Trehalose Suppresses Lysosomal Anomalies in Supporting Cells of Oocytes and Maintains Female Fertility

Supporting cells of oocytes, i.e., cumulus cells, control oocyte quality, which determines fertilization success. Therefore, the transformation of mature and immature cumulus cells (MCCs and ICCs, respectively) into dysmature cumulus cells (DCCs) with dead characteristics deteriorates oocyte quality. However, the molecular basis for this transformation remains unclear. Here, we explored the link between autophagic decline and cumulus transformation using cumulus cells from patients with infertility, female mice, and human granulosa cell-derived KGN cell lines. When human cumulus cells were labeled with LysoTracker probes, fluorescence corresponding to lysosomes was enhanced in DCCs compared to that in MCCs and ICCs. Similarly, treatment with the autophagy inhibitor chloroquine elevated LysoTracker fluorescence in both mouse cumulus cells and KGN cells, subsequently suppressing ovulation in female mice. Electron microscopy analysis revealed the proliferation of abnormal lysosomes in chloroquine-treated KGN cells. Conversely, the addition of an autophagy inducer, trehalose, suppressed chloroquine-driven problematic lysosomal anomalies and ameliorated ovulation problems. Our results suggest that autophagy maintains the healthy state of the supporting cells of human oocytes by suppressing the formation of lysosomes. Thus, our results provide insights into the therapeutic effects of trehalose on female fertility.     

Prolyl Isomerase Pin1 Expression in the Spinal Motor Neurons of Patients With Sporadic Amyotrophic Lateral Sclerosis

Background and PurposeAmyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Selective deficiency of edited adenosine deaminase acting on RNA 2 (ADAR2), a key molecule in the acquisition of Ca²⁺ resistance in motor neurons, has been reported in sporadic ALS (sALS) spinal motor neurons. Since ADAR2 activity is positively regulated by prolyl isomerase Protein never in mitosis gene A interacting-1 (Pin1), a known phosphorylation-dependent peptidyl-prolyl cis/trans isomerase, we investigated Pin1 expression in spinal motor neurons in sALS.MethodsSpecimens of the spinal cord were obtained from the lumbar region in eight sALS patients and age-matched five controls after postmortem examinations. The specimens were double stained with anti-Pin1 and anti-TAR DNA-binding protein of 43 kDa (TDP-43) antibodies, and examined under a fluorescence microscope.ResultsThis study analyzed 254 and 422 spinal motor neurons from 8 sALS patients and 5 control subjects, respectively. The frequency of motor neurons with high cytoplasmic Pin1 expression from the spinal cord did not differ significantly between sALS specimens without cytoplasmic TDP-43 inclusions and control specimens. However, in sALS specimens, neurons for which the Pin1 immunoluminescence intensity in the cytoplasm was at least twice that in the background were more common in specimens with cytoplasmic TDP-43 inclusions (p<0.05 in χ² test).ConclusionsIn sALS, neurons with higher expression levels of Pin1 levels had more TDP-43 inclusions. Despite the feedback mechanism between Pin1 and ADAR2 being unclear, since Pin1 positively regulates ADAR2, our results suggest that higher Pin1 expression levels in motor neurons with TDP-43 pathology from sALS patients represent a compensatory mechanism.     

Pharmacokinetics and metabolism of NO-1886, a lipoprotein lipase-promoting agent,in cynomolgus monkey

1. The study was conducted to investigate the pharmacokinetics and metabolism of NO-1886 (diethyl 4-[(4-bromo-2-cyanophenyl) carbamoyl] benzylphosphonate) in cynomolgus monkeys.

2. After single intravenous administration of NO-1886 at a dose of 3?mg?kg^?1, the total clearance (CL_tot), area under the plasma concentration–time curve (AUC_0–t), half-life (t_1/2), and volume of distribution (V_d) in cynomolgus monkeys were 531?ml?h^?1?kg^?1, 5.63?µg?h?ml^?1, 0.96?h and 679?ml?kg^?1, respectively. The AUC_0–t for oral administration of NO-1886 (3?mg?kg^?1) was 4.23?µg?h?ml^?1 and the bioavailability was 75%.

3. M-2 (ethyl 4-[(4-bromo-2-cyanophenyl) carbamoyl] benzylphosphonate) and M-3 (4-[(diethoxy-phosphoryl) methyl)] benzoic acid) were present as metabolites in plasma and urine. In faeces, M-2 was present but M-3 was not.

4. The major metabolite of NO-1886 in liver S9 or microsomes was M-2 in the presence of NADPH. On the other hand, M-3 was formed in the absence of NADPH in liver S9 or microsomes and its formation was inhibited by bis-(?p-nitrophenyl) phosphate (BNPP) in liver S9, suggesting that the formation of M-3 was catalysed by carboxylesterase.

5. The findings suggest that the main metabolic pathway of NO-1886 in cynomolgus monkeys is the O-deethylation of NO-1886 to M-2, as in rats and humans, and that the hydrolysis of the amide bond is a minor metabolic pathway.     

Effect of vitamin E in gastric mucosal injury induced by ischaemia-reperfusion in nitric oxide-depleted rats

BACKGROUND: Neutrophil infiltration and lipid peroxide accumulation are involved in reperfusion-induced gastric mucosal injury in nitric oxide-depleted rats. AIM: To assess the effect of vitamin E on this injury. METHODS: After ischaemia-reperfusion, the total area of erosions, lipid peroxide contents in gastric mucosa, and gastric neutrophil accumulation were compared between nitric oxide-depleted rats with deficient, normal, and increased vitamin E intake over 8 weeks. Thiobarbituric acid-reactive substances and tissue-associated myeloperoxidase activity were measured in gastric mucosa as indices of lipid peroxidation and neutrophil infiltration. RESULTS: The total area of erosions was significantly increased in the vitamin E-deficient group compared with the sufficient-intake and vitamin-supplemented groups. Both thiobarbituric acid-reactive substances and myeloperoxidase activity also were significantly increased in the vitamin E-deficient group compared with others. The total area of erosions closely paralleled the increases in both thiobarbituric acid-reactive substances and myeloperoxidase activity. CONCLUSION: These results indicate that the inhibition of lipid peroxidation and interference with neutrophil infiltration by vitamin E may be responsible for its cytoprotective effect in ischaemia-reperfusion.     

Bilaminar structure of the developing stapedial footplate in the mouse--a histological study using a light microscope

Normal development of the stapes, especially the bilaminar structure of the footplate, in the mouse was investigated histologically. On day 14 of pregnancy, the footplate had an easily distinguishable lamina consisting of two layers. This bilaminar structure was seen clearly as a pale and loose portion composed of mesenchymal cells in the vestibular side adjacent to the original footplate in the stapedial ring. The authors considered this structure to be the same structure as the lamina stapedialis in the developing human stapes. A dualistic theory of the developmental origin of the footplate in the mouse is proposed.     

Microbial contamination of dialysate and its prevention in haemodialysis units

At the haemodialysis centres of nine hospitals in Japan, microbial contamination of treated water (reverse osmosis method), acid and bicarbonate concentrates, and dialysate was investigated. Among these fluids used in haemodialysis, the dialysate was most frequently contaminated and had the highest concentration of bacteria. Of 40 dialysate samples analysed, 42.5% showed a bacterial count of more than 2000cfu/mL, which was above the Association for the Advancement of Medical Instrumentation (AAMI) standard. However, among the 40 samples from 20 dialysis machines, all six dialysate samples from three dialysis machines that used an ultrafiltration membrane in the circuit before the entrance of the dialysate into the dialyser, showed a bacterial count of < or =10 cfu/mL. In addition, when an ultrafiltration membrane was used in the circuit before the entrance of the dialysate into the dialyser for four dialysis machines showing dialysate samples contaminated with 10(4)-10(5)cfu/mL the bacterial count in dialysate samples from these machines became zero. Because dialysis machines are susceptible to microbial contamination, it is necessary to take measures such as placing an ultrafiltration membrane into the circuit before the entrance of dialysate into the dialyser.     

Purification and partial characterization of fimbriae of Vibrio cholerae O1

Fimbriae of Vibrio cholerae O1 were purified from a strain of the classical biotype, Inaba serotype (Bgd 17), and a strain of the El Tor biotype, Ogawa serotype (K23), grown on TCG agar medium by the following procedure; homogenization of the cell suspension to detach fimbriae, ultracentrifugation to remove remaining cells and their debris, concentration of the supernatant containing fimbriae with ultrafiltration, and 20 to 40% sucrose linear gradient centrifugation of the concentrated material. The fimbriae in both preparations were flexible, long fibres readily distinguishable under the electron microscope from those of CFA/I, CFA/II seen in ETEC strains. Their structural subunit was a protein of 16 kdaltons. Fimbriae isolated from both serotypes and biotypes shared antigenic determinants.