Bombyxin F1 Gene: Structure and Expression of a New Bombyxin Family Gene That Forms a Pair with Bombyxin B10 Gene

金沢大学理工研究域自然システム学系Bombyxin F1 gene, a new bombyxin family gene, has been identified. The F1 gene forms a pair with bombyxin B10 gene with an opposite transcriptional orientation and the gene pair F1/B10 is located between bombyxin gene pairs B9/C1 and A7/B7 in a bombyxin gene cluster. The nucleotide sequence of the F1 gene and its deduced amino acid sequence deviate moderately from those characterized previously for the family-A, family-B, family-C, family-D, and family-E bombyxin genes; the bombyxin F1 gene and preprobombyxin F1 share no more than 62% and 53% sequence identities with other bombyxin members, respectively. Harr-plot analysis indicated that the spacer of the F1/B10 gene pair has low sequence similarity with that of other bombyxin gene pairs characterized. The bombyxin F1 mRNA in Bombyx mori brain was shown to locate in four pairs of medial neurosecretory cells, which also produce other bombyxin family mRNAs. Genomic Southern hybridization indicated that the Bombyx haploid genome contains a single copy of the family-F bombyxin gene

Changes in cardiac sympathetic nerve innervation and activity in pathophysiologic transition from typical to end-stage hypertrophic cardiomyopathy

金沢大学大学院医学系研究科Left ventricular (LV) systolic function in hypertrophic cardiomyopathy (HCM) is usually normal. Late in the disease, however, LV systolic dysfunction and dilatation are recognized. Although abnormalities in cardiac sympathetic nerve activity in patients with HCM have been demonstrated using 123I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy, the changes of cardiac sympathetic nerve activity throughout the clinical course from typical to end-stage HCM are unclear. The objective of this study was to evaluate the relationship between abnormalities on 123I-MIBG myocardial scintigraphy and pathophysiologic changes in patients with HCM. Methods: We performed 123I-MIBG scintigraphy on 46 patients with HCM and 18 age-matched control subjects. The patients were categorized into 3 groups: 28 patients with normal LV systolic function (group A), 9 patients with LV systolic dysfunction (group B), and 9 patients with LV systolic dysfunction and dilatation (group C). With planar 123I-MIBG imaging, the heart-to-mediastinum ratio for early and delayed acquisitions and the washout rate were calculated. With SPECT, polar maps of the LV myocardium were divided into 20 segments. The regional uptake and washout rate were calculated from semiquantitative 20-segment bull\u27s-eye analysis. Results: The early uptake was significantly lower in group C than in the control group (P < 0.01). The washout rate was progressively higher in group A, group B, and group C (P < 0.01). Reduced regional early uptake was found in 2.9 ± 3.4 (group A), 4.1 ± 4.7 (group B), and 7.4 ± 4.3 (group C) segments, respectively. In group C, regional early uptake was significantly reduced, predominantly in the interventricular septal wall, and regional washout rate was increased in the apex and lateral wall. Conclusion: These results suggest that cardiac sympathetic nerve abnormalities in patients with HCM may advance with development of LV systolic dysfunction and dilatation and that 123I-MIBG myocardial scintigraphy may be a useful tool for the evaluation of pathophysiologic changes in HCM

Heterogeneous Nucleation of Protein Crystals on Fluorinated Layered Silicate

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface

Development of Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of Human Norovirus Major Capsid Protein

Human Norwalk viruses (HuNoVs), the most common etiological agents of acute gastroenteritis, are genetically diverse RNA viruses that frequently cause mass food poisoning internationally. Although nucleic acid detection methods, such as reverse transcription&ndash;quantitative polymerase chain reaction (RT-qPCR), are the gold standard for the diagnosis of norovirus infection, alternative methods are needed for the specific and sensitive viral protein detection for rapid diagnosis and surveillance. In this study, we developed a robust and high-throughput targeted proteomic assay workflow to directly detect the VP1 major capsid protein of HuNoVs. A parallel reaction monitoring (PRM) assay using a high-resolution mass spectrometer was used to detect representative peptides derived from VP1 in six different HuNoV genotypes. An optimized protocol using synthesized heavy isotope-labeled peptides as internal standards was also used to simultaneously genotype and quantify the VP1 protein in human stool specimens. This method is expected to become a new tool for studying the molecular epidemiology of HuNoV and to shed new light on targeted proteomics in clinical practice

Nuclear translocation of MLKL enhances necroptosis by a RIP1/RIP3-independent mechanism in H9c2 cardiomyoblasts

Accumulating evidence suggests that necroptosis of cardiomyocytes contributes to cardiovascular diseases. Lethal disruption of the plasma membrane in necroptosis is induced by oligomers of mixed lineage kinase domain-like (MLKL) that is translocated to the membrane from the cytosol. However, the role played by cytoplasmic-nuclear shuttling of MLKL is unclear. Here, we tested the hypothesis that translocation of MLKL to the nucleus promotes the necroptosis of cardiomyocytes. Activation of the canonical necroptotic signaling pathway by a combination of TNF-α and zVAD (TNF/zVAD) increased nuclear MLKL levels in a RIP1-activity-dependent manner in H9c2 cells, a rat cardiomyoblast cell line. By use of site-directed mutagenesis, we found a nuclear export signal sequence in MLKL and prepared its mutant (MLKL-L280/283/284A), though a search for a nuclear import signal was unsuccessful. MLKL-L280/283/284A localized to both the cytosol and the nucleus. Expression of MLKL-L280/283/284A induced necroptotic cell death, which was attenuated by GppNHp, an inhibitor of Ran-mediated nuclear import, but not by inhibition of RIP1 activity or knockdown of RIP3 expression. GppNHp partly suppressed H9c2 cell death induced by TNF/zVAD treatment. These results suggest that MLKL that is translocated to the nucleus via RIP1-mediated necroptotic signaling enhances the necroptosis of cardiomyocytes through a RIP1-/RIP3-independent mechanism