Visualizing and Modulating Mitophagy for Therapeutic Studies of Neurodegeneration.

Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.

Enhanced Expression of miR-199b-5p Promotes Proliferation of Pancreatic ß-Cells by Down-Regulation of MLK3.

BACKGROUND: The initiation of ß-cell proliferation to recover reduced ß-cell mass is considered as one of the attractive therapeutic approaches for type 1 and 2 diabetes. In this study, we investigated the involvement of miRNAs in ß-cell proliferation. METHODS: Global miRNA array analysis of pancreas tissue was carried out using a 60% partial pancreatectomy (PPx) rodent model, which is a well-characterized model for pancreatic regeneration with accelerated proliferation of ß-cells. To explore miRNAs with mitogenic activity on ß-cells, precursors and antisense oligonucleotides (ASOs) for miRNAs were transfected into a primary islet monolayer cell cultures isolated from adult rats in order to modify their expression and proliferation of ß-cells. RESULTS: We found that miR-199b-5p, which was up-regulated 2.6 times in the pancreas of the PPx treated group, significantly enhanced the proliferation of ß-cells when its precursor was over-expressed. Target genes of miR-199b-5p were investigated and Mixed lineage kinase-3 (MLK3) was identified as one of the candidates since its expression was down-regulated through an interaction with miR-199b-5p and siRNA treatment for MLK3 enhanced the proliferation of ß-cells. CONCLUSION: Our data suggest that the up-regulation of miR-199b-5p enhances proliferation of ß-cells at least in part through down-regulation of MLK3.

Development and validation of sensitive sandwich ELISAs for two investigational nonapeptide metastin receptor agonists, TAK-448 and TAK-683.

TAK-448 and TAK-683, investigational agents with potential utility in the treatment of prostate cancer, are potent low molecular weight metastin receptor agonists consisting of nine amino acids. Monoclonal antibodies (mAbs) against these agents were developed to facilitate their evaluation in preclinical studies. Six mAbs were obtained from four immunogens. Three mAbs recognized the C-terminal of TAK-683 and TAK-448, two recognized the N-terminal of TAK-683, and one recognized the N-terminal of TAK-448. Using various combinations of these six mAbs, sandwich ELISAs for TAK-448 and TAK-683 were developed. These assays were highly sensitive, specific, and accurate. The detection limit for TAK-448 and TAK-683 was 3 and 5 pg/mL, respectively, and there was no interference from rat plasma, rat metastin, or analogs of TAK-448/TAK-683. Recovery achieved ≤±10% with intra-/inter-day assay precision coefficient of variation <10%. The assay demonstrated high stability and sample pre-treatment was not required. Each assay detected the dose-dependent concentration of TAK-448 and TAK-683 in blood 24h after a single intravenous administration of 0.1 and 1mg/kg doses. In conclusion, sensitive sandwich ELISAs were developed to detect the small peptides TAK-448 and TAK-683. The novel assays reliably quantified these nonapeptides in rat plasma, and thus will be useful for preclinical studies of these agents. This methodology may be applicable to the development of similar assays for other short peptides.

Retinoic acids exhibit anti-fibrotic activity through the inhibition of 5-lipoxygenase expression in scleroderma fibroblasts.

The pathogenesis of systemic sclerosis (SSc) is not fully understood and there is no effective treatment for this disease. Retinoic acid (RA) can modulate connective tissue metabolism, exhibit anti-fibrotic activity, and improve the clinical symptoms of SSc. However, the mechanisms by which RA elicits its anti-fibrotic actions remain to be determined. The aim of this study was to elucidate the underlying mechanisms by which RA exerts beneficial effects on scleroderma. Cultured skin fibroblasts from patients with scleroderma were treated with RA and their effect on the expression of 5-lipoxygenase (LOX), transforming growth factor (TGF)-ß1, connective tissue growth factor (CTGF), type I and type III collagen was tested by reverse transcription polymerase chain reaction (RT-PCR) and western immunoblotting. The effect of MK886, a 5-LOX-specific inhibitor, on the expression of TGF-ß1, CTGF, type I and type III collagen was also examined by RT-PCR. In cultured scleroderma fibroblasts, the expression of 5-LOX was elevated compared with normal human dermal fibroblasts. RA significantly inhibited the expression of 5-LOX and of TGF-ß1, CTGF, type I and type III collagen. We further found that the expression of TGF-ß1, CTGF and type I and type III collagen mRNA was inhibited by MK886 in scleroderma fibroblasts. In vitro, RA reduced 5-LOX expression in scleroderma fibroblasts and downregulated TGF-ß1 and CTGF expression, leading to the inhibition of type I and type III collagen synthesis. Our results indicate that the clinical effects of RA on scleroderma are, at least in part, attributable to the reduction of 5-LOX expression and the subsequent suppression of TGF-ß1 and CTGF expression that results in the blockade of collagenogenesis.

Primary squamous cell carcinoma of the frontal sinus.

We report a 74-year-old Japanese man with squamous cell carcinoma (SCC) originating in the frontal sinus. It presented as a cutaneous nodule on his right forehead. Magnetic resonance imaging (MRI) revealed invasion of the anterior wall of the ethmoid sinus, the frontal bone, and possibly the meninx by a frontal sinus carcinoma. Despite right fronto craniotomy with en bloc resection followed by two courses of radiation therapy and chemotherapy with 5-fluorouracil and nedaplatin or TS-1 he died of disease-related causes 20 months later. Herein, we present a detailed description of this patient and a review of the published work.

Bidens pilosa suppresses interleukin-1beta-induced cyclooxygenase-2 expression through the inhibition of mitogen activated protein kinases phosphorylation in normal human dermal fibroblasts.

Bidens pilosa (BP) Linn. var. radiata is a plant used in traditional folk medicine. It is clinically effective in various diseases; the pathogenesis of most of these involves cyclooxygenase (COX)-2. To investigate the mechanism on which the clinical effectiveness of BP is based, we examined its effects on COX-2 expression and its major product, prostaglandin (PG)E(2), under conditions of inflammation. We induced inflammation in normal human dermal fibroblasts with interleukin (IL)-1beta and examined the effects of BP on COX-2 expression and PGE(2) production using Western blotting and competitive enzyme immunoassay, respectively. The functional involvements of mitogen activated protein kinases (MAPK) ERK1/2, p38, and JNK in COX-2 expression were also examined by Western blotting. IL-1beta-induced COX-2 expression was regulated by MAPK pathways, especially by p38. BP inhibited the phosphorylation of MAPKs, COX-2 expression, and subsequent PGE(2) production. The physiological activities and clinical effectiveness of BP observed under diverse conditions may be partly attributable to its ability to inhibit MAPK, mainly p38, activity, COX-2 expression, and subsequent PGE(2) production.

SNP genotyping in the beta(2)-adrenergic receptor by electronic microchip assay, DHPLC, and direct sequencing.

The beta(2)-adrenergic receptor (beta2AR) is the key target for the beta(2)-agonist drugs used for bronchodilation in asthma and chronic obstructive pulmonary disease. To detect four SNPs with amino acid variations at positions -47T/C (CysBUP19Arg), 46A/G (Gly16Arg), 79C/G (Gln27Glu), and 491C/T (Thr164Ile) in the beta 2AR gene, we used the electronic microchip assay, denaturing high-performance liquid chromatography (DHPLC), and direct sequencing. Genomic DNA samples were obtained from the blood of 84 Japanese healthy volunteers. The agreement rates of the first data set with the final data (allele calls) were 99.7% (332/333), 99.2% (246/248), and 96.7% (329/340). The percentages of no allele designation (ND) were 2.06% (7/340), 2.75% (7/255), and 0.00% (0/340) for the electronic microchip assay, DHPLC, and direct sequencing, respectively. Furthermore, we found three samples that had a novel haplotype.

Determination of a novel haplotype of beta2-adrenergic receptor in the Japanese population by the combination of the electronic microchip assay using the NanoChip system with allele-specific PCR.

The beta(2)-adrenergic receptor (B2AR) is a G protein-coupled cell surface receptor that is the key target for the beta(2)-agonist drugs used for bronchodelation in asthma and chromic obstructive pulmonary disease. To detect four SNPs with amino acid variations in the B2AR gene, we used the electronic microchip assay (NanoChip system), DHPLC and sequencing. Genomic DNA samples were obtained from the blood of 84 Japanese healthy volunteers. In sum, the agreement rates of the first data set with the final agreement data (allele calls) were 99.7% (328/329), 99.2% (243/245) and 96.7% (325/336). The percentages of no allele designation (ND) were 2.06% (7/336), 2.75% (7/252), and 0.00% (0/336) for the NanoChip system, DHPLC, and sequencing, respectively. As a result of SNP genotyping, we found three samples that might have a novel haplotype. Furthermore we identified the novel haplotype by a simple technique combining the NanoChip system and allele-specific PCR. These results indicated that NanoChip system was the useful method for clinical SNP genotyping and/or haplotyping because of its accuracy, simplicity and versatility.