were measured using the d-ROM test. Interviews about dietary habits were performed using the Food Frequency Questionnaire established by the Kagawa Nutrition University. RESULTS: ROM levels were 429.7 ± 95.2 Carr U for Murun subjects, whereas Japanese people (n = 220, 21–98 y) showed 335.3 ± 59.8 (p < 0.001). The levels of serum malondialdehyde-modified low-density lipoprotein-cholesterol and urinary 8-hydroxydeoxyguanosine were also high. ROM levels correlated with body fat ratio and inversely correlated with handgrip strength. Handgrip strength in the subjects over 45 years decreased more rapidly than that of age-matched Japanese. Murun subjects ate larger amounts of meat, fat, milk and flour and dairy products than Japanese, but less vegetables or fruit. Serum vitamin A and E levels were the same as Japanese references, but vitamin C levels were lower. CONCLUSION: Murun subjects may be in high oxidative stress, which may have a relationship with early ageing and several diseases, ultimately resulting in their short life span. In order to increase antioxidant capacity and suppress overproduction of ROM, antioxidant food intake is recommended

Endobronchial Argon Plasma Coagulation for the Palliation of Recurrent Tracheobronchial Adenoid Cystic Carcinoma

This is a non-final version of an article published in final form in Journal of Bronchology, 14(4):278-280, October 2007.The standard treatment of adenoid cystic carcinoma (ACC) of the airway is surgery, if possible, because survival rate is better than other treatments including radiotherapy. Although ACC shows frequent recurrence during the long-term follow-up unless there has been a complete resection (negative surgical margin), no further treatments are recommended. This report describes how argon plasma coagulation using flexible bronchoscopy has been successfully employed in the treatment of ACC after conventional therapy in 1 case of recurrence after surgery and 2 cases of inoperable patients. All of the patients are alive and healthy more than 6 years after diagnosis.ArticleJournal of Bronchology. 14(4):278-280 (2007)journal articl

Effect of amino acid mixtures on nasal allergic responses induced by toluene diisocyanate in mice

We studied the effect of various amino acid mixtures on nasal allergy induced by the intranasal application of toluene diisocyanate (TDI) in mice. In Experiment 1 (Exp. 1), mice were fed a 25% casein, soy protein isolate (SPI), egg white protein (EW) or gluten diet. In Experiment 2 (Exp. 2), mice were fed a 25% amino acid mixture diets patterned after casein (AA-casein), SPI (AA-SPI), EW protein (AA-EW) or gluten (AA-gluten). In Experiment 3 (Exp. 3) we modified the glutamine/glutamic acid (Gln/Glu) concentrations in the amino acid mixtures. Mice were fed a 25% AA-SPI, low Gln/Glu AA-SPI (LG-AA-SPI), AA-EW or high Gln/Glu AA-EW (HG-AA-EW) diet. At the 5th week, mice were divided into sensitized (sen-) and non-sensitized (ns-) groups. The mice in sensitized groups were treated with two courses of intranasal application of toluene diisocyanate (TDI) in ethyl acetate for 5 consecutive days, separated by 9 days rest. The non-sensitized groups of mice were treated with a vehicle. Nine days after the second sensitization, all mice were provoked by TDI. Nasal responses and serum IgE concentration were studied. The findings of Exp. 1 showed that the sen-EW group exhibited a lower body weight gain, higher nasal symptom score and higher IgE concentration than the other sensitized groups. The findings of Exp. 2 showed that the sen-EW group had a lower body weight gain, higher nasal symptom score and higher IgE concentration than the other sensitized groups. In Exp. 3, the AA-EW group showed a higher total nasal score and IgE concentration than the HG-AA-EW group, however, the findings of LG-AA-SPI and AA-SPI were similar. These findings demonstrated that amino acid mixtures affect nasal allergy induced by the intranasal application of TDI in mice

Third quantization of f(R)f(R)-type gravity

We examine the third quantization of $f(R)$-type gravity, based on its effective Lagrangian in the case of a flat Friedmann-Lemaitre-Robertson-Walker metric. Starting from the effective Lagrangian, we execute a suitable change of variable and the second quantization, and we obtain the Wheeler-DeWitt equation. The third quantization of this theory is considered. And the uncertainty relation of the universe is investigated in the example of $f(R)$-type gravity, where $f(R)=R^2$. It is shown, when the time is late namely the scale factor of the universe is large, the spacetime does not contradict to become classical, and, when the time is early namely the scale factor of the universe is small, the quantum effects are dominating.Comment: 9 pages, Arbitrary constants in (4.19) are changed to arbitrary functions of $\varphi$. Conclusions are not changed. References are added. Typos are correcte

Increased Interleukin-8 in Epithelial Lining Fluid of Collapsed Lungs During One-Lung Ventilation for Thoracotomy

The present study was designed to evaluate inflammatory changes in collapsed lungs during one-lung ventilation using the assistance of a bronchoscopic microsampling probe. Serial albumin and interleukin (IL)-8 concentrations in epithelial lining fluid (ELF) were measured in seven patients undergoing resection of lung tumors. The samples were taken after induction of anesthesia (baseline), 30 min after one-lung ventilation was started (point 2), just before resuming two-lung ventilation (point 3), and 30 min after two-lung ventilation was restarted (point 4). The albumin and IL-8 concentrations in ELF were significantly increased at point 2 and point 3, respectively, and remained to be high, compared to the baseline. The increase in IL-8 at point 3 was correlated with the interval of one-lung ventilation; however, none developed specific acute lung injury. These findings suggest that inflammatory changes can occur on the epithelium of a collapsed lung even in patients who underwent successful and standard thoracic surgery.ArticleINFLAMMATION. 35(6):1844-1850 (2012)journal articl

The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay

<p>Abstract</p> <p>Background</p> <p>SPO11 is a key protein for promoting meiotic recombination, by generating chromatin locus- and timing-specific DNA double-strand breaks (DSBs). The DSB activity of SPO11 was shown by genetic analyses, but whether SPO11 exerts DSB-forming activity by itself is still an unanswered question. DSB formation by SPO11 has not been detected by biochemical means, probably because of a lack of proper protein-folding, posttranslational modifications, and/or specific SPO11-interacting proteins required for this activity. In addition, plants have multiple SPO11-homologues.</p> <p>Results</p> <p>To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a <it>Drosophila </it>bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of <it>Arabidopsis </it>AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in <it>Arabidopsis</it>, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells.</p> <p>Conclusions</p> <p>The <it>Drosophila </it>bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function.</p

Endobronchial argon plasma coagulation for the management of post-intubation tracheal stenosis

The definitive version is available at www.blackwell-synergy.com.ArticleRespirology. 11(5):659-662 (2006)journal articl