A Common Complement C3 Variant Is Associated with Protection against Wet Age-Related Macular Degeneration in a Japanese Population

博士（医学）神戸大

A case of rhabdomyolysis in which levetiracetam was suspected as the cause

AbstractSeveral studies have reported rhabdomyolysis induced by various drugs but not by the antiepileptic drug levetiracetam. We present a case of suspected levetiracetam-induced rhabdomyolysis. A 29-year-old woman was hospitalized for generalized tonic–clonic seizure and given levetiracetam for the first time. One day after starting levetiracetam, she developed myalgia, particularly backache, and weakness in both lower limbs. Based on her clinical symptoms and blood test results indicating hyperCKemia, our diagnosis was levetiracetam-induced rhabdomyolysis. Withdrawal of levetiracetam immediately improved the clinical symptoms and hyperCKemia. This first report of suspected levetiracetam-induced rhabdomyolysis provides important information for treating patients early in levetiracetam administration

Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability.

Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational β-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The β-lysyl EF-P modification pathway is conserved among only 26-28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl-EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli β-lysyl-EF-P. The possible reasons for the unique requirement of rhamnosyl-EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others

Genetic incorporation of non-canonical amino acid photocrosslinkers in Neisseria meningitidis: New method provides insights into the physiological function of the function-unknown NMB1345 protein.

Although whole-genome sequencing has provided novel insights into Neisseria meningitidis, many open reading frames have only been annotated as hypothetical proteins with unknown biological functions. Our previous genetic analyses revealed that the hypothetical protein, NMB1345, plays a crucial role in meningococcal infection in human brain microvascular endothelial cells; however, NMB1345 has no homology to any identified protein in databases and its physiological function could not be elucidated using pre-existing methods. Among the many biological technologies to examine transient protein-protein interaction in vivo, one of the developed methods is genetic code expansion with non-canonical amino acids (ncAAs) utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair from Methanosarcina species: However, this method has never been applied to assign function-unknown proteins in pathogenic bacteria. In the present study, we developed a new method to genetically incorporate ncAAs-encoded photocrosslinking probes into N. meningitidis by utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair and elucidated the biological function(s) of the NMB1345 protein. The results revealed that the NMB1345 protein directly interacts with PilE, a major component of meningococcal pili, and further physicochemical and genetic analyses showed that the interaction between the NMB1345 protein and PilE was important for both functional pilus formation and meningococcal infectious ability in N. meningitidis. The present study using this new methodology for N. meningitidis provides novel insights into meningococcal pathogenesis by assigning the function of a hypothetical protein

Strategies for <i>efp</i> deletion from the <i>N</i>. <i>meningitidis</i> genome.

<p>(A) The <i>efp</i> allele in the <i>N</i>. <i>meningitidis</i> genome can be disrupted, but only in the presence of a plasmid containing the wild-type <i>N</i>. <i>meningitidis efp</i> gene. (B, C) The IncQ plasmid pHT1139, containing P<i>tac-</i>TTG<i>-efp-lacI</i>^<i>q</i>, was transformed into <i>N</i>. <i>meningitidis</i> H44/76 cells. Subsequently, the DNA fragment bearing the erythromycin resistance gene (<i>ermC</i>) or the <i>earP</i>-<i>efp</i>(<i>R32opal</i>) gene was introduced into the <i>efp</i> allele within the <i>N</i>. <i>meningitidis</i> H44/76/pHT1139 genome, to obtain the <i>N</i>. <i>meningitidis</i> strains HT1913/pHT1139 (B, left) and HT1914/pHT1139 (C, left), respectively. In these strains, the <i>efp</i> gene expression can be controlled by IPTG, and EF-P can be inducibly produced in the presence of IPTG. (B, C, right) Growth of the <i>N</i>. <i>meningitidis</i> HT1913/pHT1139 and HT1914/pHT1139 cells, with and without IPTG. Both of the <i>N</i>. <i>meningitidis</i> cells lack the <i>efp</i> gene in the genome, but contain an inducible copy of the <i>efp</i> gene in the IncQ plasmid.</p

Rhamnosyl modification of the recombinant EF-P(<i>Nm</i>) by EarP(<i>Nm</i>).

<p>(A) Coexpression of EF-P(<i>Nm</i>) with EarP(<i>Nm</i>) in <i>E</i>. <i>coli</i> cells. Lane 1, molecular mass standards; lane 2, crude extract of <i>E</i>. <i>coli</i> cells producing EF-P(<i>Nm</i>); lane 3, crude extract of <i>E</i>. <i>coli</i> cells producing EF-P(<i>Nm</i>) and EarP(<i>Nm</i>); lane 4, crude extract of <i>E</i>. <i>coli</i> cells producing EarP(<i>Nm</i>); lane 5, molecular mass standards; lane 6, the recombinant EF-P(<i>Nm</i>) purified from the cells producing EF-P(<i>Nm</i>); lane 7, the recombinant EF-P(<i>Nm</i>) purified from the cells producing EF-P(<i>Nm</i>) and EarP(<i>Nm</i>); lane 8, purified EarP(<i>Nm</i>). (B) PMF analysis of the modified and unmodified EF-P(<i>Nm</i>). The recombinant EF-P(<i>Nm</i>) proteins were purified from the cells producing EF-P(<i>Nm</i>), with or without EarP(<i>Nm</i>). After digestion with AspN and API, the PMF analysis of the peptides was performed. (C) MS/MS analyses of the recombinant EF-P(<i>Nm</i>) and the recombinant EF-P(<i>Nm</i>) modified with EarP(<i>Nm</i>). After digestion with AspN and API, the EF-P(<i>Nm</i>) peptides with masses of 662.35 Da (GGRSSAK) and 808.4 Da (GGR*SSAK, R* designates the modified Arg32) were subjected to the MS/MS analysis. The sequence can be read from the annotated b (blue) or y (red) ion series; the b2, b3, b4, b5, y1, y2, y3, and y4 ions from the peptide “GGRSSAK” and the b2, b3, y1, y2, y3, and y4 ions from the peptide “GGR*SSAK” were observed.</p