1,10-Phenanthroline- or Electron-Promoted Cyanation of Aryl Iodides

A 1,10-phenanthroline-promoted cyanation of aryl iodides has been developed. 1,10-Phenanthroline worked as an organocatalyst for the reaction of aryl iodides with tetraalkylammonium cyanide to afford aryl cyanides. A similar reaction occurred through an electroreductive process

Dibutyryl cyclic adenosine monophosphate attenuates lung injury caused by cold preservation and ischemia-reperfusion

AbstractObjective: Dibutyryl adenosine 3`,5`cyclic monophosphate (db-cAMP) is a membrane-permeable analog of adenosine 3`,5`cyclic monophosphate (cAMP). We examined the effect of db-cAMP against lung injury caused by cold preservation and ischemia-reperfusion. Methods: Rats were divided into three groups (each n = 6) according to the presence or absence of db-cAMP in the preservative solution and cold ischemia (4° C for 15 hours). In the fresh group, the lung was flushed with the preservative solution and reperfusion was performed immediately. In the control group and the db-cAMP group, the lung was flushed either with the solution or with a combination of the solution plus db-cAMP, respectively, and preserved at 4° C for 15 hours. The lung was reperfused for 60 minutes in an ex vivo rat lung perfusion model. Results: The shunt ratios of the reperfused lung in the db-cAMP group were 4.0% ± 1.6% and 3.4% ± 1.2% 10 and 60 minutes, respectively, after the initiation of reperfusion, being as low as those in the fresh group and significantly lower than those in the control group (p < 0.01). The wet/dry weight ratio of the lung tissue after reperfusion was 5.99 ± 1.50 in the db-cAMP group, which was similar to that in the fresh group (5.45 ± 0.23) and significantly lower than that in the control group (14.20 ± 3.43) (p < 0.01). Electron microscopic examination showed less damage in the pulmonary arterial endothelium in the db-cAMP group. Conclusions: We conclude that db-cAMP attenuates the lung injury by cold preservation and ischemia-reperfusion, at least partly by protection of the vascular endothelium. (J Thorac Cardiovasc Surg 1997;114:635-42

Casein kinase 1γ acts as a molecular switch for cell polarization through phosphorylation of the polarity factor Tea1 in fission yeast

Fission yeast undergoes growth polarity transition from monopolar to bipolar during G2 phase, designated NETO (New End Take Off). It is known that NETO onset involves two prerequisites, the completion of DNA replication and attainment of a certain cell size. However, the molecular mechanism remains unexplored. Here, we show that casein kinase 1γ, Cki3 is a critical determinant of NETO onset. Not only did cki3∆ cells undergo NETO during G1‐ or S‐phase, but they also displayed premature NETO under unperturbed conditions with a smaller cell size, leading to cell integrity defects. Cki3 interacted with the polarity factor Tea1, of which phosphorylation was dependent on Cki3 kinase activity. GFP nanotrap of Tea1 by Cki3 led to Tea1 hyperphosphorylation with monopolar growth, whereas the same entrapment by kinase‐dead Cki3 resulted in converse bipolar growth. Intriguingly, the Tea1 interactor Tea4 was dissociated from Tea1 by Cki3 entrapment. Mass spectrometry identified four phosphoserine residues within Tea1 that were hypophosphorylated in cki3∆ cells. Phosphomimetic Tea1 mutants showed compromised binding to Tea4 and NETO defects, indicating that these serine residues are critical for protein–protein interaction and NETO onset. Our findings provide significant insight into the mechanism by which cell polarization is regulated in a spatiotemporal manner.T.K. was the recipient of a JSPS fellowship (PD) and was partly supported by ‘Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation’ from JSPS. This work was supported by Cancer Research UK (T.T. and A.P.S) and the Ministry of Education, Culture, Sports, Science and Technology (D.H.)

EFFECT OF FERULIC ACID ON PIG OOCYTES

The value of laboratory and genetically-modified pigs is becoming increasingly clear; however, their in vitro development remains inefficient. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls, and which exhibits antioxidant effects in vitro. Trans-FA is known to improve sperm viability and motility; however, its effects on porcine oocytes are unknown. Our aim was to investigate the effects of trans-FA supplementation during in vitro maturation on the meiotic and developmental competence of porcine oocytes. Oocytes were matured either without (control) or with trans-FA (10, 100 and 1,000 µM), fertilized, and cultured in vitro for 7 days. The maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher than that of controls (65.0%; P<0.05). The fertilization rate of oocytes matured with 10 µM trans-FA (57.4%) was also significantly higher than that of controls (32.7%) and oocytes cultured with other concentrations (33.1% and 22.7% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher than that of controls (2.3%; P<0.05). Our results suggest that in vitro maturation with 10 µM trans-FA is beneficial for the in vitro production of porcine embryos and has the potential to improve production system

EMBRYONIC MOSAICISM BY MICROINJECTION

Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/μl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/μl each (20 ng/μl group) or 100 ng/μl each (100 ng/μl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/μl group was significantly higher (P < 0.05) than that in the 20 ng/μl group. Although no blastocysts from the 20 ng/μl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/μl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed

Characterization of Mouse Tissue Kallikrein 5

Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (KIk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling