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Aging is associated with the dysfunction of the blood-brain barrier (BBB), which comprises brain microvessel endothelial cells (BMECs), astrocytes, and pericytes. Pericytes are present at intervals along the walls of the brain capillaries and play a key role in maintaining BBB integrity. Accumulation of senescent cells and the senescence-associated secretory phenotype (SASP) in the brain facilitate the development of age-related neurodegenerative diseases with BBB dysfunction. However, the ability of pericytes to support BBB integrity and their correlation with cellular senescence or aging remain unknown. Here, we investigated cellular senescence in pericytes focusing on its impact on BBB function using BBB models comprising intact BMECs co-cultured with senescent pericytes, which were obtained through a serial passage or isolated from 18-month-old rats. To assess BBB function, transendothelial electrical resistance (TEER) and permeability of sodium fluorescein (Na-F) were studied. Both serially passaged pericytes (in passage 4, 7, and 10) and aged pericytes isolated from 18-month-old rats showed decreased TEER and enhanced permeability of BMECs to Na-F compared to that of normal pericytes (passage 2 or young). Furthermore, serially passaged and aged pericytes showed characteristic features of cellular senescence, including increased β-galactosidase activity, cell cycle arrest, enhanced expression of mRNA, and SASP factors. However, the senescence-induced mRNA expression profile of pericyte markers varied between serially passaged and aged pericytes. Hence, in vitro serial passages and isolation from naturally aged rodents differently influenced genetic and biochemical features of senescent brain pericytes. We conclude that senescent brain pericytes can induce BBB dysfunction and those isolated from aged rodents retain the senescence-specific properties. Our findings provide an alternative tool to investigate the senescence in brain pericytes in vitro., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: Recent randomized phase III trial has shown significant benefit in overall survival (OS) for patients with advanced cervical cancer by adding bevacizumab to conventional chemotherapy. The aim of this study was to evaluate the prognostic impact for Japanese recurrent, persistent, or metastatic cervical cancer patients where bevacizumab was added to paclitaxel plus carboplatin., Materials and Methods: A retrospective analysis was performed on 90 patients with recurrent, persistent, or metastatic cervical cancer mainly treated by paclitaxel plus carboplatin between 2005 and 2019 at our hospital. Data for the following clinicopathological variables were analyzed: (1) bevacizumab use; (2) histology; (3) disease presentation; (4) performance status; (5) prior chemotherapy containing platinum agent; (6) pelvic disease; (7) prior pelvic radiotherapy; (8) location of target lesions. Survival analysis was performed using Kaplan-Meier curves, log-rank tests, Wilcoxon tests, and Cox proportional hazards models combined with propensity score matching., Results: Adding bevacizumab to paclitaxel plus carboplatin showed significantly increased complete response to compared with that of non-users. In a Cox regression hazard model, bevacizumab use tended to show better OS though without statistically significance. After propensity score matching, adding bevacizumab to paclitaxel plus carboplatin showed a significant better OS by univariate analysis using Wilcoxon test, not by log-rank test., Conclusion: Adding bevacizumab to paclitaxel plus carboplatin showed a limited prognostic impact for recurrent, persistent or advanced cervical cancer patients in the real world. Further effective second-line treatments are needed to prolong OS of patients with recurrent, persistent or advanced cervical cancer., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

Orthopedic and dental implants coated with fibroblast growth factor-2 (FGF-2)-calcium phosphate composite layers promote dermis formation, bone formation, and angiogenesis because of the biological activity of FGF-2. Enhancing the biological activity of FGF-2 in the composite layers is important for its wider application in orthopedics and dentistry. This study incorporated low-molecular-weight heparin (LMWH) into the FGF-2-calcium phosphate composite layers and clarified the enhancing effects of LMWH on the biological activity of FGF-2 in the composite layers in vitro. LMWH-FGF-2-calcium phosphate composite layers were successfully formed on zirconia in supersaturated calcium phosphate solutions. The composite layers comprised continuous and macroscopically homogeneous layers and particles smaller than 500 nm in size composed of amorphous calcium phosphate. The amounts of Ca and P deposited on zirconia remained almost unchanged with the addition of LMWH under the presence of FGF-2 in the supersaturated calcium phosphate solution. The LMWH in the supersaturated calcium phosphate solution increased the stability of FGF-2 in the solution and the amount of FGF-2 in the composite layers. The LMWH in the composite layers increased the mitogenic and endothelial tube-forming activities of FGF-2, and FGF-2 activity of inducing osteogenic differentiation gene expression pattern in the composite layers. Our results indicate that the enhanced biological activity of FGF-2 in the LMWH-FGF-2-calcium phosphate composite layers is attributed to an LMWH-mediated increase in the amount of FGF-2, which maintains its biological activity in the supersaturated calcium phosphate solution and the composite layers. The LMWH-FGF-2-calcium phosphate composite layer is a promising coating for orthopedic and dental implants. STATEMENT OF SIGNIFICANCE: Orthopedic and dental implants coated with fibroblast growth factor-2 (FGF-2)-calcium phosphate composite layers promote dermis formation, bone formation, and angiogenesis because of the biological activity of FGF-2. Enhancing the biological activity of FGF-2 in the layers is important for wider its application in orthopedics and dentistry. This study demonstrates the enhancing effects of low-molecular-weight heparin (LMWH) contained within LMWH-FGF-2-calcium phosphate composite layers on the biological activity of FGF-2 in vitro. Our results indicate that the enhanced biological activity of FGF-2 within the composite layers arises from an LMWH-mediated increase in the amount of FGF-2, which maintains its biological activity in the LMWH-FGF-2-calcium phosphate composite layers and supersaturated calcium phosphate solutions used for coating the composite layers., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2022. Published by Elsevier Ltd.)

In predicting the hepatic elimination of compounds, the extended clearance concept has proven useful. Yet, its experimental proof was scarce partly due to the lack of models with the controlled expression of transporters. Here, the uptake and efflux transporters [NTCP (SLC10A1) and BSEP (ABCB11), respectively] were doubly and transiently expressed in MDCKII cells by electroporation-based transfection (with the BSEP plasmid amount varied and with the NTCP plasmid fixed), achieving the activity levels of NTCP and BSEP comparable to those of sandwich cultured human hepatocytes. The biliary excretion clearance for taurocholate increased proportionally to the BSEP expression level. Under the same conditions, the basal-to-apical transcellular clearance of taurocholate displayed an initial increase, and a subsequent plateau, indicating that the basolateral uptake of taurocholate became rate-limiting. The doubly transfected MDCKII cells were also used to kinetically analyze the inhibitory effects of rifampicin on BSEP and NTCP. The obtained results showed a bell-shaped profile for cell-to-medium concentration ratios over a range of rifampicin concentrations, which were quantitatively captured by kinetic modeling based on the extended clearance concept. The present study highlights the utility of the transient, tunable transporter expression system in delineating the rate-determining process and providing mechanistic insights into intracellular substrate accumulation., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Cell morphology is related to proliferation and differentiation. We previously reported that cell attachment area of rat mesenchymal stem cells (MSCs) is negatively correlated with their osteogenic differentiation level on osteoconductive hydroxyapatite (HAp) with various microstructures. In this study, the correlation between the cell attachment area and osteogenic differentiation level was investigated on substrates without osteoconductive property using tissue culture polystyrene (TCPS), and 3 mol% yttria-stabilized zirconia (3Y-TZP) with or without surface periodic microstructures. It was found that the osteogenic differentiation level after 3 weeks of culture increased with a decrease in cell attachment area after 3 h of culture. The square of the correlation coefficient between cell attachment area and osteocalcin secretion content was 0.845 among the three types of substrates. Thus, the negative correlation between cell attachment area and differentiation level is confirmed even when cultured on substrates without osteoconductive property. These findings suggest that the correlation between the cell attachment area of rat MSCs and osteogenic differentiation level could also apply to various types of substrate, regardless of osteoconductive property., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Several reports have implicated myo-inositol (MI) in myelin formation. We hypothesized that MI is involved in this process through facilitating the biosynthesis of ethanolamine plasmalogens (PlsEtns), which are the major component of myelin membranes, and essential for myelin formation and function. Excessive MI urinary excretion possibly causes PlsEtn deficiency, leading to demyelinating diseases including dementia., Methods: We examined the association between cognitive impairment, serum levels of PlsEtn, and baseline levels of urinary MI excretion, in the enrollment of 55 memory clinic outpatients and 107 cognitively normal elderly., Results: Serum PlsEtns were independently associated with cognitive impairment, and significantly reduced in memory clinic outpatients, especially in those with high urinary MI, as compared to normal elderly. On the other hand, there was no direct association between urinary MI and cognitive impairment, but urinary MI was significantly associated with serum hemoglobin A1c and amyloid β 1-40. The interaction between PlsEtn and urinary MI for cognitive impairment was statistically confirmed, and their combined usage improved diagnosis of cognitive impairment., Conclusions: We proposed the involvement of MI and PlsEtn in cognitive impairment pathology. In conclusion, serum PlsEtn may be useful in detecting cognitive decline among elderly with hyperglycemia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The metazoan cell membrane is highly organized. Maintaining such organization and preserving membrane integrity under different conditions are accomplished through intracellular tethering to an extensive, flexible protein network. Spectrin, the principal component of this network, is attached to the membrane through the adaptor protein ankyrin, which directly bridges the interaction between β-spectrin and membrane proteins. Ankyrins have a modular structure that includes two tandem ZU5 domains. The first domain, ZU5A, is directly responsible for binding β-spectrin. Here, we present a structure of the tandem ZU5 repeats of human erythrocyte ankyrin. Structural and biophysical experiments show that the second ZU5 domain, ZU5B, does not participate in spectrin binding. ZU5B is structurally similar to the ZU5 domain found in the netrin receptor UNC5b supramodule, suggesting that it could interact with other domains in ankyrin. Comparison of several ZU5 domains demonstrates that the ZU5 domain represents a compact and versatile protein interaction module., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The extracellular signal-regulated kinase (ERK) signaling pathway is constitutively activated in many human tumor cell types. Given the cytoprotective role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although blockade of ERK signaling alone did not induce substantial cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the ERK pathway is constitutively activated. The synergistic activation of c-Jun NH(2)-terminal kinase by the combination of an ERK pathway inhibitor and a microtubule-destabilizing agent appeared to be responsible, at least in part, for this effect. These results suggest that administration of the combination of an ERK pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells with constitutive activation of the ERK pathway.

Accumulating evidence suggests that several ATP-binding cassette (ABC) transporters mediate the elimination of anticancer drugs from cancer cells and thereby confer drug resistance. SN-38-selected PC-6/SN2-5H human lung carcinoma cells were shown to overexpress ABCG2 with the reduced intracellular accumulation of SN-38, the active metabolite of irinotecan. We have recently demonstrated that plasma membrane vesicles prepared from those cells transported SN-38 in an ATP-dependent manner, and it was suggested that ABCG2 is involved in the active extrusion of SN-38 from cancer cells. In the present study, we have cloned the cDNA of ABCG2 from PC-6/SN2-5H human lung carcinoma cells, expressed ABCG2 in Sf9 insect cells, and characterized its function. Sequence analysis has revealed that the cloned ABCG2 has an arginine at the amino acid position 482, as does the wild type. Expression of the cloned ABCG2 in Sf9 cell membranes was detected by immunoblotting with the BXP-21 antibody. Contrary to our expectation, however, ATPase activity in the cell membranes expressing ABCG2 was stimulated by neither SN-38 nor rhodamine 123. It is suggested that there is a partner protein of ABCG2 required for heterodimer formation to exhibit transport activity toward SN-38.

Background: Little is known about the biological nature of T4 esophageal carcinoma growth signals and host defenses., Methods: Paraffin-embedded sections from 78 patients with T2 to T4 esophageal squamous cell carcinoma who underwent operation were analyzed with immunohistochemistry., Results: Positive cyclin A showed a significantly greater increase in T4 tumors than in those of other stages, and negative p27 showed a significantly greater decrease in T4 tumors than in large T3 stage tumors (tumor size > or = 4.0 cm). Patients with low-grade tumor-infiltrating lymphocyte (TIL) density showed a significantly greater decrease in T4 than in T2. The combination of p27 and cyclin A was a significant independent prognostic factor among T and N factors in multivariate analysis. TIL density was an independent prognostic factor among immunonutritional variables such as serum albumin concentration and the number of total blood lymphocytes., Conclusions: T4 esophageal squamous cell carcinoma has a poor prognosis, which is associated with increased p27-negative and cyclin A-positive growth signals in the tumor and with low TIL density in the host.

Secretin is known to stimulate the flow of bicarbonate-rich bile from the bile-duct epithelium, but has no effect on hepatocytes. To investigate the effects of secretin on bile production during intrahepatic cholestasis, we infused secretin into rats with taurocholate-induced cholestasis. Secretin was given at 0.25 and 0.50 units.min-1.kg-1 to Wistar rats that simultaneously received a continuous infusion of taurocholic acid at above its maximum hepatic transport capacity to produce cholestasis. When taurocholic acid was infused at doses of 1.4 and 1.6 mumol.min-1.100 g b.w.-1, bile volume decreased in control rats. In contrast, the simultaneous infusion of secretin significantly increased bile flow and the biliary excretion of bile acids and bicarbonate. The serum taurocholic acid level at the end of the experiment was significantly lower in the secretin-treated groups than in the control group. These findings indicate that secretin prevents taurocholate-induced cholestasis and may enhance the biliary excretion of bile acids.

When 0.25 mumol of hydrocortisone succinate sodium (HC) was administered to 15-day-old fertile eggs, almost all lenses of chick embryos treated with HC for 48 hr were classified as cataract stage IV-V (95%). A triple application of potassium pyrroloquinoline quinone (PQQ) (1.25 mumol/egg) at 3, 10 and 20 hr after HC treatment showed a preventive effect against the HC-induced cataract formation (I:45%, II:25%, III: 30%). PQQ also prevented the decline of GSH in the lens caused by HC. The decline of GSH in liver 24 hr after HC administration was prevented by PQQ. These data indicate that PQQ can modify HC-induced effects and that the preventive effect of PQQ against HC-induced decline of hepatic GSH seemed to influence HC-induced events in lens.