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A recent study showed that a cocktail of three small molecules, Y-27632, A83-01, and CHIR99021 (YAC), converts mature hepatocytes (MHs) into proliferative bipotent cells that can be induced into MHs and cholangiocytes in rats. However, when we reproduced these experiments, it was found that bipotent cells may be derived from resident liver progenitor cells (LPCs), whose proliferative activity was promoted by YAC. A simple and efficient sorting scheme was also developed in this study to harvest high-purity and high-yield LPCs. The inducible bipotency of purified LPCs was verified; in addition, they were found to spontaneously differentiate into hepatocytes and cholangiocytes due to changes in proliferative status even without induction. Moreover, during the differentiation process, some hepatocytes spontaneously reconverted to LPCs under certain conditions, such as the release of contact inhibition. These findings may improve our understanding of LPCs and provide a cell source for regenerative medicine., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The physiologically based pharmacokinetics (PBPK) model is a major mechanistic approach for predicting human pharmacokinetics (PK) using drug-specific and physiological parameters but has been difficult to use for human PK prediction with acceptable accuracy. Here, we report a newly developed PBPK approach that incorporates the mechanism of albumin-mediated membrane penetration in the liver and interspecies correlation for unbound tissue fractions. To verify the utility of our PBPK approach, we used 12 drugs that are mainly eliminated by hepatic metabolism to compare the prediction accuracy with a conventional PBPK approach and to observe human PK parameters. We found the predictive accuracy for total clearance (CL tot ), distribution volume at the steady state (V ss ), elimination half-life (t 1/2 ), and plasma concentration at the last measurable time point (C last ) of our PBPK approach to show better absolute average fold error and percentage within 2-fold error (1.6-1.8 and 67%-92%, respectively) compared with values obtained from the conventional PBPK approach (2.1-2.4 and 42%-67%, respectively). As our approach can use parameters obtained in early drug screening, it could help accelerate successful nomination of drug candidates by optimizing the pharmacokinetics of new chemical entities by directly using predicted human PK profiles., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Objectives: In this study, we aimed to examine the possible association between liver fibrosis and loss of skeletal muscle mass (SM) in community-dwelling older adults with no history of liver diseases., Methods: A total of 2,028 older adults (mean age, 69.8 ± 5.2 years) who had not received any treatment for liver diseases and had participated in a comprehensive health survey for community residents in Wakayama, Japan were included in this study. We carried out bioelectrical impedance analysis to estimate the SM of the whole body including the arms, legs, and trunk of the subjects. Liver fibrosis was evaluated by calculating the Fib4 index based on the subject's age, AST level, ALT level, and platelet counts., Results: The subjects were divided into three groups according to cutoff values of the Fib4 index (low: <1.30, medium: 1.30-2.66, high: ≥2.67). The SM index (kg/m 2 ) was the lowest among subjects in the high-Fib4-index category, followed by the medium- and low-Fib4-index categories. This dose-response reduction in the SM index was more pronounced among individuals with lower blood albumin level (low nutrition) and in those with more sedentary behavior (physical inactivity). Among the selected 262 subjects who underwent SM measurement twice with an interval of 3 years, the subjects with a high Fib4 index showed greater reduction in the SM index than those with medium and low Fib4 indices. Multiple regression analysis revealed that the Fib4 index was significantly associated with the SM index, independent of age, sex, albumin level, sedentary behavior, diabetes mellitus, alcohol intake, and smoking status., Conclusions: The present findings suggest that the potential progression of liver fibrosis is associated with the excessive loss of SM among apparently healthy older adults without any treatment for liver diseases.

Accurate prediction of human pharmacokinetics (PK) is important for the choice of promising compounds in humans. As the predictability of human PK by an empirical approach is low for drugs with species-specific PK, the utility of a physiologically based pharmacokinetic (PBPK) model was verified using 16 hepatically metabolized reference drugs. After the prediction method for total clearance (CL tot ) and distribution volume at steady state (Vd ss ) in the conventional PBPK model had been optimized, plasma concentrations following a single oral administration of each reference drug to healthy volunteers were simulated, and the prediction accuracy for human PK was compared between empirical approaches and the optimized PBPK model. In the drugs with low species-specific CL tot , there was little difference in predictability for maximum concentration (C max ), time to maximum plasma concentration (T max ), and area under the curve (AUC) (absolute average fold error: 1.3-2.4). In contrast, the optimized PBPK model predicted C max and AUC of the drugs with high species-specific CL tot with lower absolute average fold error (C max and AUC: 2.8 and 3.2, respectively) than those of the empirical approach (C max and AUC: 2.6-4.9 and 3.9-10.7, respectively). Therefore, the optimized PBPK model is useful for human PK prediction of drugs with species-specific CL tot ., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Hepatocellular carcinoma (HCC), the fifth most common cancer type and the third highest cause of cancer death worldwide, develops in different types of liver injuries, and is mostly associated with cirrhosis. However, non-alcoholic fatty liver disease often causes HCC with less fibrosis, and the number of patients with this disease is rapidly increasing. The high mortality rate and the pathological complexity of liver diseases and HCC require blood biomarkers that accurately reflect the state of liver damage and presence of HCC., Methods and Findings: Here we demonstrate that a circulating protein, apoptosis inhibitor of macrophage (AIM) may meet this requirement. A large-scale analysis of healthy individuals across a wide age range revealed a mean blood AIM of 4.99 ± 1.8 µg/ml in men and 6.06 ± 2.1 µg/ml in women. AIM levels were significantly augmented in the younger generation (20s-40s), particularly in women. Interestingly, AIM levels were markedly higher in patients with advanced liver damage, regardless of disease type, and correlated significantly with multiple parameters representing liver function. In mice, AIM levels increased in response to carbon tetrachloride, confirming that the high AIM observed in humans is the result of liver damage. In addition, carbon tetrachloride caused comparable states of liver damage in AIM-deficient and wild-type mice, indicating no influence of AIM levels on liver injury progression. Intriguingly, certain combinations of AIM indexes normalized to liver marker score significantly distinguished HCC patients from non-HCC patients and thus could be applicable for HCC diagnosis., Conclusion: AIM potently reveals both liver damage and HCC. Thus, our results may provide the basis for novel diagnostic strategies for this widespread and fatal disease.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) has RNA-dependent RNA polymerase (RdRp) activity. Because NS5B recognizes various RNA motifs besides the HCV genome, NS5B has the potential of interacting with host RNA molecules. In this study, an RNA pool enriched with the 3'-UTR sequences was generated and mRNA molecules with high affinity binding to NS5B were selected by iterative selection. Among the high binding mRNA 3'-UTR segments, we analyzed the housekeeping ribosomal protein S4, X-linked [RPS4X] mRNA 3'-UTR and the 3'-UTR of galectin-1 (GAL-1) mRNA, which is known to be one of the genes upregulated in HCV-infected liver cells and to have a wide spectrum of biological properties. By means of IP-RT-PCR, it was demonstrated that both of the mRNA molecules bind to NS5B in the cytoplasm. Interestingly, GAL-1 and RPS4X mRNA can serve as templates for NS5B RdRp, suggesting these RNA molecules are regulated in vivo by NS5B., (© 2013 Published by Elsevier Inc.)

Background and Aim: Low levels of serum adiponectin have been reported to be associated with obesity, diabetes, and non-alcoholic steatohepatitis (NASH), as well as several malignancies. Adiponectin knockout (KO) mice have been reported to cause insulin resistance and neointimal formation of the artery. We used adiponectin KO mice fed a high fat (HF) diet, and investigated the effect of adiponectin on the progression of steatohepatitis and carcinogenesis in vivo., Methods: Adiponectin KO mice and wild type (WT) mice were fed a HF diet or normal chow for the periods of 24 and 48 weeks. The HF diet contained 60% of calories from fat., Results: The adiponectin KO mice on the HF diet showed obesity, marked elevation of serum transaminase levels, and hyperlipidemia. At 24 weeks, hepatic expression of tumor necrosis factor-alpha and procollagen alpha (I) was higher in KO mice as compared with WT mice. At 48 weeks, liver triglyceride contents in KO mice on normal chow were significantly higher than those in WT mice. Hepatocyte ballooning, spotty necrosis, and pericellular fibrosis around central veins were observed in KO mice on the HF diet. The pericellular fibrosis was more severe in KO mice on the HF diet than that in WT mice (1.62% vs 1.16%, P = 0.033). Liver adenoma and hyperplastic nodules developed in a KO mouse on the HF diet at 48 weeks (12.5%, n = 1/8), whereas no tumor was detected in WT mice (n = 10)., Conclusions: Adiponectin may play a protective role in the progression of NASH in the early stages by suppressing tumor necrosis factor-alpha expression and liver fibrosis.

Adiponectin, one of the insulin-sensitizing adipokines, has been shown to activate fatty acid oxidation in liver and skeletal muscle, thus maintaining insulin sensitivity. However, the precise roles of adiponectin in fatty acid synthesis are poorly understood. Here we show that adiponectin administration acutely suppresses expression of sterol regulatory element-binding protein (SREBP) 1c, the master regulator which controls and upregulates the enzymes involved in fatty acid synthesis, in the liver of +Lepr(db)/+Lepr(db) (db/db) mouse as well as in cultured hepatocytes. We also show that adiponectin suppresses SREBP1c by AdipoR1, one of the functional receptors for adiponetin, and furthermore that suppressing either AMP-activated protein kinase (AMPK) via its upstream kinase LKB1 deletion cancels the negative effect of adiponectin on SREBP1c expression. These data show that adiponectin suppresses SREBP1c through the AdipoR1/LKB1/AMPK pathway, and suggest a possible role for adiponectin in the regulation of hepatic fatty acid synthesis.

Insulin receptor substrate (Irs) mediates metabolic actions of insulin. Here, we show that hepatic Irs1 and Irs2 function in a distinct manner in the regulation of glucose homeostasis. The PI3K activity associated with Irs2 began to increase during fasting, reached its peak immediately after refeeding, and decreased rapidly thereafter. By contrast, the PI3K activity associated with Irs1 began to increase a few hours after refeeding and reached its peak thereafter. The data indicate that Irs2 mainly functions during fasting and immediately after refeeding, and Irs1 functions primarily after refeeding. In fact, liver-specific Irs1-knockout mice failed to exhibit insulin resistance during fasting, but showed insulin resistance after refeeding; conversely, liver-specific Irs2-knockout mice displayed insulin resistance during fasting but not after refeeding. We propose the concept of the existence of a dynamic relay between Irs1 and Irs2 in hepatic insulin signaling during fasting and feeding.

Doxorubicin, a potent anticancer drug, is effective against a wide range of human neoplasms. However, the clinical uses of doxorubicin have been limited due to its serious cardiotoxic effects, which are likely the result of generation of free radicals and lipid peroxidation. S-Allylcysteine (SAC), an organosulfur compound purified from garlic, has been reported to have antioxidant and radical scavenging effects. Thus, we examined the effect of SAC on doxorubicin toxicity in mice. Severe doxorubicin toxicity was induced in mice by a single intraperitoneal injection (15 mg/kg body weight). SAC (30 mg/kg) was injected intraperitoneally daily for 5 days, starting two days prior to the administration of doxorubicin. Body weight was measured every alternate day. A measurement of serum creatine phosphokinase (CPK) and a histopathological analysis of the heart and liver was performed 6 days after the administration of doxorubicin. Death of any of the animals was recorded during the observation period. Doxorubicin injection induced a mortality rate of 58%, with SAC treatment reducing the doxorubicin-induced mortality rate to 30%. The severe body weight loss caused by doxorubicin (13%) was also significantly attenuated by SAC treatment (9%). Although an elevation of the level of serum CPK was observed following doxorubicin injection (5472 +/- 570 i.u./L), treatment with SAC significantly reduced the level of CPK (1923 +/- 635 i.u./L). Histological analysis demonstrated that heart and liver damage was significantly less severe in SAC treated mice than in mice receiving only doxorubicin. These results suggest that SAC research may ultimately lead to a resolution of the adverse effects of doxorubicin treatment in cancer chemotherapy.

To examine the effects of Sho-saiko-to extract on liver regeneration, Sho-saiko-to extract (0.75%, 1.5% or 3%) was administered to 70% partial hepatectomized rats with dimethylnitrosamine-induced liver-injury. S phase cell number, liver retinoid levels, hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-beta) levels in each intraorgan were measured as indicators of liver regeneration. Three to seven days after hepatectomy, HGF and TGF-beta levels of the liver and spleen of the Sho-saiko-to extract groups were significantly different from the levels of the ordinary food group (P < 0.05-0.1). HGF levels in the Sho-saiko-to extract groups were approximately 1.3-1.8 times higher in the liver and approximately 1.8-2.1 times higher in the spleen compared with the levels found in the ordinary food group. TGF-beta levels in the Sho-saiko-to extract groups were approximately 0.38-0.47 times the level in the liver and 0.58-0.77 times the level in the spleen of the ordinary food group. There was no difference in HGF and TGF-beta levels of the kidney and lung between the Sho-saiko-to extract group and the ordinary food group. There was a significant and positive correlation between HGF level and S phase cell number in the liver (r = 0.826, P < 0.01). There was a significant and negative correlation between TGF-beta level and the retinoid level in the liver (r = -0.696, P < 0.01). In addition, the levels of the active constituents of Sho-saiko-to extract (glycyrrhetic acid, baicalin and baicalein) showed high values in the liver and spleen of partial hepatectomized rats, and increased from the third day after partial hepatectomy. These results show that Sho-saiko-to extract induces liver regeneration by increasing the production of HGF and suppressing the production of TGF-beta in the liver and spleen of partial hepatectomized rats. It was considered that the increase in the Sho-saiko-to extract active constituent levels in the liver and spleen greatly influences this action.

We previously reported that a host cell glycoprotein, gp180, binds duck hepatitis B virus particles, and is encoded by a member of the carboxypeptidase gene family (Kuroki, K., Eng, F., Ishikawa, T., Turck, C., Harada, F., Ganem, D., 1995. gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family. J. Biol. Chem. 270, 15022-15028). After that report, carboxypeptidase D (CPD) was subsequently purified from bovine pituitary and characterized as a novel carboxypeptidase E (CPE)-like enzyme, with many characteristics in common with duck gp180 (Song, L., Fricker, L.D., 1995. Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. J. Biol. Chem. 270, 25007-25013). CPD is now supposed to play an important role in a secretory pathway. To clarify the function of gp180 further, we have isolated and analyzed human and mouse homologues of duck gp180. cDNA clones derived from human HepG2 cells and mouse livers have been isolated on the basis of homology to the duck gp180. The suggested open reading frames of the human and mouse cDNA encode 1380 and 1377 amino acid proteins, respectively and have three carboxypeptidase homologous domains (A, B, and C). Domains A and B have completely conserved the residues known to have the enzymatic activity of carboxypeptidase, but domain C in each cDNA does not. Northern blotting revealed a ubiquitous tissue distribution of human gp180 mRNA with several transcript species. Expression of human gp180 cDNA in transfected 293Tcells exhibited carboxypeptidase activity upon radiometric assay. The human and mouse homologues of duck gp180 have many characteristics in common with bovine CPD. Fluorescence in-situ hybridization reveals that the gene encoding human gp180 is located in region 17q11.2.

In the present studies, we analyzed the effects of acute endotoxemia on hepatocyte nitric oxide production and functional activity. Treatment of rats with 5 mg/kg of lipopolysaccharide (LPS), which induces acute endotoxemia, caused an increase in nitric oxide production in the liver, as measured by electron paramagnetic spin trapping, which was evident within 6 hours. This was associated with expression of inducible nitric oxide synthase (iNOS) messenger (m) RNA in hepatocytes and in sinusoidal cells throughout the liver lobule. Acute endotoxemia also caused alterations in hepatic structure, including hypertrophy, vacuolization, and chromosomal emargination, however these changes were not apparent for 24 to 48 hours. Hepatocytes isolated from endotoxemic rats released increased amounts of nitric oxide, measured by nitrite production, in response to interferon gamma (gamma-IFN) alone or in combination with LPS, tumor necrosis factor alpha, macrophage-colony stimulating factor, granulocyte/macrophage-colony stimulating factor, or hepatocyte growth factor. These results show that hepatocytes are sensitized by acute endotoxemia to respond to inflammatory mediators and growth factors. Increased nitrite production by hepatocytes was due to increased expression of iNOS mRNA and protein and was correlated with the time following induction of acute endotoxemia. Thus, cells isolated 48 hours after induction of acute endotoxemia released significantly more nitrite than cells recovered after 6 hours, a response that was not due to alterations in hepatocyte viability. Hepatocytes isolated from endotoxemic rats also exhibited a marked increase in proliferative capacity when compared with cells from control rats. Nitric oxide production by hepatocytes in vitro was associated with inhibition of cell growth and protein synthesis, which was reversed by the nitric oxide synthase inhibitor, NG-monomethyl-l-arginine (L-NMMA). Agarose gel electrophoresis showed extensive cytoplasmic DNA fragmentation in hepatocytes treated with LPS and gamma-IFN, a characteristic of apoptosis, which was also reversed by L-NMMA. These results, together with our findings that treatment of rats with an inhibitor of nitric oxide synthase partially reversed the structural alterations in the liver associated with acute endotoxemia suggest that nitric oxide may contribute to the pathophysiologic response to this bacterially derived toxin.

Fusion of Golgi membranes isolated from rabbit liver with liposomes was studied by lipid mixing of fluorescent lipid analogues and internal content mixing and by electron microscopic observation of transfer of horseradish peroxidase from liposomes into Golgi membranes. A monoclonal antibody was used to confirm fusion of Golgi membranes but not other contaminating vesicles. Fusion was rapid and efficient, reaching about 20% of the maximum after a 5-min incubation using small or large unilamellar dioleoylphosphatidylcholine vesicles. The fusion was dependent on temperature, decreasing at lower temperatures, and becoming nearly zero below 10 degrees C. The addition of ATP, GTP, cytosolic factors, or N-ethylmaleimide did not affect fusion. Treatments of Golgi membranes with 0.1 M Na2CO3 or 1 M KCl did not cause any changes in fusion. However, treatment with proteases inhibited fusion. These results suggest that Golgi integral membrane protein(s) are involved in fusion. Changing the medium to an isoosmotic substance, sucrose, in place of KCl or NaCl inhibited fusion. The binding assay of fluorescent liposomes to Golgi membranes showed that lowering the temperature or replacing salts with sucrose did not affect binding. However, treatment of Golgi membranes with proteases inhibited binding. Addition of phosphatidylserine or phosphatidylethanolamine to dioleoylphosphatidylcholine liposomes caused a 2-fold increase in binding and fusion. Fusion between Golgi membranes by themselves did not occur. These results provide some information on the mechanism of intracellular vesicular transport.

We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol-stabilized bovine-brain microtubules without microtubule-associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential-interference-contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine-labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient. The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N-ethylmaleimide, brefeldin A, or GTP-gamma-S. The association is mediated by trypsin-sensitive peripheral protein(s) on the Golgi membranes.

An inhibitor of protein kinases, staurosporine (ssp), was found to affect the endocytic pathway of asialoglycoproteins subsequent to endocytosis in monolayer cultures of rat hepatocytes. The effect of 5 or 10 microM staurosporine on the internalization of a synthetic ligand (galBSA-HRP: bovine serum albumin exposing galactose, horseradish peroxidase conjugates) prebound to the cell surface was minimal. The presence of 5, 7, or 10 microM ssp during a 1-h chase period resulted in the ligand remaining in a low density (1.04-1.05 g/ml), nonlysosomal subcellular fraction in a Percoll gradient. The ligand, arrested by 7 microM ssp, was further processed to the lysosome during subsequent incubation in the absence of ssp. Cells maintained the ability to internalize ligand at 37 degrees C for 1 h in the presence of these concentrations of ssp. During a 1-h continuous uptake of 0-50 micrograms/ml nonlabeled ligand, the presence of 7 microM ssp did not cause any decrease in the amount of asialoglycoprotein receptor at the cell surface, which indicates receptor recycling occurred normally. These results suggest a possible involvement of protein kinase(s), which can be inhibited by ssp, in the delivery of endocytosed ligand to the lysosome, but not in ligand endocytosis and receptor recycling.

A new cell line derived from a woodchuck hepatitis surface antigen-positive woodchuck hepatocellular carcinoma has been established and named T3-HEP-W1. This new cell line was established directly from a primary woodchuck hepatocellular carcinoma. Adaptation of the cells to the in vitro culture condition was completed after 3 months, with the doubling time of 24 hr. The morphologic features of the cell by light microscopy were of an epithelial type. The modal chromosome number was 100. Ornithine and tyrosine aminotransferase activities were detected. Production of albumin was negative. Integration of woodchuck hepatitis virus DNA was demonstrated by Southern blot analysis, although the secretion of woodchuck hepatitis surface antigen was not detected. T3-HEP-W1 is quite different from the previously reported WH257GE10 cell line and provides another in vitro model for the study of human hepatocellular carcinoma related to hepatitis B virus.

Both androgen and estrogen receptors were studied in human hepatocellular carcinoma and noncancerous liver tissue surrounding it. Androgen receptor was detected in the cytosol and/or nucleosol of 4 of 8 cancerous tissues and 1 of 6 noncancerous tissues. The levels of androgen receptor in hepatocellular carcinomas ranged from 3.4 to 37.6 fmoles per mg protein with dissociation constants (Kd) of 0.226 - 51.3 X 10(-9) M. That in the surrounding noncancerous tissue was 2.1 fmoles per mg protein with Kd of 0.941 X 10(-9) M. Estrogen receptor was detected in the cytosol of 1 of 7 cancerous tissues, while it was detected in the cytosol and/or nucleosol of 3 of 7 noncancerous tissues. The level of estrogen receptor in hepatocellular carcinoma was 4.9 fmoles per mg protein with Kd of 1.20 X 10(-9) M, and those in the surrounding noncancerous tissues ranged from 2.6 to 1,073 fmoles per mg protein with Kd of 0.223 - 3.15 X 10(-9) M. The results suggest that the expression of androgen receptor may be augmented in association with malignant transformation of hepatocytes while the expression of estrogen receptor may be rather suppressed and that some of hepatocellular carcinomas may be androgen-dependent.

Acute-phase response of mRNAs for serum amyloid P component (SAP), C-reactive protein (CRP) and prealbumin was examined in C57BL/6 mouse liver by hybridization to specific cDNA probes. Although the level of SAP mRNAs in the unstimulated mouse was about one-tenth of that of CRP mRNAs, it increased up to 60-fold during the first 20 hr, and returned gradually to the original level at 69 hr after the administration of Escherichia coli lipopolysaccharide. On the other hand, the level of CRP mRNA rapidly increased up to 6-fold during the first 4 hr, and reverted to the original level as early as at 20 hr. In contrast, the level of mRNA for prealbumin decreased to about 0.5-fold during the first 20 hr, recovered and increased up to 1.6-fold of the original level during 32 to 69 hr.

Effect of bilayer membrane curvature of substrate phosphatidylcholine and inhibitor phosphatidylserine on the activity of phosphatidylcholine exchange protein has been studied by measuring transfer of spin-labeled phosphatidylcholine between vesicles, vesicles and liposomes, and between liposomes. The transfer rate between vesicles was more than 100 times larger than that between vesicles and liposomes. The transfer rate between liposomes was still smaller than that between vesicles and liposomes and nearly the same as that in the absence of exchange protein. The markedly enhanced exchange with vesicles was ascribed to the asymmetric packing of phospholipid molecules in the outer layer of the highly curved bilayer membrane. The inhibitory effect of phosphatidylserine was also greatly dependent on the membrane curvature. The vesicles with diameter of 17 nm showed more than 20 times larger inhibitory activity than those with diameter of 22 nm. The inhibitory effect of liposomes was very small. The size dependence was ascribed to stronger binding of the exchange protein to membranes with higher curvatures. The protein-mediated transfer from vesicles to spiculated erythrocyte ghosts was about four times faster than that to cup-shaped ghosts. This was ascribed to enhanced transfer to the highly curved spiculated membrane sites rather than greater mobility of phosphatidylcholine in the spiculated ghost membrane.