Biol. Pharm. Bull. [JOURNAL]
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200 papers
Errata for Biological and Pharmaceutical Bulletin.
Biol Pharm Bull · 2026 · PMID 41765406 · Publisher ↗
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Inhibitory Effects of Lansoprazole and Omeprazole on the CYP2C19-Mediated Metabolism of Arachidonic Acid to Epoxyeicosatrienoic Acids: Implications for Cardiovascular Risk.
Kobayashi R, Shibata K, Hayakawa D … +5 more , Nishimura Y, Watanabe A, Momo K, Gouda H, Nobe K
Biol Pharm Bull · 2026 · PMID 41741166 · Publisher ↗
Proton pump inhibitors (PPIs), such as lansoprazole and omeprazole, are associated with an increased risk of cardiovascular events; however, the underlying mechanisms remain unclear. We investigated the inhibitory effect...
Proton pump inhibitors (PPIs), such as lansoprazole and omeprazole, are associated with an increased risk of cardiovascular events; however, the underlying mechanisms remain unclear. We investigated the inhibitory effects of lansoprazole and omeprazole on the formation of cardioprotective epoxyeicosatrienoic acids (EETs) and their downstream metabolites, dihydroxyeicosatrienoic acids (DHETs), from arachidonic acid catalyzed by CYP enzymes. In vitro incubation studies were conducted using human liver microsomes (HLMs), recombinant CYP2C19 (rCYP2C19), and recombinant CYP2C9 (rCYP2C9). IC values and R values (a clinical drug-drug interaction risk predictor) were estimated. In HLMs, lansoprazole inhibited the formation of 5,6-, 8,9-, and 11,12-EETs. Omeprazole showed limited inhibition in comparison. In experiments using recombinant enzymes, lansoprazole and omeprazole strongly inhibited EET formation via rCYP2C19, but not rCYP2C9. Lansoprazole exhibited more potent inhibition than omeprazole. Notably, R values for lansoprazole in rCYP2C19 exceeded 1.1 for total EET and DHET formation. Molecular docking analysis suggested that both PPIs may bind to CYP2C19 in a similar pose to a known inhibitor. Docking scores also supported the stronger inhibitory potential of lansoprazole compared to omeprazole. In conclusion, lansoprazole and omeprazole suppress EET formation primarily via CYP2C19 inhibition. The predicted high risk (R > 1.1) for lansoprazole observed in the rCYP2C19 assay suggests a potential mechanism for the increased cardiovascular risk. Further clinical studies are warranted to validate these findings.
Ninjurin-1 Negatively Regulates Humoral and Cellular Immune Responses Induced by the Saponin-Based Adjuvant Quil-A in Mice.
Shao M, Takahama M, Takemura N … +1 more , Saitoh T
Biol Pharm Bull · 2026 · PMID 41741165 · Publisher ↗
Adjuvants are co-administered with antigens to enhance vaccine-induced protection. Saponins are plant-derived compounds with adjuvant properties, some of which are used in licensed vaccines. Macrophages and dendritic cel...
Adjuvants are co-administered with antigens to enhance vaccine-induced protection. Saponins are plant-derived compounds with adjuvant properties, some of which are used in licensed vaccines. Macrophages and dendritic cells (DCs) exposed to saponin-based adjuvants have been reported to exhibit NLRP3-dependent interleukin-1 beta (IL-1β) release, and NLRP3 signaling has been shown to limit their adjuvant activity. Saponin-based adjuvants also induce plasma membrane rupture (PMR) and the release of high-molecular-weight intracellular molecules; however, the molecular mechanisms that mediate PMR and its impact on adjuvant-induced immune responses remain unclear. Here, we investigated the involvement of Ninjurin-1 (NINJ1), a key executor of PMR, in Quil-A-induced PMR and its immunological consequences. Upon stimulation with the saponin mixture Quil-A, peritoneal macrophages and bone marrow-derived dendritic cells (BMDCs) showed NLRP3-independent PMR but NLRP3-dependent IL-1β release. Quil-A-induced PMR was almost completely suppressed in Ninj1 peritoneal macrophages and BMDCs compared with wild-type cells, whereas IL-1β release remained unaffected by NINJ1 deficiency. Immunization with Quil-A and ovalbumin (OVA) increased OVA-specific serum immunoglobulin G (IgG), IgG2b, and IgG2c levels in Ninj1 mice compared with wild-type mice. Splenocytes from Ninj1 mice produced higher levels of interferon-gamma upon stimulation with class I- and class II-restricted OVA peptides than those from wild-type mice. Ninj1 mice also showed a higher frequency of OVA-bearing cells, particularly monocyte-derived DCs, in the draining lymph nodes. These results demonstrate that NINJ1 is critical for Quil-A-induced PMR and that NINJ1-mediated PMR negatively regulates Quil-A-induced humoral and cellular immune activation by restricting antigen delivery via antigen-presenting cells.
Interlaboratory Method Validation for Determination of Polysorbate 80 for Analysis of mAbs.
Yu X, Wang F, Yu C … +1 more , Wang L
Biol Pharm Bull · 2026 · PMID 41741148 · Publisher ↗
Polysorbate 80 (PS80) is a widely used surfactant in therapeutic monoclonal antibodies (mAbs) because it prevents mAb aggregation and stabilizes the drug product. Various analytical methods have been published and are av...
Polysorbate 80 (PS80) is a widely used surfactant in therapeutic monoclonal antibodies (mAbs) because it prevents mAb aggregation and stabilizes the drug product. Various analytical methods have been published and are available for measuring PS80, each with its own limitations. In this study, an HPLC method employing an evaporative light scattering detector (ELSD) was developed for multiproduct analysis of PS80. Method validation was conducted across multiple laboratories using different types of instruments and ELSD detectors. The method was validated using various immunoglobulin G (IgG) mAb molecules and different sources of PS80, with the results showing that it can accurately measure PS80 concentrations ranging from 0.05 to 0.5 mg/mL. Robustness tests demonstrated that the method tolerates up to 160 mg/mL mAb interference, through either sample dilution or protein precipitation. This method can be recommended as a release/quality control method for PS80 concentration determination in therapeutic mAbs.
Functional Transport Properties of Human Zinc Transporter 1: Kinetics and pH-Dependency.
Yoshioka Y, Miyaji T
Biol Pharm Bull · 2026 · PMID 41730615 · Publisher ↗
Intracellular zinc (Zn) homeostasis is essential for physiological and pathological processes and is strictly regulated by Zn transporters. Zinc transporter 1 (ZnT1) is a ubiquitously expressed plasma membrane-localized...
Intracellular zinc (Zn) homeostasis is essential for physiological and pathological processes and is strictly regulated by Zn transporters. Zinc transporter 1 (ZnT1) is a ubiquitously expressed plasma membrane-localized Zn transporter that exports Zn from the cytoplasm to the extracellular space. However, the functional transport properties regarding kinetics and driving forces of ZnT1 remain debatable. In this study, we established a cell-free proteoliposome assay system and demonstrated that ZnT1 transports Zn with high affinity in pH-dependent and pH-independent manners. The K and V of pH-dependent Zn transport were 0.40 μM and 15.13 nmol/min/mg protein, and those of pH-independent Zn transport were 0.52 μM and 8.88 nmol/min/mg protein (low concentrations of Zn), 3.02 μM and 17.59 nmol/min/mg protein (high concentrations of Zn), respectively, suggesting biphasic kinetic components of Zn transport. Even without pH gradient formation, ZnT1 exhibits potent Zn transport activity. In pH dependency, Zn transport activity was higher at an inside pH of 6.0 than at 6.5-7.5 for proteoliposomes, despite the same ΔpH of 0.5-1.5. The Zn transport activity decreased at an outside pH of 8.0, despite an increase in ΔpH. Although previous studies have proposed that ZnT1-mediated Zn transport activity is driven by a calcium (Ca) gradient and not by a pH gradient, Ca does not enhance Zn transport activity in the presence or absence of a pH gradient. These results strongly suggest that ZnT1 protein transports Zn optimally at a specific pH and exports excess intracellular Zn even without ΔpH.
Identification of Inhibitors for eIF5A2-Dependent Translation Elongation by Monitoring the Translational Efficiency of Polyproline Motif in Mitochondrial Fission Regulator 1.
Suzuki M, Komeno M, Yasumura R … +8 more , Miwa A, Enomoto M, Kotani H, Matsumoto K, Akimoto K, Takahashi C, Igarashi K, Higashi K
Biol Pharm Bull · 2026 · PMID 41730614 · Publisher ↗
We previously reported that eukaryotic translation initiation factor 5A2 (eIF5A2), rather than eIF5A1, is important for the proliferation of HeLa S3 and MDA-MB-231 cells, despite the 84% amino acid sequence identity betw...
We previously reported that eukaryotic translation initiation factor 5A2 (eIF5A2), rather than eIF5A1, is important for the proliferation of HeLa S3 and MDA-MB-231 cells, despite the 84% amino acid sequence identity between eIF5A1 and eIF5A2. In addition, individual upregulated genes, including mitochondrial fission regulator 1 (MTFR1), which has a proline-rich motif, by eIF5A2 were different from those in eIF5A1. Thus, eIF5A2-dependent translational elongation is a promising target for cancer treatment with minimal side effects. In this study, we constructed a high-throughput screening system to identify eIF5A2-dependent translation elongation inhibitors by monitoring the translation efficiency of the MTFR1-luciferase fusion protein in HeLa S3 cells. Orlistat and andrographolide (AGP) were suggested as inhibitors of eIF5A2-dependent translation elongation among 1744 compounds from libraries. In addition to the findings related to AGP, the present study revealed that orlistat and the silencing of eIF5A2 suppressed the invasive activity of MDA-MB-231 cells. Downregulation of heparanase 1 expression, but not of matrix metalloproteinase-2 (MMP2) and MMP9, by eIF5A2 silencing was similar to that by treatment with orlistat and AGP, suggesting that orlistat and AGP were, at least in part, capable of attenuating eIF5A2-dependent translation elongation through the repression of eIF5A2 expression. Based on these observations, monitoring the translational efficiency of MTFR1 synthesis may be useful for identifying new eIF5A2 inhibitors.
Evaluation of Readthrough Efficiency of Negamycin Derivatives against Nonsense Mutations in Muscular Dystrophy Genes.
Omura N, Taguchi A, Hamada K … +3 more , Konno S, Taniguchi A, Hayashi Y
Biol Pharm Bull · 2026 · PMID 41730613 · Publisher ↗
Recently, "readthrough compounds" have attracted attention as a promising approach to treat human hereditary diseases caused by nonsense mutations. These compounds enable ribosomes to bypass a premature termination codon...
Recently, "readthrough compounds" have attracted attention as a promising approach to treat human hereditary diseases caused by nonsense mutations. These compounds enable ribosomes to bypass a premature termination codon (PTC) introduced into mRNA by a nonsense mutation, thereby restoring the expression of full-length functional proteins. We performed a structure-activity relationship study focusing on (+)-negamycin, a known readthrough compound, and identified potent derivatives, TCP-304 and TCP-306, featuring a cyclopropane moiety. In this study, we investigated how the nature of PTCs and their surrounding nucleotide sequences influence the readthrough activity of these negamycin derivatives, using nonsense mutation sequences derived from Duchenne muscular dystrophy and congenital muscular dystrophy genes. In cell-based reporter assay systems, TCP-306 exhibited potent readthrough efficiency against several nonsense mutation sequences containing the TGA-A. Moreover, its sequence preference differed from that of the aminoglycoside G418, a representative readthrough compound that preferentially induces readthrough at TGA-C sequences, suggesting that TCP-306 may serve as an alternative therapeutic option for muscular dystrophies associated with TGA-A nonsense mutations. Overall, this study provides valuable insights for the development of readthrough drugs for hereditary diseases such as muscular dystrophy caused by nonsense mutations.
Alteration of Fatty Acid Composition of Phospholipids in EA.hy926 Endothelial Cells Is Reflected in Microvesicles during TNF-α Stimulation.
Ishibashi K, Tanaka Y, Kushida R … +3 more , Ohkita R, Saito M, Atsumi GI
Biol Pharm Bull · 2026 · PMID 41708091 · Publisher ↗
The fatty acid composition of phospholipids in the plasma membrane affects cell function; however, these findings have primarily been observed in vitro because there are no suitable tools for monitoring phospholipid fatt...
The fatty acid composition of phospholipids in the plasma membrane affects cell function; however, these findings have primarily been observed in vitro because there are no suitable tools for monitoring phospholipid fatty acid composition of the plasma membrane in vivo. In this study, we used elaidate as a trigger for altering fatty acid composition in phospholipids and determined whether such changes are reflected in microvesicles (MVs) released from the plasma membrane. Persistent exposure of the human endothelial cell line EA.hy926 to elaidate altered the intracellular fatty acid composition of phospholipids; however, the changes occurring in whole cells were not reflected in the MV fraction during the basal state. Following stimulation with tumor necrosis factor-α, elaidate-mediated alterations in fatty acid composition of the intracellular phospholipids were observed in the MV fraction. Because the alteration of fatty acid composition of intracellular phospholipids is reflected in the plasma membrane, MVs may serve as a useful tool for monitoring the phospholipid fatty acid composition of the plasma membrane under limited conditions.
Risk Factor of Persistent Hypotension Associated with 5-Aminolevulinic Acid: A Single-Institution Retrospective Study.
Okada N, Kimura H, Ozaki M … +5 more , Tamura M, Ochi F, Sasaki T, Takimoto T, Kitahara T
Biol Pharm Bull · 2026 · PMID 41708090 · Publisher ↗
5-Aminolevulinic acid (5-ALA), an amino acid precursor of protoporphyrin IX, is used in the photodynamic diagnosis (PDD) of bladder cancer because it emits fluorescence at specific wavelengths. Severe hypotension has bee...
5-Aminolevulinic acid (5-ALA), an amino acid precursor of protoporphyrin IX, is used in the photodynamic diagnosis (PDD) of bladder cancer because it emits fluorescence at specific wavelengths. Severe hypotension has been reported in patients undergoing 5-ALA-based PDD. Previous studies have mainly focused on hypotension occurring immediately after 5-ALA administration. Persistent hypotension lasting beyond the day of administration has also been reported, indicating a need for continued postoperative management. However, the risk factors associated with persistent 5-ALA-induced hypotension remain unclear. This retrospective study aimed to identify the risk factors for persistent hypotension following 5-ALA administration. Among 263 patients who received 5-ALA for PDD of bladder cancer at Yamaguchi University Hospital between April 2018 and March 2022, 183 developed hypotension and were included in the analysis. Patients were classified into a persistent hypotension group (n = 30), comprising those with continued hypotension the following day, and a nonpersistent group (n = 153). Baseline demographics and clinical characteristics were comparable between the groups. In contrast, preoperative hemoglobin levels were significantly lower in the persistent group (p < 0.05). Multivariate logistic regression analysis identified preoperative hemoglobin levels as an independent risk factor for persistent hypotension (odds ratio, 0.76; 95% confidence interval, 0.60-0.97). A hemoglobin concentration of 12.9 g/dL was determined as the cutoff value for predicting the incidence of persistent hypotension using receiver-operating characteristic curve analysis. Although further validation is required, these findings suggest that the preoperative hemoglobin level may serve as a potential indicator for risk stratification of persistent hypotension induced by 5-ALA.
Development and Validation of a Risk Score Predicting Medication Non-adherence in Patients with Heart Failure.
Ishida T, Kawada K, Jobu K … +13 more , Hamada T, Kubo T, Hyohdoh Y, Okazaki M, Sagawa T, Kawai K, Nakaoka Y, Yabe T, Furuno T, Yamada E, Kawano Y, Kitaoka H, Hamada Y
Biol Pharm Bull · 2026 · PMID 41708089 · Publisher ↗
In this study, we aimed to develop a clinically applicable risk score using demographic and clinical data available at hospital admission for decompensated heart failure (HF) to identify patients at high risk of medicati...
In this study, we aimed to develop a clinically applicable risk score using demographic and clinical data available at hospital admission for decompensated heart failure (HF) to identify patients at high risk of medication non-adherence after discharge. In total, 795 patients with HF were enrolled from a prospective, multicenter cohort study. A logistic regression model identified independent predictors of medication non-adherence leading to hospitalization, and we derived a risk score from the partial regression coefficients of these factors. Hospitalization due to medication non-adherence occurred in 6.4% of patients. Independent predictors included dementia (odds ratio [OR]: 2.79; 95% confidence interval [CI]: 1.38-5.65), male sex (OR: 2.50; 95% CI: 1.30-4.79), current smoking (OR: 2.17; 95% CI: 1.07-4.41), non-assisted living (OR: 2.14; 95% CI: 1.17-3.94), and prior HF hospitalization (OR: 1.91; 95% CI: 1.05-3.46). The predictive model showed good discrimination (area under the receiver operating characteristic curve = 0.704). Patients with higher risk scores (4-6 points) had significantly higher rates of all-cause and cardiovascular mortality over 2 years. In conclusion, this pragmatic, admission-based risk score enables early identification of patients at risk for medication non-adherence and may support targeted interventions to improve long-term HF outcomes. Incorporating this score into routine admission processes may help prioritize adherence support for high-risk patients, guide multidisciplinary care planning, and reduce post-discharge complications in HF management.
Oxaliplatin-Induced Liver Toxicity: Hepatic Transglutaminase 7 Upregulation Associates with Oxidative Stress, Inflammation, and Apoptosis.
Alowss HM, Almami IS, Gomaa HF
Biol Pharm Bull · 2026 · PMID 41692438 · Publisher ↗
Transglutaminases (TGs) are calcium-dependent enzymes that cross-link proteins, contributing to apoptosis, extracellular matrix (ECM) stabilization, and inflammation. While TG2 has been extensively studied in hepatic inj...
Transglutaminases (TGs) are calcium-dependent enzymes that cross-link proteins, contributing to apoptosis, extracellular matrix (ECM) stabilization, and inflammation. While TG2 has been extensively studied in hepatic injury, the role of TG7 in oxaliplatin-induced liver responses remains unclear. Oxaliplatin, a third-generation platinum chemotherapeutic, effectively treats solid tumors but can induce hepatic stress through oxidative and pro-inflammatory signaling. Adult rats received intraperitoneal oxaliplatin (10 mg/kg weekly) for 6 weeks. qRT-PCR, immunohistochemistry (IHC), immunofluorescence (IF), and a TG activity assay assessed hepatic TG7 expression, localization, and activity. Oxidative stress indicators (serum malondialdehyde [MDA] and reduced glutathione [GSH]) and pro-inflammatory cytokine transcription (CASP3, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α)) were evaluated. Oxaliplatin exposure markedly increased TG7 mRNA and protein levels, elevated TG enzymatic activity, raised MDA (+49.4%), depleted GSH (-18.6%), and upregulated CASP3, IL-6, and TNF-α. DNA fragmentation and microscopic observations from IHC- and IF-processed sections were consistent with apoptosis-associated DNA degradation and subtle stress-related structural variations. Immunostaining revealed altered TG7 distribution within hepatocytes and sinusoidal regions. In this oxaliplatin-exposed rat liver model, TG7 upregulation and increased TG activity were associated with oxidative stress, inflammatory cytokine induction, and apoptotic signaling. These findings identify TG7 as a stress-associated marker during oxaliplatin exposure and support further studies to clarify its mechanistic role and evaluate its potential as utility as a biomarker under chemotherapy-associated hepatic stress conditions.
Enhanced Therapeutic Efficacy of Photodynamic Therapy Using Hybrid Liposomes Containing Indocyanine Green in a Triple-Negative Breast Cancer Mouse Model.
Okumura M, Otsuka H, Takai J … +3 more , Goto K, Matsumoto Y, Ichihara H
Biol Pharm Bull · 2026 · PMID 41692435 · Publisher ↗
Triple-negative breast cancer (TNBC) is characterized by the absence of hormone receptors and human epidermal growth factor receptor 2 (HER2) expression, rendering hormone therapy and HER2-targeted treatments ineffective...
Triple-negative breast cancer (TNBC) is characterized by the absence of hormone receptors and human epidermal growth factor receptor 2 (HER2) expression, rendering hormone therapy and HER2-targeted treatments ineffective. Consequently, conventional chemotherapy remains the primary therapeutic option, despite its severe side effects and poor prognosis. Hybrid liposomes (HL) composed of phospholipids and polyethylene glycol (PEG)-based surfactants have been reported to have therapeutic effects on various cancers without containing anticancer drugs. In this study, we investigated whether photodynamic therapy (PDT) with HL containing indocyanine green (HL/ICG) enhanced the therapeutic effect of HL alone in a mouse model of subcutaneous implantation of triple-negative breast cancer (TNBC) cells in vivo. HL/ICG selectively accumulated in tumors in mice implanted with 4T1-Luc cells, a TNBC cell line. Histological analysis of resected tumor tissues following HL/ICG-mediated PDT revealed a significant increase in cells positive for oxidative stress markers, indicating elevated intracellular oxidative damage. Additionally, a marked presence of apoptotic cells was observed, suggesting that PDT effectively induced programmed cell death in tumor tissues. These results indicate that PDT with HL/ICG induces oxidative stress-mediated apoptosis in tumors derived from 4T1-Luc cells and significantly enhances the therapeutic efficacy of HL alone in vivo, highlighting its potential as a promising strategy for the treatment of TNBC.
Development and Clinical Application of a Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Method for Patients with Graft-versus-Host Disease to Quantify the Plasma Concentrations of Ruxolitinib and Posaconazole.
Kumondai M, Hayashi N, Sato Y … +13 more , Kobayashi D, Otsuki A, Ueki Y, Onishi Y, Tsukamoto T, Yoshikawa K, Hayakawa Y, Sato Y, Sato T, Sato M, Kikuchi M, Maekawa M, Mano N
Biol Pharm Bull · 2026 · PMID 41692434 · Publisher ↗
Graft-versus-host disease (GVHD) is a clinically significant problem with high mortality that is gradually increasing. Ruxolitinib (RUX) is the only drug used for steroid-refractory GVHD treatment and is thereby crucial....
Graft-versus-host disease (GVHD) is a clinically significant problem with high mortality that is gradually increasing. Ruxolitinib (RUX) is the only drug used for steroid-refractory GVHD treatment and is thereby crucial. Therapeutic drug monitoring of RUX may be effective because of the relationship between the plasma RUX concentration and treatment outcomes. Posaconazole (PCZ) has also been the recent focus of combined treatment with RUX owing to its pharmacokinetics. We established a simultaneous LC-tandem MS (LC-MS/MS) method and performed plasma drug concentration measurements and monitoring using clinical laboratory values for both RUX and PCZ. We also compared our technique to a simple LC-MS/MS method for clinical application. Moreover, the utility of the automated pretreatment LC-MS/MS (auto-LC-MS/MS) method was tested for further applications. The simultaneous quantification LC-MS/MS method satisfied analytical validation criteria under clinical conditions. Our method demonstrated linearity over the range of 0.3-500 ng/mL for RUX and 3-5000 ng/mL for PCZ, with intra- and inter-day precision and accuracy within ±15%. A possible correlation between plasma RUX concentration and kidney injury was observed in 1 of the 6 patients. Notably, plasma PCZ concentrations were decreased by changing the administration route. Moreover, the plasma concentration levels obtained using the auto-LC-MS/MS method were highly concordant with those obtained using the LC-MS/MS method. The validated LC-MS/MS method was found to be useful in clinical applications; thus, further research into its applications in clinical practice is desirable.
Ouabain Suppresses CD63 Loading into Extracellular Vesicles via Na/K-ATPase-Dependent Localization to Autophagosomes.
Adachi H, Yoshida T, Itoh RE … +1 more , Oneyama C
Biol Pharm Bull · 2026 · PMID 41692433 · Publisher ↗
Extracellular vesicles (EVs), including exosomes, mediate intercellular communication by transferring lipids, proteins, and nucleic acids. However, the mechanisms determining selective cargo loading into EVs remain poorl...
Extracellular vesicles (EVs), including exosomes, mediate intercellular communication by transferring lipids, proteins, and nucleic acids. However, the mechanisms determining selective cargo loading into EVs remain poorly understood. Here, we identify the cardiac glycoside ouabain as a selective inhibitor of CD63 loading into EVs. Using a luciferase-based high-throughput assay with CD9- and CD63-tagged reporter cells, ouabain was found to specifically suppress CD63 loading into EVs without affecting CD9 loading into EVs. Ouabain, Na/K-ATPase inhibitor, did not suppress EV secretion but markedly decreased CD63 incorporation. Other cardiac glycoside with strong Na/K-ATPase inhibitory activity, such as bufalin, exhibited similar effects, whereas weak inhibitors did not. Ouabain induced the internalization of Na/K-ATPase (ATP1A1) with CD63, resulting in the disappearance of CD63 from the plasma membrane. Furthermore, ouabain activated autophagy and promoted the colocalization of CD63 with autophagosomes, thereby selectively inhibiting the loading of CD63 into EVs. These effects required both Na/K-ATPase-dependent endocytosis and autophagy, as rapamycin-induced autophagy alone did not remove surface CD63. Our findings reveal a previously unrecognized mechanism in which cardiac glycoside regulates EV cargo composition by coupling Na/K-ATPase-mediated endocytosis with autophagy. Given that endogenous and therapeutic cardiac glycosides are implicated in cardiovascular and cancer biology, this mechanism may broadly influence EV-mediated intercellular communication and represent a potential target for modulating EV functions.
Intracellular Signaling Pathways for Erythropoietin-Induced Cell Proliferation in Primary Cultured Hepatocytes.
Moteki H, Ogihara M, Kimura M
Biol Pharm Bull · 2026 · PMID 41672545 · Publisher ↗
The mechanisms by which erythropoietin (EPO) promotes hepatocyte proliferation in primary cultures of adult rat hepatocytes were studied. EPO stimulated cell proliferation in a time- and dose-dependent manner, significan...
The mechanisms by which erythropoietin (EPO) promotes hepatocyte proliferation in primary cultures of adult rat hepatocytes were studied. EPO stimulated cell proliferation in a time- and dose-dependent manner, significantly increasing the number of hepatocyte nuclei and DNA synthesis. EPO-induced hepatocyte proliferation was completely suppressed by specific inhibitors targeting Janus kinase 2 (JAK 2), phospholipase C (PLC), protein kinase C (PKC), intracellular Ca mobilization, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), and mammalian target of rapamycin (mTOR). In contrast, inhibition of signal transducer and activator of transcription 3/5 (STAT 3/5) or granule secretion had no effect, indicating that EPO acts through a pathway distinct from the classical JAK2-STAT signaling pathway. Western blot analysis showed rapid phosphorylation of ERK 2, but not ERK 1, following EPO stimulation. In addition, EPO induced phosphorylation of PLC and C-rapidly accelerated fibrosarcoma (C-Raf), with PKC acting downstream of PLC and upstream of C-Raf. In contrast, intracellular Ca concentration and activated Ras were transiently increased in hepatocytes after EPO stimulation, and EPO-induced activated Ras was significantly suppressed by the specific PKC inhibitor GF109203X. These results indicate that EPO engages the JAK2/PLC/PKC-Ca signaling cascade, leading to the sequential activation of Ras, C-Raf, and ERK2, ultimately promoting hepatocyte proliferation in vitro.
A Pilot Study to Develop an Ultrasensitive Thio-NAD Cycling ELISA for Neisseria gonorrhoeae Detection.
Chen PK, Yang TY, Li JW … +5 more , Tang HJ, Chen YW, Chang YC, Lu PL, Ito E
Biol Pharm Bull · 2026 · PMID 41656091 · Publisher ↗
Neisseria gonorrhoeae and its increasing antimicrobial resistance have raised global health concerns, creating an urgent need for more advanced diagnostics to contain the spread. We conducted a preclinical trial using an...
Neisseria gonorrhoeae and its increasing antimicrobial resistance have raised global health concerns, creating an urgent need for more advanced diagnostics to contain the spread. We conducted a preclinical trial using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) with Thio-nicotinamide-adenine dinucleotide (NAD) cycling for the detection of N. gonorrhoeae. Three independent researchers conducted the initial experiments to obtain calibration curves. The inter-assay reproducibility across the 3 investigators yielded coefficients of variation of 11% when measuring 500000 CFU/mL, confirming the consistent performance of the ultrasensitive ELISA. Specificity against common clinical microorganisms was also assessed. Performance was first evaluated in simulated urine, vaginal fluid, and saliva. A comparative evaluation of 90 clinical isolates was then performed using a conventional ELISA method. The ultrasensitive ELISA produced consistent calibration curves (R = 0.99) with limits of detection of 1.1 × 10 to 1.8 × 10 CFU/mL. No cross-reactivity to common pathogens was observed. The assay results revealed linearity in simulated fluids, with a modestly reduced sensitivity in urine and vaginal fluid. At 50000 CFU/mL, the ultrasensitive ELISA generated signals >50-fold stronger than the p-nitrophenyl phosphate (pNPP) ELISA. For clinical isolates, the ultrasensitive ELISA identified 78.9% as positive compared with 30.0% by pNPP ELISA (p = 0.0005). The ultrasensitive ELISA detected N. gonorrhoea with high sensitivity and specificity across sample types, consistently outperforming the conventional ELISA, and demonstrating the potential of this approach as a cost-effective diagnostic alternative.
C4ST-1 Overexpression Suppresses Osteoblast Differentiation via a Wnt/β-Catenin-p53 Axis.
Koike T, Nadanaka S, Kitagawa H
Biol Pharm Bull · 2026 · PMID 41656089 · Publisher ↗
Disruption of osteoblast differentiation can lead to severe bone diseases, such as osteoporosis and osteosclerosis. Our previous study showed that a reduced 4S/6S ratio promotes osteoblast differentiation, linking this s...
Disruption of osteoblast differentiation can lead to severe bone diseases, such as osteoporosis and osteosclerosis. Our previous study showed that a reduced 4S/6S ratio promotes osteoblast differentiation, linking this specific chondroitin sulfate (CS) modification to bone pathology. This study investigated the effect of forced elevation of the 4S/6S ratio on differentiation. C4ST-1 was found to exhibit overexpression, increasing the 4S/6S ratio, significantly suppressing osteoblast differentiation, as evidenced by reduced Akp2 gene expression and ALP activity. This inhibition was far more potent than that caused by the enzymatic removal of CS. Notably, treating C4ST-1-overexpressing cells with chondroitinase reduced differentiation inhibition to a level similar to that in mock cells treated with chondroitinase. These results suggest that the strong inhibition was due to the excessive 4-sulfated CS produced by C4ST-1, implying a mechanism distinct from the inhibition caused by a lack of CS. To elucidate this mechanism, we investigated the potential feedback loop. The increase in 4-sulfated CS from C4ST-1 overexpression enhanced Wnt3a expression, which upregulated p53 expression. Pharmacological inhibition of β-catenin and p53 partially restored Akp2 expression, confirming their roles as key mediators. We propose that a high 4S/6S ratio activates a Wnt/β-catenin-p53 axis, where p53 acts as a brake on differentiation. Our findings highlight the critical role of CS sulfation patterns in fine-tuning the osteoblast fate.
The Daily Medication Frequency at Which Participants Begin to Perceive Dosing as Excessive: A Questionnaire-Based Study Using the Personal Health Record Infrastructure via Electronic Medication Notebooks.
Imai S, Asano M, Shimizu Y … +10 more , Kizaki H, Tsuchiya M, Ito Y, Tsuchiya M, Kuriyama R, Yoshida N, Shimada M, Sando T, Ishijima T, Hori S
Biol Pharm Bull · 2026 · PMID 41656088 · Publisher ↗
Maintaining medication adherence remains a major clinical challenge, as higher daily dosing frequencies are often associated with decreased adherence. Although once-daily regimens are generally preferred, specific patien...
Maintaining medication adherence remains a major clinical challenge, as higher daily dosing frequencies are often associated with decreased adherence. Although once-daily regimens are generally preferred, specific patient preferences regarding dosing frequency are unclear, despite implications for improving patient satisfaction and optimizing pharmacotherapy. Here, we evaluated the frequency at which patients begin to perceive dosing as excessive. A web-based questionnaire survey using the personal health record infrastructure of electronic medication notebooks was administered between July 20 and 26, 2023. Eligible participants were aged ≥20 years and had received oral tablets or capsules within 90 d of the survey. The questionnaire consisted of 6 items, including whether participants felt that the daily medication frequency was excessive. A multivariate logistic regression analysis was performed to identify the frequency beyond which patients perceive dosing as excessive, adjusting for confounders, such as medication formulation, number of medications, and patient characteristics. Of 1478 respondents, 1236 were included in the analysis. In total, 28.9% of participants reported that their medication frequency felt excessive. In the multivariate logistic regression analysis, twice-daily or more frequent dosing was significantly associated with the perception of excessiveness, using once-daily dosing as the reference. Twice-daily dosing is the frequency beyond which patients are significantly more likely to perceive medication as excessive. Our findings emphasize the importance of simplifying dosing regimens.
Regulation of ER-Resident Transcription Factor NFE2L1 in HEK293 Cells.
Yasui M, Nagae I, Murase R … +4 more , Uchio-Yamada K, Kandeel M, Tsujita T, Oh-Hashi K
Biol Pharm Bull · 2026 · PMID 41656087 · Publisher ↗
Nuclear factor erythroid-derived 2 like 1 (NFE2L1) is reported to be embedded in the endoplasmic reticulum (ER) membrane and subsequently undergo N-glycosylation at several asparagine residues as well as other ER-residen...
Nuclear factor erythroid-derived 2 like 1 (NFE2L1) is reported to be embedded in the endoplasmic reticulum (ER) membrane and subsequently undergo N-glycosylation at several asparagine residues as well as other ER-resident factors including cAMP response element binding protein 3 (CREB3)/ATF6 family members. In this study, we investigated the regulation of NFE2L1 protein expression by treating wild-type HEK293 cells and HEK293 cells deficient in selected ER-associated degradation (ERAD) factors with various reagents. NFE2L1 protein expression in wild-type HEK293 cells was negligible, but MG132/bortezomib treatment induced Endo H-resistant two bands. Suppressor/enhancer of lin-12-like (SEL1L)/hydroxymethylglutaryl-CoA (HMG-CoA) reductase degradation 1 (Hrd1) loss increased NFE2L1 protein expression without any stimuli. In these deficient cells, the band shift of NFE2L1 by MG132 was mostly suppressed. Treatment with the valosin containing protein (VCP) inhibitor CB-5083 increased NFE2L1 expression, but deficiencies in other ERAD-associated factors (ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2), thioredoxin domain-containing protein 11 (TXNDC11), gp78, ring finger protein 5 (RNF5), ring finger protein 185 (RNF185), and USP19) did not affect its expression. Comparing the stability of the two intrinsic NFE2L1, which increases with proteasome inhibition, the higher molecular weight form corresponding to full-length form, was more unstable. Therefore, we constructed NFE2L1 genes with mutations in the site where NFE2L1 is cleaved by DDI2 and in the four asparagine residues where N-glycosylation occurs, and found that the high molecular weight form, especially a hypoglycosylated mutant, tended to be more unstable. Taken together, this study using several ERAD disordered models shows that the regulation of NFE2L1 is different in some ways from the regulation of CREB3/ATF6 family, and these findings implicate the diversity of N-glycosylated protein regulation in the ER.
Induction of Hepatocyte Growth Factor Production by Quercetin.
Akimoto Y, Katsube A, Miyamae Y … +1 more , Shigemori H
Biol Pharm Bull · 2026 · PMID 41656086 · Publisher ↗
Hepatocyte growth factor (HGF) exhibits mitogenic, motogenic, and morphogenic activities. Enhancing HGF production could serve as a therapeutic approach for organ regeneration, wound healing, and embryogenesis. Notably,...
Hepatocyte growth factor (HGF) exhibits mitogenic, motogenic, and morphogenic activities. Enhancing HGF production could serve as a therapeutic approach for organ regeneration, wound healing, and embryogenesis. Notably, HGF demonstrates therapeutic potential in the treatment of neurodegenerative diseases. In this study, we found that quercetin promotes HGF production in normal human dermal fibroblasts (NHDF) at low concentrations. cAMP response element binding protein (CREB), a transcription factor, is phosphorylated. Additionally, this activity may result from quercetin's interaction with the β2 adrenaline receptor (β2AR). Further pharmacological analysis suggested that HGF production is promoted via PKA pathway. In conclusion, quercetin shows potential as a drug for treating organ-related diseases, including neurodegenerative disorders, by enhancing HGF production.
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