Mitochondrial LETM1 drives ionic and molecular clock rhythms in circadian pacemaker neurons

The mechanisms that generate robust ionic oscillation in circadian pacemaker neurons are under investigation. Here, we demonstrate critical functions of the mitochondrial cation antiporter leucine zipper- EF-hand-containing transmembrane protein 1 (LETM1), which exchanges K+/H+ in Drosophila and Ca2+/H+ in mammals, in circadian pacemaker neurons. Letm1 knockdown in Drosophila pacemaker neurons reduced circadian cytosolic H+ rhythms and prolonged nuclear PERIOD/TIMELESS expression rhythms and locomotor activity rhythms. In rat pacemaker neurons in the hypothalamic suprachiasmatic nucleus (SCN), circadian rhythms in cytosolic Ca2+ and Bmal1 transcription were dampened by Letm1 knockdown. Mitochondrial Ca2+ uptake peaks late during the day were also observed in rat SCN neurons following photolytic elevation of cytosolic Ca2+. Since cation transport by LETM1 is coupled to mitochondrial energy synthesis, we propose that LETM1 integrates metabolic, ionic, and molecular clock rhythms in the central clock system in both invertebrates and vertebrates

Molecular genetic epidemiology of homozygous familial hypercholesterolemia in the Hokuriku district of Japan

金沢大学医学系研究科Aim: Familial hypercholesterolemia (FH) is caused by mutations of FH genes, i.e. LDL-receptor (LDLR), PCSK9 and apolipoprotein B (ApoB) gene. We evaluated the usefulness of DNA analysis for the diagnosis of homozygous FH (homo-FH), and studied the frequency of FH in the Hokuriku district of Japan. Methods: Twenty-five homo-FH patients were recruited. LDLR mutations were identified using the Invader assay method. Mutations in PCSK9 were detected by PCR-SSCP followed by direct sequence analysis. Results: We confirmed 15 true homozygotes and 10 compound heterozygotes for LDLR mutations. Three types of double heterozygotes for LDLR and PCSK9 were found. No FH patients due to ApoB mutations were found. The incidences of homo-FH and hetero-FH in the Hokuriku district were 1/171,167 and 1/208, respectively. Conclusions: Our observations underlined the value of FH gene analysis in diagnosing homo-FH and confirmed extraordinarily high frequency of FH in the Hokuriku district of Japan. © 2010 Elsevier Ireland Ltd

Novel mutations of cholesteryl ester transfer protein (CETP) gene in Japanese hyperalphalipoproteinemic subjects

Thesis of Ohtani, Rumiko / 大谷 留珠子 博士学位論文(金沢大学 / 大学院医薬保健学総合研究科)Background: The half of hyperalphalipoproteinemia (HALP) in Japan is caused by CETP gene mutations. Other than two prevalent mutations (D442G and Intron 14 splicing donor site +. 1G>A), some rare CETP mutations are found in Japanese HALP subjects. Methods: CETP gene analysis of genomic DNA from subjects was performed by restriction fragment length polymorphism (RFLP) and sequencing analysis. Mutations which were suspected to cause a splicing defect or a protein secretion defect were investigated in COS-1 cells transfected with a CETP minigene construct or a cDNA expression vector. Results: Each of three subjects was identified as a carrier of CETP gene mutation of a compound heterozygote of c.653_654delGGinsAAAC and Intron 14 splicing donor site +. 1G>A, a heterozygote of c.658G>A or a homozygote of L261R. The c.658G>A mutation was located at the last nucleotide of exon 7, and it was confirmed to cause splicing abnormality revealed by the CETP minigene analysis. The L261R CETP was not secreted to conditioned media of the cells. Conclusions: Three novel CETP gene mutations are responsible for HALP by CETP deficiency. It is predicted that there are more rare CETP gene mutations in Japanese, and these multiple rare mutations alone or a combination with each of prevalent mutations is responsible for mild-to-moderate or marked HALP, respectively. © 2011 Elsevier B.V

Effect of ultrasonic irradiation on γ-Fe2O3 formation by co-precipitation method with Fe3+ salt and alkaline solution

The effect of ultrasonic irradiation on direct maghemite (γ-Fe2O3) preparation by a co-precipitation method with Fe3+ salt (Fe(NO3)3) and an excess amount of alkaline (KOH) solution without going through the conventional magnetite (Fe3O4) formation route was explored in comparison with impeller stirring. The preparation procedure for obtaining iron oxide nanoparticles was designed using the sequential processes of precipitation, decantation, drying and thermal dehydration, and ultrasonic irradiation or impeller stirring was done during the precipitation process. γ-ferric oxyhydroxide (γ-FeOOH) was partially formed in addition to α-ferric oxyhydroxide (α-FeOOH) and thermally dehydrated to γ-Fe2O3 and hematite (α-Fe2O3) by ultrasonic-assisted co-precipitation of Fe3+ salt and the excess KOH solution, whereas only α-FeOOH and α-Fe2O3 were synthesized by impeller stirring. The difference between the products of the two methods was explained by the Lamer model associated with the nucleation and growth of FeOOH. Magnetization increased as the crystallite diameter decreased, which is estimated to facilitate partial formation of magnetic γ-Fe2O3. Magnetization was enhanced by a lower ultrasonic frequency due to the stronger shock wave induced by the cavitation effect.This fulltext is available in Dec. 2022

Effect of ultrasonic irradiation on γ-Fe2O3 formation by co-precipitation method with Fe3+ salt and alkaline solution

The effect of ultrasonic irradiation on direct maghemite (γ-Fe2O3) preparation by a co-precipitation method with Fe3+ salt (Fe(NO3)3) and an excess amount of alkaline (KOH) solution without going through the conventional magnetite (Fe3O4) formation route was explored in comparison with impeller stirring. The preparation procedure for obtaining iron oxide nanoparticles was designed using the sequential processes of precipitation, decantation, drying and thermal dehydration, and ultrasonic irradiation or impeller stirring was done during the precipitation process. γ-ferric oxyhydroxide (γ-FeOOH) was partially formed in addition to α-ferric oxyhydroxide (α-FeOOH) and thermally dehydrated to γ-Fe2O3 and hematite (α-Fe2O3) by ultrasonic-assisted co-precipitation of Fe3+ salt and the excess KOH solution, whereas only α-FeOOH and α-Fe2O3 were synthesized by impeller stirring. The difference between the products of the two methods was explained by the Lamer model associated with the nucleation and growth of FeOOH. Magnetization increased as the crystallite diameter decreased, which is estimated to facilitate partial formation of magnetic γ-Fe2O3. Magnetization was enhanced by a lower ultrasonic frequency due to the stronger shock wave induced by the cavitation effect

Histamine Regulates Molecular Clock Oscillations in Human Retinal Pigment Epithelial Cells via H1 Receptors

Vertebrate eyes are known to contain circadian clocks, but their regulatory mechanisms remain largely unknown. To address this, we used a cell line from human retinal pigment epithelium (hRPE-YC) with stable coexpression of reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+ concentrations (YC3.6). We observed concentration-dependent increases in cytosolic Ca2+ concentrations after treatment with histamine (1–100 µM) and complete suppression of histamine-induced Ca2+ mobilizations by H1 histamine receptor (H1R) antagonist d-chlorpheniramine (d-CPA) in hRPE-YC cells. Consistently, real-time RT-PCR assays revealed that H1R showed the highest expression among the four subtypes (H1–H4) of histamine receptors in hRPE-YC cells. Stimulation of hRPE-YC cells with histamine transiently increased nuclear localization of phosphorylated Ca2+/cAMP-response element-binding protein that regulates clock gene transcriptions. Administration of histamine also shifted the Bmal1-luciferase rhythms with a type-1 phase-response curve, similar to previous results with carbachol stimulations. Treatment of hRPE-YC cells with d-CPA or with more specific H1R antagonist, ketotifen, blocked the histamine-induced phase shifts. Furthermore, an H2 histamine receptor agonist, amthamine, had little effect on the Bmal1-luciferase rhythms. Although the function of the in vivo histaminergic system within the eye remains obscure, the present results suggest histaminergic control of the molecular clock via H1R in retinal pigment epithelial cells. Also, since d-CPA and ketotifen have been widely used (e.g., to treat allergy and inflammation) in our daily life and thus raise a possible cause for circadian rhythm disorders by improper use of antihistamines

The Cholinergic Pathway and MitoKATP Induce UCP4 Expression Involved in Neuroprotection of FN Stimulation in Rats

Background Electrical stimulation of the cerebellar fastigial nucleus (FN) reduces the infarct size induced by middle cerebral artery occlusion in rats. FN stimulation confers long‐lasting protection from brain injury; however, its underlying mechanism is not yet understood. We aimed to elucidate the mechanism by which FN stimulation exerts neuroprotection. We hypothesized that the neuroprotective effect of FN stimulation involves activation of cholinergic pathways, which increases reactive oxygen species (ROS) production by opening mitochondrial K+ATP channels, thus leading to an increase in UCP4 (uncoupling protein 4) expression and subsequent neuroprotection. Methods FN stimulation was performed for 1 hour in rats. The UCP4 protein and mRNA levels were measured by western blot, dot blot, and in situ hybridization. Carbachol was applied following UCP4‐promoter tdTomato reporter vector transfection of the rat primary cortical cell culture (in vitro) and rat brain (in vivo). We observed cellular UCP4 expression using fluorescence microscopy. UCP4 expression in the cell culture in response to diazoxide application was determined by a reverse transcription‐polymerase chain reaction and western blotting. Results Whereas FN stimulation increased UCP4 protein and mRNA levels, carbachol administration induced UCP4 expression in vitro and in vivo. The attenuation of this effect by atropine suggests that FN‐induced UCP4 expression involves the cholinergic pathway. The opening of mitochondrial K+ATP channels with diazoxide increased the production of ROS and led to increased UCP4 expression. In contrast, quenching ROS with superoxide dismutase reversed the effect of diazoxide on UCP4 expression. Therefore, the opening of mitochondrial K+ATP channels increased ROS production, which subsequently enhanced UCP4 expression and attenuated ROS generation. Conclusion Neuroprotective effect of FN stimulation involves activation of the cholinergic pathways, which increases ROS production by opening mitochondrial K+ATP channels, leading to increased expression of neuroprotective UCP4