Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nɛ-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification

SummaryPyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNAPyl. In this study, Nɛ-(tert-butyloxycarbonyl)-L-lysine (BocLys) and Nɛ-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNAPyl by PylRS. Crystal structures of a PylRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PylRS requires an Nɛ-carbonyl group bearing a substituent with a certain size. A PylRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PylRS. Furthermore, the structure-based Y306A mutation of PylRS drastically increased the in vitro aminoacylation activity for Nɛ-benzyloxycarbonyl-L-lysine (ZLys). A PylRS with both the Y306A and Y384F mutations enabled the large-scale preparation (>10 mg per liter medium) of proteins site-specifically containing Nɛ-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation

Mechanisms of the inhibition of reverse transcription by unmodified and modified antisense oligonucleotides

AbstractWe demonstrated that unmodified and modified (phosphorothioate) oligonucleotides prevent cDNA synthesis by AMV or HIV reverse transcriptases. Antisense oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA fragment bound to the antisense oligonucleotide by the reverse transcriptase-associated RNase H activity. However, the phosphorothioate oligomer inhibited polymerization by binding to the AMV RT rather than to the template RNA, whereas there was no competitive binding of the phosphorothioate oligomer on the HIV RT during reverse transcription

Twenty barrel in situ pipe gun type solid hydrogen pellet injector for the Large Helical Device

A 20 barrel solid hydrogen pellet injector, which is able to inject 20 cylindrical pellets with a diameter and length of between 3.0 and 3.8 mm at the velocity of 1200 m/s, has been developed for the purpose of direct core fueling in LHD (Large Helical Device). The in situ pipe gun concept with the use of compact cryo-coolers enables stable operation as a fundamental facility in plasma experiments. The combination of the two types of pellet injection timing control modes, i.e., pre-programing mode and real-time control mode, allows the build-up and sustainment of high density plasma around the density limit. The pellet injector has demonstrated stable operation characteristics during the past three years of LHD experiments

Subsequent Rupture after Clip on Wrap Method for Ruptured Dissecting Aneurysm of the Distal Middle Cerebral Artery : A case report and review of the literature

Dissecting aneurysms rarely occur in the middle cerebral artery (MCA). Furthermore, recurrent subarachnoid hemorrhage from ruptured dissecting aneurysms is rare with no published report of subsequent rupture after treatment by the clip on wrap method. The case reported is a 41-year-old man with subarachnoid hemorrhage. Angiography demonstrated aneurysm at the right M2 portion. We performed surgery to prevent rebleeding during the acute stage. Surgery revealed a discolored protrusion of the arterial wall of the lesion. The lesion was clipped on wrapping with Bemsheet. At one month postoperatively, angiography showed neither aneurysmal formation nor stenosis at the right M2, but after 5 months a subsequent rupture occurred. Angiography demonstrated pseudoaneurysm formation at the same portion of the right M2. The lesion was trapped with anastomosis of the superficial temporal artery (STA) to the MCA. He was discharged following a good recovery. The rate of subsequent rupture in ruptured dissecting aneurysm in the MCA is 14.3 0n published papers. Acute surgery must be performed to prevent the risk of re-rupture. In our single case report, trapping was effective treatment

Genetic Encoding of 3-Iodo-l-Tyrosine in Escherichia coli for Single-Wavelength Anomalous Dispersion Phasing in Protein Crystallography

SummaryWe developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-l-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-l-tyrosine. The 1.7 Å crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-l-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-l-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Å resolutions by SAD phasing at CuKα and CrKα wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation

Effect of Revascularization on Headache Associated with Moyamoya Disease in Pediatric Patients

Episodic headache is common in childhood moyamoya disease (MMD). The onset, mechanism, cause of headache and the effect of revascularization surgery on headache are not yet clear. We studied 10 cases of children (7 boys and 3 girls) younger than 18 years who underwent revascularization for MMD between 2009 and 2013. We evaluated frequency of headache and cerebral blood flow changes by single photon emission computed tomography brain imaging with [I123]-labeled iofetamine (IMP­SPECT) before and after surgery. Patients’ ages ranged from 0 to 15 years at onset and 2 to 17 years at the time of surgery, mean age being 6.7 and 8.0 years respectively. 9 of 10 patients presented with ischemic symptoms and 8 had headache. 5 patients underwent indirect bypass and 5 underwent combined direct and indirect bypass. Cerebral blood flow improvement was obtained in 14 of the 15 cerebral hemispheres revascularized mean follow-up duration was 32.9 months. All the patients had good outcomes with improvement of ischemic neurological deficits. Headache improved in 7 (87.5%) of 8 patients. Headache in pediatric moyamoya disease is associated with change in cerebral hemodynamics. Revascularization including combined direct bypass and indirect techniques may be required to reduce headache in patients with MMD

Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNATyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNATyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNATyr. The endogenous TyrRS and tRNATyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNATyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion

Targeted single-cell gene induction by optimizing the dually regulated CRE/loxP system by a newly defined heat-shock promoter and the steroid hormone in Arabidopsis thaliana

Multicellular organisms rely on intercellular communication systems to organize their cellular functions. In studies focusing on intercellular communication, the key experimental techniques include the generation of chimeric tissue using transgenic DNA recombination systems represented by the CRE/loxP system. If an experimental system enables the induction of chimeras at highly targeted cell(s), it will facilitate the reproducibility and precision of experiments. However, multiple technical limitations have made this challenging. The stochastic nature of DNA recombination events, especially, hampers reproducible generation of intended chimeric patterns. Infrared laser-evoked gene operator (IR-LEGO), a microscopic system that irradiates targeted cells using an IR laser, can induce heat shock-mediated expression of transgenes, for example, CRE recombinase gene, in the cells. In this study, we developed a method that induces CRE/loxP recombination in the target cell(s) of plant roots and leaves in a highly specific manner. We combined IR-LEGO, an improved heat-shock-specific promoter, and dexamethasone-dependent regulation of CRE. The optimal IR-laser power and irradiation duration were estimated via exhaustive irradiation trials and subsequent statistical modeling. Under optimized conditions, CRE/loxP recombination was efficiently induced without cellular damage. We also found that the induction efficiency varied among tissue types and cellular sizes. The developed method offers an experimental system to generate a precisely designed chimeric tissue, and thus, will be useful for analyzing intercellular communication at high resolution in roots and leaves

A NIN-LIKE PROTEIN mediates nitrate-induced control of root nodule symbiosis in Lotus japonicus

Legumes and rhizobia establish symbiosis in root nodules. To balance the gains and costs associated with the symbiosis, plants have developed two strategies for adapting to nitrogen availability in the soil: plants can regulate nodule number and/or stop the development or function of nodules. Although the former is accounted for by autoregulation of nodulation, a form of systemic long-range signaling, the latter strategy remains largely enigmatic. Here, we show that the Lotus japonicus NITRATE UNRESPONSIVE SYMBIOSIS 1 (NRSYM1) gene encoding a NIN-LIKE PROTEIN transcription factor acts as a key regulator in the nitrate-induced pleiotropic control of root nodule symbiosis. NRSYM1 accumulates in the nucleus in response to nitrate and directly regulates the production of CLE-RS2, a root-derived mobile peptide that acts as a negative regulator of nodule number. Our data provide the genetic basis for how plants respond to the nitrogen environment and control symbiosis to achieve proper plant growth