BMAL1 positively correlates with genes regulating steroidogenesis in human luteinized granulosa cells.

In brief: In this study, we examined the relationship between BMAL1 expression and the genes regulating steroid biosynthesis in human luteinized granulosa cells. BMAL1 function is crucial for steroid production and proper ovarian function, highlighting the importance of circadian clock regulation in female reproductive health. Abstract: Human luteinized granulosa cells were collected to analyze circadian clock gene expression and its effect on the genes regulating steroid biosynthesis. We used siRNA to knock down the expression of BMAL1 in KGN cells. We measured the expression levels of genes regulating steroid biosynthesis and circadian clock RT-qPCR. We demonstrated that BMAL1 expression positively correlates with genes regulating steroid biosynthesis (CYP11A1, CYP19A1, STAR, and ESR2). The knockdown of BMAL1 in KGN cells revealed a significant decrease in steroid synthase expression. In contrast, when BMAL1 was overexpressed in KGN and HGL5 cells, we observed a significant increase in the expression of steroid synthases, such as CYP11A1 and CYP19A1. These results indicated that BMAL1 positively controls 17ß-estradiol (E2) secretion in granulosa cells. We also demonstrated that dexamethasone synchronization in KGN cells enhanced the rhythmic alterations in circadian clock genes. Our study suggests that BMAL1 plays a critical role in steroid biosynthesis in human luteinized granulosa cells, thereby emphasizing the importance of BMAL1 in the regulation of reproductive physiology.

Identifying subgroup characteristics of adult ambulance users with nonurgent medical conditions in Japan: A population-based observational study.

Aim: In Japan, approximately 60% of adult ambulance users are diagnosed with minor injuries or diseases in the emergency department and thus do not require hospitalization. This study aimed to determine the distinct subgroup (segment) characteristics of adult ambulance users with nonurgent medical conditions by interpreting quantitatively derived segments through the segmentation approach. Methods: This population-based observational study used the ambulance transportation and request call records databases of the Higashihiroshima Fire Department, Japan, between January 1, 2016, and December 31, 2020. The participants were ambulance users aged 18-64 years diagnosed with minor injuries or diseases in the emergency department (defined as adult ambulance users with nonurgent medical conditions). A soft clustering method was used to divide the participants based on 13 variables. Results: This analysis included 5,982 adult ambulance users. Six segments were obtained: (1) "users with neurological diseases or other injuries occurring late at night on weekdays"; (2) "users injured or involved in fire accidents, with increased on-scene time and multiple hospital inquiries"; (3) "users transferred between hospitals"; (4) "users with acute illnesses and transported from home"; (5) "users involved in motor vehicle accidents"; and (6) "users transferred to hospitals outside of the area during the daytime on weekdays." Conclusion: These findings indicate that adult ambulance users with nonurgent medical conditions can be divided into distinct segments using population-based ambulance records. Further research is warranted to address the ambulance user needs of each segment.

Treatment of lethal acetylsalicylic acid poisoning without hemodialysis.

Case: A woman aged in her 20s ingested approximately 99 g acetylsalicylic acid, and was transported to our hospital 2 h later. She was lucid, but complained of hearing loss and tinnitus. We performed gastric lavage and gave her activated charcoal several times. We attempted to maintain the urinary pH at 7.5 and output above 100 mL/h while preparing for urgent hemodialysis. Outcome: It was revealed after discharge that the blood concentration of acetylsalicylic acid was 103.8 mg/dL on admission (lethal dose level) and had decreased to 35.4 mg/dL by the next morning. The half-life was 8.5 h. Conclusion: Hemodialysis is strongly recommended for patients who take a lethal dose of acetylsalicylic acid. However, by carefully evaluating the vital signs and urinary output and pH, while preparing for emergency hemodialysis, we consider that it is possible to treat acetylsalicylic acid poisoning by alkaline diuresis and critical supportive care.

Possible involvement of endogenous opioid system located downstream of α7 nicotinic acetylcholine receptor in mice with physical dependence on nicotine.

We previously reported that nicotine (NIC)-induced analgesia was elicited in part by activation of the endogenous opioid system. Moreover, it is well known that NIC has physical-dependence liability, but its mechanism is unclear. Therefore, we examined whether physical dependence on NIC was mediated by activation of the endogenous opioid system in ICR mice. We evaluated increased serum corticosterone (SCS) as an indicator of NIC withdrawal, as it is a quantitative indicator of naloxone (opioid receptor antagonist, NLX)-precipitated morphine withdrawal in mice. In this study, NLX precipitated an SCS increase in mice receiving repeated NIC, by a dose-dependent mechanism, and correlated with the dose and number of days of repeated NIC administration. When an opioid receptor antagonist (naltrexone) was concomitantly administered with repeated NIC, the NLX-precipitated SCS increase was not elicited. Concomitant administration of the α7 nicotinic acetylcholine receptor (nAChR) antagonist (methyllycaconitine) with repeated NIC, but not the α4ß2 nAChR antagonist (dihydro-ß-erythroidine), did not elicit an SCS increase by NLX. Thus, a physical dependence on NIC was in part mediated by the activation of the endogenous opioid system, located downstream of α7 nAChR.

Pharmacological relationship between nicotinic and opioid systems in analgesia and corticosterone elevation.

AIMS: Although a pharmacological relationship is known to exist between nicotine and morphine, the exact mechanisms are unclear. Here, we investigated crosstalk between the endogenous opioid system and nicotinic acetylcholine receptors (nAChRs), specifically in nicotine-induced analgesia and activation of the hypothalamic-pituitary-adrenal (HPA) axis. MAIN METHODS: Nicotine and morphine were administered subcutaneously to mice and the effects of these drugs on analgesia and serum corticosterone (SCS) levels were evaluated by the tail-pinch method and fluorometric assay, respectively. KEY FINDINGS: Both nicotine and morphine produced analgesia and SCS increase after a single injection. Nicotine-induced analgesia was prevented by both mecamylamine (MEC; 1mg/kg) and naloxone (NLX; 1mg/kg), and also by repeated administration of morphine or nicotine. Morphine-induced analgesia was prevented by NLX, but not MEC, and by repeated administration of morphine, but not nicotine. Conversely, the nicotine-induced increase in SCS level was prevented by MEC, but not NLX. Morphine-induced SCS increase was prevented by NLX, but not MEC. Moreover, nicotine-induced analgesia was suppressed by dihydro-ß-erythroidine (DHßE; an antagonist for the α4ß2 nAChR) or methyllycaconitine (MLA; an antagonist for the α7 nAChR). The nicotine-induced increase in SCS level was suppressed by DHßE, but not MLA. SIGNIFICANCE: Nicotine-induced analgesia may involve the endogenous opioid system through crosstalk with nicotinic pathways. However, the relationship between these systems does not extend to cooperative actions in nicotine-induced HPA-axis activation.

MUC1 mucin is a negative regulator of toll-like receptor signaling.

MUC1 (MUC1 in humans and Muc1 in nonhuman species) is a transmembrane mucin-like glycoprotein expressed in epithelial cells lining various mucosal surfaces as well as hematopoietic cells. Recently, we showed that Muc1(-/-) mice exhibited greater inflammatory responses to Pseudomonas aeruginosa or its flagellin compared with their wild-type littermates, and our studies with cultured cells revealed that MUC1/Muc1 suppressed the Toll-like receptor (TLR) 5 signaling pathway, suggesting its anti-inflammatory role. Here we demonstrate that other TLR signaling (TLR2, 3, 4, 7, and 9) is also suppressed by MUC1/Muc1. The results from this study suggest that MUC1/Muc1 may play a crucial role during airway infection and inflammation by various pathogenic bacteria and viruses.

Phosphatidic acid metabolism regulates the intracellular trafficking and retrotranslocation of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is transported to the plasma membrane from endoplasmic reticulum (ER) through the Golgi. Crucial to these trafficking events is the role of not only the proteinous factors but also the membrane lipids. However, the involvement of lipids, such as phospholipids, on the regulation of CFTR trafficking has been largely unexplored. Here, we show that the inhibition of phospholipase D (PLD)-mediated phosphatidic acid (PA) formation by 1-butanol inhibited the maturation and export of CFTR from the ER. Exogenously added PA reversed these effects. Moreover, knock down of PLD1 by small interfering RNA decreased the expression of mature CFTR. Interestingly, sustaining the level of PA, by the addition of excess PA in the presence of PA phosphatase inhibitor, attenuated the transport of CFTR from the Golgi to plasma membrane and the retrograde transport of DeltaF508 CFTR to the cytoplasm, a necessary step for the ER-associated degradation of DeltaF508 CFTR. These results indicated that the metabolism of PA modulated the intracellular dynamics and trafficking of CFTR.

Deterioration of glycemic control during octreotide LAR treatment in an acromegalic Japanese patient with type 2 diabetes mellitus.

We report a case showing deterioration of glycemic control during octreotide long-acting release (LAR) treatment in an acromegalic Japanese patient with type 2 diabetes mellitus. The patient did not show much improvement of insulin sensitivity (QUICKI; 0.33 before treatment, 0.35 during octreotide LAR treatment), and showed a significant reduction in early insulin secretion (insulinogenic index; 0.28 before treatment, 0.08 during octreotide LAR treatment) on 75 g oral glucose tolerance test (75gOGTT), despite decreases in GH and IGF-I levels during the course of octreotide LAR treatment. Postoperatively, both insulin sensitivity and early insulin secretion on 75gOGTT were improved (QUICKI 0.59, insulinogenic index 0.35). There are some reports that insulinogenic index is lower in most Japanese patients with type 2 diabetes mellitus and that early insulin secretions are significantly suppressed after administration of octreotide LAR. Although the influence of octreotide LAR on glucose metabolism varies among individuals, it is necessary to manage the deterioration of glucose tolerance during octreotide LAR treatment in acromegalic Japanese patients with decreased insulinogenic index.

[A case of thoracic empyema with gas formation associated with Streptococcus dysgalactiae subsp. equisimilis].

A 63-year-old woman suffering from dyspnea since 1 week earlier underwent a combination therapy of oral corticosteroid and ciclosporin for rheumatic arthritis. Chest radiography showed plural effusion with gas formation in the right thorax. Empyema was diagnosed based on the specimen from pleural effusion. After a chest tube was emplaced to remove and wash out pus, antibiotics were started and empyema improved immediately. Streptococcus dysgalactiae subsp. equisimilis, but not aerobic bacilli, was detected from the pus by repeated culture. Insofar as we know this is the first case of empyema with gas formation associated with Streptococcus dysgalactiae subsp. equisimilis.

Calreticulin negatively regulates the cell surface expression of cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl- channel at the plasma membrane, and its malfunction results in cystic fibrosis, the most common lethal genetic disease in Caucasians. Quality control of CFTR is strictly regulated by several molecular chaperones. Here we show that calreticulin (CRT), which is a lectin-like chaperone in the endoplasmic reticulum (ER), negatively regulates the cell surface CFTR. RNA interference-based CRT knockdown induced the increase of CFTR expression. Consistently, this effect was observed in vivo. CRT heterozygous (CRT+/-) mice had a higher endogenous expression of CFTR than the wild-type mice. Moreover, CRT overexpression induced cell surface expression of CRT, and it significantly decreased the cell surface expression and function of CFTR. CRT overexpression destabilized the cell surface CFTR by enhancing endocytosis, leading to proteasomal degradation. Deletion of the carboxyl domain of CRT, which results in its ER export, increased the negative effect and enhanced the interaction with CFTR. Thus, CRT in the post-ER compartments may act as a negative regulator of the cell surface CFTR.

Characterization of the trafficking pathway of cystic fibrosis transmembrane conductance regulator in baby hamster kidney cells.

To examine the unknown trafficking pathway of the cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER), we utilized baby hamster kidney cells stably expressing CFTR fused with green fluorescent protein. CFTR trafficking from the ER was visualized and analyzed by immunocytochemical analyses. Here we show that CFTR was exported from the ER to the cis-Golgi and early endosome, suggesting that CFTR transport in the early secretory pathway may utilize a non-conventional pathway. This CFTR trafficking pathway may be a target for pharmacological modulation that selectively stimulates CFTR transport.