Simultaneous Detection of Both Single Nucleotide Variations and Copy Number Alterations by Next-Generation Sequencing in Gorlin Syndrome

Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions.     

Occurrence of a novel strain of Scirtothrips dorsalis (Thysanoptera: Thripidae) in Japan and development of its molecular diagnostics

Scirtothrips dorsalis Hood is a cosmopolitan and polyphagous thrips species. Recently, a novel strain of S. dorsalis attacking capsicum crops was found in Japan. A molecular phylogenetic analysis using mitochondrial cytochrome c oxidase subunit I sequences revealed that the capsicum-associated populations were genetically different from Japanese native strains and were closely related to Southeast Asian populations. We named the capsicum-associated populations “strain C” and the Japanese native ones “strain YT”. A total of 10 haplotypes were found in strain C and 26 in strain YT. To differentiate the two strains, we developed a multiplex-PCR method using the ribosomal ITS2 region.     

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

(R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.     

Development of an Ultra-Rapid Diagnostic Method Based on Heart-Type Fatty Acid Binding Protein Levels in the CSF of CJD Patients

Creutzfeldt-Jakob disease (CJD) is a transmissible, fatal, neurodegenerative disease in humans. Recently, various drugs have been reported to be useful in the treatment of CJD; however, for such treatments to be useful it is essential to rapidly and accurately diagnose CJD. 124 CJD patients and 87 with other diseases causing rapid progressive dementia were examined. Cerebral spinal fluid (CSF) from CJD patients was analyzed by 2D-PAGE and the protein expression pattern was compared with that from healthy subjects. One of three CJD-specific spots was found to be fatty acid binding protein (FABP), and heart-type FABP (H-FABP) was analyzed as a new biochemical marker for CJD. H-FABP ELISA results were compared between CJD patients and patients with other diseases (n = 211). Visual readout accuracy of the Rapicheck^® H-FABP test panel for CSF was analyzed using an independent measure of CSF H-FABP concentration. The distribution of H-FABP in the brains of CJD patients was examined by immunohistochemistry. ELISA sensitivity and specificity were 90.3% and 92.9%, respectively, and Rapicheck^® H-FABP sensitivity and specificity were 87.9% and 96.0%, respectively. ELISA and Rapicheck^® H-FABP assays provided comparable results for 14-3-3 protein and total tau protein. Elevated H-FABP levels were associated with an accumulation of abnormal prion protein, astrocytic gliosis, and neuronal loss in the cerebral cortices of CJD patients. In conclusion, Rapicheck^® H-FABP of CSF specimens enabled quick and frequent diagnosis of CJD. H-FABP represents a new biomarker for CJD distinct from 14-3-3 protein and total tau protein.     

Structure–activity relationship of a novel pentapeptide with cancer cell growth‐inhibitory activity

We previously reported that yamamarin, a pentapeptide with an amidated C‐terminus (DILRG‐NH₂) isolated from larvae of the silkmoth, and its palmitoylated analog (C16‐DILRG‐NH₂) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure–activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C‐terminal part (‐RG‐NH₂) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG‐NH₂ and C16‐DILRG‐NH₂ revealed that the N‐terminal palmitoyl group of C16‐DILRG‐NH₂ did not affect the conformation of the C‐terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Understanding substrate misrecognition of hydrogen peroxide dependent cytochrome P450 from Bacillus subtilis

Cytochrome P450_BSβ, a H₂O₂-dependent cytochrome P450 catalyzing the hydroxylation of long-alkyl-chain fatty acids, lacks the general acid–base residue around the heme, which is indispensable for the efficient generation of the active species using H₂O₂. On the basis of the crystal structure of the palmitic acid bound form of cytochrome P450_BSβ, it was suggested that the role of the general acid–base function was provided by the carboxylate group of fatty acids. The participation of the carboxylate group of the substrate was supported by the fact that cytochrome P450_BSβ can catalyze oxidations of nonnatural substrates such as styrene and ethylbenzene in the presence of a series of short-alkyl-chain carboxylic acids as a dummy molecule of fatty acid. We refer to a series of short-alkyl-chain carboxylic acids as a “decoy molecule”. As shown here, we have clarified the crystal structure of the decoy-molecule-bound form and elucidated that the location of its carboxylate group is virtually the same as that of palmitic acid in the heme cavity, indicating that the carboxylate group of the decoy molecule serves as the general acid–base catalyst. This result further confirms that the role of the acid–base function is satisfied by the carboxylate group of the substrates. In addition, the structure analysis of the substrate-free form has clarified that no remarkable structural change is induced by the binding of the decoy molecule as well as fatty acid. Consequently, whether the carboxylate group is positioned in the active site provides the switching mechanism of the catalytic cycle of cytochrome P450_BSβ.     

A bacterial elongation factor G homologue exclusively functions in ribosome recycling in the spirochaete Borrelia burgdorferi

Translation elongation factor G (EF‐G) in bacteria plays two distinct roles in different phases of the translation system. EF‐G catalyses the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post‐termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF‐G are distributed over two different EF‐G paralogues in human mitochondria. In the present study, we show that the same division of roles of EF‐G is also found in bacteria. Two EF‐G paralogues are found in the spirochaete Borrelia burgdorferi, EF‐G1 and EF‐G2. We demonstrate that EF‐G1 is a translocase, while EF‐G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF‐G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF‐3) is present in the reaction. These results indicate that two B. burgdorferi EF‐G paralogues are close relatives to mitochondrial EF‐G paralogues rather than the conventional bacterial EF‐G, in both their phylogenetic and biochemical features.