Direct and Specific Effect of Sevoflurane Anesthesia on rat Per2 Expression in the Suprachiasmatic Nucleus

Background

Our previous studies revealed that application of the inhalation anesthetic, sevoflurane, reversibly repressed the expression of Per2 in the mouse suprachiasmatic nucleus (SCN). We aimed to examine whether sevoflurane directly affects the SCN.

Methods

We performed in vivo and in vitro experiments to investigate rat Per2 expression under sevoflurane-treatment. The in vivo effects of sevoflurane on rPer2 expression were examined by quantitative in situ hybridization with a radioactively-labeled cRNA probe. Additionally, we examined the effect of sevoflurane anesthesia on rest/activity rhythms in the rat. In the in vitro experiments, we applied sevoflurane to SCN explant cultures from Per2-dLuc transgenic rats, and monitored luciferase bioluminescence, representing Per2 promoter activity. Bioluminescence from two peripheral organs, the kidney cortex and the anterior pituitary gland, were also analyzed.

Results

Application of sevoflurane in rats significantly suppressed Per2 expression in the SCN compared with untreated animals. We observed no sevoflurane-induced phase-shift in the rest/activity rhythms. In the in vitro experiments, the intermittent application of sevoflurane repressed the increase of Per2-dLuc luminescence and led to a phase delay in the Per2-dLuc luminescence rhythm. Sevoflurane treatment did not suppress bioluminescence in the kidney cortex or the anterior pituitary gland.

Conclusion

The suppression of Per2-dLuc luminescence by sevoflurane in in vitro SCN cultures isolated from peripheral inputs and other nuclei suggest a direct action of sevoflurane on the SCN itself. That sevoflurane has no such effect on peripheral organs suggests that this action might be mediated through a neuron-specific cellular mechanism or a regulation of the signal transduction between neurons.     

Genetic Variation of Human Papillomavirus Type 16 in Individual Clinical Specimens Revealed by Deep Sequencing

Viral genetic diversity within infected cells or tissues, called viral quasispecies, has been mostly studied for RNA viruses, but has also been described among DNA viruses, including human papillomavirus type 16 (HPV16) present in cervical precancerous lesions. However, the extent of HPV genetic variation in cervical specimens, and its involvement in HPV-induced carcinogenesis, remains unclear. Here, we employ deep sequencing to comprehensively analyze genetic variation in the HPV16 genome isolated from individual clinical specimens. Through overlapping full-circle PCR, approximately 8-kb DNA fragments covering the whole HPV16 genome were amplified from HPV16-positive cervical exfoliated cells collected from patients with either low-grade squamous intraepithelial lesion (LSIL) or invasive cervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novo assembly of the full-length HPV16 genome sequence for each of 7 specimens (5 LSIL and 2 ICC samples). Subsequent alignment of read sequences to the assembled HPV16 sequence revealed that 2 LSILs and 1 ICC contained nucleotide variations within E6, E1 and the non-coding region between E5 and L2 with mutation frequencies of 0.60% to 5.42%. In transient replication assays, a novel E1 mutant found in ICC, E1 Q381E, showed reduced ability to support HPV16 origin-dependent replication. In addition, partially deleted E2 genes were detected in 1 LSIL sample in a mixed state with the intact E2 gene. Thus, the methods used in this study provide a fundamental framework for investigating the influence of HPV somatic genetic variation on cervical carcinogenesis.     

Modeling Alzheimer’s Disease with iPSCs Reveals Stress Phenotypes Associated with Intracellular Aβ and Differential Drug Responsiveness

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Highlights? Familial and sporadic Alzheimer’s patient iPSC-derived neural cells were analyzed ? Intracellular Aβ oligomers accumulate in lines from some patients ? Aβ oligomer accumulation is associated with ER and oxidative stress ? DHA-alleviated ER and oxidative stresses improve cell viability     

Multiple Regulation of Rad51-Mediated Homologous Recombination by Fission Yeast Fbh1

Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCF^Fbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.     

Nitric oxide exerts protective effects against bleomycin-induced pulmonary fibrosis in mice

Background

Increased expression of nitric oxide synthase (NOS) and an increase in plasma nitrite plus nitrate (NOx) have been reported in patients with pulmonary fibrosis, suggesting that nitric oxide (NO) plays an important role in its development. However, the roles of the entire NO and NOS system in the pathogenesis of pulmonary fibrosis still remain to be fully elucidated. The aim of the present study is to clarify the roles of NO and the NOS system in pulmonary fibrosis by using the mice lacking all three NOS isoforms.

Methods

Wild-type, single NOS knockout and triple NOS knockout (n/i/eNOS^−/−) mice were administered bleomycin (BLM) intraperitoneally at a dose of 8.0 mg/kg/day for 10 consecutive days. Two weeks after the end of the procedure, the fibrotic and inflammatory changes of the lung were evaluated. In addition, we evaluated the effects of long-term treatment with isosorbide dinitrate, a NO donor, on the n/i/eNOS^−/− mice with BLM-induced pulmonary fibrosis.

Results

The histopathological findings, collagen content and the total cell number in bronchoalveolar lavage fluid were the most severe/highest in the n/i/eNOS^−/− mice. Long-term treatment with the supplemental NO donor in n/i/eNOS^−/− mice significantly prevented the progression of the histopathological findings and the increase of the collagen content in the lungs.

Conclusions

These results provide the first direct evidence that a lack of all three NOS isoforms led to a deterioration of pulmonary fibrosis in a BLM-treated murine model. We speculate that the entire endogenous NO and NOS system plays an important protective role in the pathogenesis of pulmonary fibrosis.     

Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B‐positive structures

Several autophagy proteins contain an LC3‐interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B‐positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP‐ALFY‐LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR‐binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.     

Effects of synaptotagmin 2 on membrane fusion between liposomes that contain SNAREs involved in exocytosis in mast cells

Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca(2+) concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca(2+) dependent, the target molecule that interacts with Ca(2+) is not clear. Synaptotagmin is a Ca(2+) sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagmin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca(2+), low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca(2+), and phosphatidylserine.     

Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane

Upon mitochondrial depolarization, Parkin, a Parkinson disease-related E3 ubiquitin ligase, translocates from the cytosol to mitochondria and promotes their degradation by mitophagy, a selective type of autophagy. Here, we report that in addition to mitophagy, Parkin mediates proteasome-dependent degradation of outer membrane proteins such as Tom20, Tom40, Tom70, and Omp25 of depolarized mitochondria. By contrast, degradation of the inner membrane and matrix proteins largely depends on mitophagy. Furthermore, Parkin induces rupture of the outer membrane of depolarized mitochondria, which also depends on proteasomal activity. Upon induction of mitochondrial depolarization, proteasomes are recruited to mitochondria in the perinuclear region. Neither proteasome-dependent degradation of outer membrane proteins nor outer membrane rupture is required for mitophagy. These results suggest that Parkin regulates degradation of outer and inner mitochondrial membrane proteins differently through proteasome- and mitophagy-dependent pathways.