Prebiotic effect of poly-D-3-hydroxybutyrate prevents dyslipidemia in obese mice.

Obesity is a global health problem caused by genetic, environmental, and psychological factors and is associated with various health disorders. As such, there is a growing focus on the prevention of obesity and related diseases. The gut microbiota plays a crucial role in these diseases and has become a therapeutic target. Prebiotics, such as poly-d-3-hydroxybutyric acid (PHB), have gained attention for their potential to alter the gut microbiota, promote beneficial bacterial growth, and alleviate obesity. In this study, we examined the prebiotic effects of PHB in obese mice. We found that, in C57BL/6N mice, PHB reduced blood lipid levels. Analysis of the intestinal microflora also revealed an increase in short-chain fatty acid-producing bacteria. When PHB was administered to obese mice, subcutaneous fat and dyslipidemia were reduced, and the number of beneficial bacteria in the intestinal microflora increased. Furthermore, fatty degradation and oxidative stress were suppressed in the liver. PHB regulates gut bacterial changes related to obesity and effectively inhibits dyslipidemia, suggesting that it could be a prebiotic agent for curing various obesity-related diseases. In summary, PHB increases the beneficial gut microbiota, leading to an alleviation of obesity-associated dyslipidemia.

The novel sustained 3-hydroxybutyrate donor poly-D-3-hydroxybutyric acid prevents inflammatory bowel disease through upregulation of regulatory T-cells.

Inflammatory bowel disease (IBD) is a chronic persistent intestinal disorder, with ulcerative colitis and Crohn's disease being the most common. However, the physio-pathological development of IBD is still unknown. Therefore, research on the etiology and treatment of IBD has been conducted using a variety of approaches. Short-chain fatty acids such as 3-hydroxybutyrate (3-HB) are known to have various physiological activities. In particular, the production of 3-HB by the intestinal microflora is associated with the suppression of various inflammatory diseases. In this study, we investigated whether poly-D-3-hydroxybutyric acid (PHB), a polyester of 3-HB, is degraded by intestinal microbiota and works as a slow-release agent of 3-HB. Further, we examined whether PHB suppresses the pathogenesis of IBD models. As long as a PHB diet increased 3-HB concentrations in the feces and blood, PHB suppressed weight loss and histological inflammation in a dextran sulfate sodium-induced IBD model. Furthermore, PHB increased the accumulation of regulatory T cells in the rectum without affecting T cells in the spleen. These results indicate that PHB has potential applications in treating diseases related to the intestinal microbiota as a sustained 3-HB donor. We show for the first time that biodegradable polyester exhibits intestinal bacteria-mediated bioactivity toward IBD. The use of bioplastics, which are essential materials for sustainable social development, represents a novel approach to diseases related to dysbiosis, including IBD.

Combination of pathological, biochemical and behavioral evaluations for peripheral neurotoxicity assessment in isoniazid-treated rats.

In drug development, assessment of non-clinical peripheral neurotoxicity is important to ensure human safety. Clarifying the pathological features and mechanisms of toxicity enables the management of safety risks in humans by estimating the degree of risk and proposing monitoring strategies. Published guidelines for peripheral neurotoxicity assessment do not provide detailed information on which endpoints should be monitored preferentially and how the results should be integrated and discussed. To identify an optimal assessment method for the characterization of peripheral neurotoxicity, we conducted pathological, biochemical (biomaterials contributing to mechanistic considerations and biomarkers), and behavioral evaluations of isoniazid-treated rats. We found a discrepancy between the days on which marked pathological changes were noted and those on which biochemical and behavioral changes were noted, suggesting the importance of combining these evaluations. Although pathological evaluation is essential for pathological characterization, the results of biochemical and behavioral assessments at the same time points as the pathological evaluation are also important for discussion. In this study, since the measurement of serum neurofilament light chain could detect changes earlier than pathological examination, it could be useful as a biomarker for peripheral neurotoxicity. Moreover, examination of semi-thin specimens and choline acetyltransferase immunostaining were useful for characterizing morphological neurotoxicity, and image analysis of semi-thin specimens enabled us to objectively show the pathological features.

Effects of low concentrations of ozone gas exposure on percutaneous oxygen saturation and inflammatory responses in a mouse model of Dermatophagoides farinae-induced asthma.

Ozone gas is widely used in hospitals as well as homes to control COVID-19 infection owing to its cost-effectiveness. Safety standard value and the tolerable value of ozone gas are set at 0.05 ppm and 0.1 ppm, respectively, in developed countries; however, this value was principally determined for healthy individuals, and the risks associated with ozone gas inhalation in patients with pulmonary diseases remains unknown. Recently, we demonstrated that 0.1 ppm ozone gas exposure significantly aggravates the symptoms of acute lung injury in mice. In the present study, we further examined the influence of ≤ 0.1 ppm ozone gas exposure on percutaneous oxygen saturation (SpO2) and pro-inflammatory responses in a mouse model of asthma. Female BALB/c mice were subjected to repetitive intranasal sensitization of Dermatophagoides farinae to generate a mouse model of asthma. Inhalation exposure of ozone gas (0.1, 0.03, 0.01 ppm), generated using an ultraviolet lamp, was performed for five consecutive days immediately before the final sacrifice. There were no abnormal findings in control mice exposed to 0.1 ppm ozone; however, 0.1 ppm ozone exposure significantly reduced the SpO2 level in asthmatic mice. Histological evaluation and gene expression analysis revealed that pro-inflammatory cytokine levels were significantly increased in mice exposed to 0.1 ppm ozone, indicating that 0.1 ppm ozone exposure affects the development of asthma symptoms. Notably, 0.03 and 0.01 ppm ozone exposure did not have any effects even in asthmatic mice. Our findings indicate that the tolerable level of ozone gas should be adjusted for individuals based on a history of respiratory disorders.

Diversity of mutations in the dystrophin gene and details of muscular lesions in porcine dystrophinopathies.

During meat inspections in pigs, dystrophinopathies are among the muscle lesions targeted for disposal. In this study, the authors examined the lesions and the distribution of dystrophin expression in 25 pigs with dystrophinopathy. In addition, complementary deoxyribonucleic acid (cDNA) sequencing and western blotting were performed in 6 of the 25 cases, all of which were characterized by degeneration, necrosis, and fat replacement of muscle fibers. Comparing the results of immunohistochemistry with anti-dystrophin antibodies that recognized at different sites in the protein, the authors noted that the loss of dystrophin expression was most pronounced in the C-terminus-recognizing antibody (19/25 cases). The authors detected 5 missense mutations and 3 types of shortened transcripts generated by the skipping of exons in the cDNA, which were associated with the pathogenesis. One missense mutation had been reported previously, whereas the remaining mutations identified had not been previously documented in pigs. In the cases with shortened transcripts, normal-sized transcripts were detected together with the defective transcripts, suggesting that these mutations were caused by splicing abnormalities. In addition, they were in-frame mutations, all of which have similar pathogeneses of Becker muscular dystrophy in humans. These cases were 6 months of age and exhibited macroscopic discoloration, fatty replacement, and muscle degeneration, suggesting that the effect of these mutations on skeletal muscle was significant.

Glucosylceramide in T cells regulates the pathology of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease in the colon characterized by excessive activation of T cells. Glycosphingolipids (GSLs) are composed of lipid rafts in cellular membranes, and their content is linked to immune cell function. In the present study, we investigated the involvement of GSLs in IBD. Microarray data showed that in IBD patients, the expression of only UDP-glucose ceramide glucosyltransferase (UGCG) decreased among the GSLs synthases. Ad libitum access to dextran sulfate sodium (DSS) resulted in decreased UGCG and glucosylceramide (GlcCer) content in mesenteric lymph nodes and T cells from the spleen. Furthermore, the knockdown of Ugcg in T cells exacerbated the pathogenesis of colitis, which was accompanied by a decrease in Treg levels. Treatment with GlcCer nanoparticles prevented DSS-induced colitis. These results suggested that GlcCer in T cells is involved in the pathogenesis of IBD. Furthermore, GlcCer nanoparticles are a potential efficacious therapeutic target for IBD patients.

Isolation and whole-genome sequencing of a novel aviadenovirus from owls in Japan.

Adenoviruses have been reported to infect a variety of birds. Here, we isolated a novel adenovirus from the liver of a dead owl chick (Bengal eagle owl; Bubo bengalensis) at a raptor-breeding facility in Japan and determined the complete genome sequence of the virus. We performed necropsies on the dead owl chicks and found that they had enlarged livers, pericardial edema, and focal necrosis of the liver tissue. Transmission electron microscopy of the liver tissue revealed a virus-like structure, appearing as paracrystalline arrays in the nucleus, and immunohistochemical staining with anti-adenovirus antibodies showed positive reactions in hepatocytes and other cells. Attempts to isolate the virus from homogenized liver tissue of a dead owl chick showed a cytopathic effect on chicken-derived cultured cells after multiple blind passages. Further, we determined the complete genome sequence of this virus and performed phylogenetic analysis, revealing that this adenovirus belongs to the genus Aviadenovirus, forming a cluster with fowl and turkey aviadenoviruses. The amino acid sequence divergence between the DNA polymerase of this virus and its closest known adenovirus relative is approximately 29%, implying that this virus can be assigned to a new species in the genus Aviadenovirus. Based on our data, this novel owl adenovirus is a likely cause of fatal infections in owls, which may threaten wild and captive owl populations. Further, this virus is unique among raptor adenoviruses in that it infects chicken-derived cultured cells, raising the importance of further investigations to evaluate interspecies transmission of this virus.

Genetic diversity, reassortment, and recombination of mammalian orthoreoviruses from Japanese porcine fecal samples.

Mammalian orthoreoviruses (MRVs) are non-enveloped double-stranded RNA viruses with a broad host range. MRVs are prevalent worldwide, and in Japan, they have been isolated from various hosts, including humans, dogs, cats, wild boars, and pigs, and they have also been found in sewage. However, Japanese porcine MRVs have not been genetically characterized. While investigating porcine enteric viruses including MRV, five MRVs were isolated from the feces of Japanese pigs using MA104 cell culture. Genetic analysis of the S1 gene revealed that the Japanese porcine MRV isolates could be classified as MRV-2 and MRV-3. Whole genome analysis showed that Japanese porcine MRVs exhibited genetic diversity, although they shared sequence similarity with porcine MRV sequences in the DDBJ/EMBL/GenBank database. Several potential intragenetic reassortment events were detected among MRV strains from pigs, sewage, and humans in Japan, suggesting zoonotic transmission. Furthermore, homologous recombination events were identified in the M1 and S1 genes of Japanese porcine MRV. These findings imply that different strains of Japanese porcine MRV share a porcine MRV genomic backbone and have evolved through intragenetic reassortment and homologous recombination events.

Macroglossia in a pig diagnosed as Becker muscular dystrophy due to dystrophin pseudoexon insertion derived from intron 26.

We report a case of Becker muscular dystrophy in a 6-month-old, mixed-breed, castrated male pig detected with macroglossia at a meat inspection center. The pig presented a severely enlarged tongue extending outside its mouth. The tongue was firm and pale with discolored muscles. Histologically, there was severe fibrosis, fatty replacement, and myofiber necrosis, degeneration, and regeneration. Immunofluorescence showed focal and severely weak labeling for dystrophin at the sarcolemma of myocytes in the tongue. Analysis of dystrophin mRNA showed a 62 base pair insertion between exons 26 and 27. The insertion was derived from intron 26. Based on these findings, we diagnosed the case as Becker muscular dystrophy-the first known muscular dystrophy case induced by pseudoexon insertion in animals.

Endothelial ganglioside GM3 regulates angiogenesis in solid tumors.

Cancer cells require oxygen and nutrients for growth, making angiogenesis one of the essential components of tumor growth. Gangliosides, constituting membrane lipid rafts, regulate intracellular signal transduction and are involved in the malignancy of cancer cells. While endothelial cells, as well as cancer cells, express vast amounts of gangliosides, the precise function of endothelial gangliosides in angiogenesis remains unclear. In this study, we focused on gangliosides of vascular endothelial cells and analyzed their functions on tumor angiogenesis. In human breast cancer, GM3 synthase was highly expressed in vascular endothelial cells as well as immune cells. Angiogenesis increased in GM3S-KO mice. In BAEC, RNA interference of GM3S showed increased cellular invasion and oxidative stress tolerance through activation of ERK. In the breast cancer model, GM3-KO mice showed an increase in tumor growth and angiogenesis. These results suggest that the endothelial ganglioside GM3 regulates tumor angiogenesis by suppressing cellular invasion and oxidative stress tolerance in endothelial cells.

Lipid-soluble polyphenols from sweet potato exert antitumor activity and enhance chemosensitivity in breast cancer.

Polyphenols are abundant in vegetables and fruit. They have been shown to have various antitumor, antioxidant, and anti-inflammatory effects. Here, we extracted the lipid-soluble fraction of polyphenols from fermented sweet potato (Ipomoea batatas). These lipid-soluble polyphenols mainly contained caffeic acid derivatives with strong antioxidant ability, which we hypothesized to affect diseases for which oxidative stress is a factor, such as cancer. We therefore investigated the antitumor and chemo-sensitizing effects of lipid-soluble polyphenols on E0771 murine breast cancer cells. The lipid-soluble polyphenols accumulated in the cells' cytoplasm due to its high lipophilicity, and reduced reactive oxygen species through its strong antioxidant activity. The lipid-soluble polyphenols also arrested the cell cycle at G0/G1 by suppressing Akt activity, and enhanced the cytotoxicity of anticancer agents. In this model, lipid-soluble polyphenols inhibited tumor growth and enhanced the efficacy of chemotherapy drugs. These results suggest the potential of lipid-soluble polyphenols as a functional food to support cancer therapy.

IgE reactivity to fish allergens from Pacific cod (Gadus macrocephalus) in atopic dogs.

BACKGROUND: IgE reactivity to fish allergens in atopic dogs, which are used as models for food allergy, has not been elucidated to date. We investigated IgE reactivity to crude extracts and purified allergens derived from the Pacific cod (Gadus macrocephalus) in atopic dogs to identify the allergenic proteins of cod. RESULTS: The levels of specific IgE to crude cod extracts were measured in the sera of 179 atopic dogs, including 27 dogs with cod allergy, using enzyme-linked immunosorbent assay (ELISA). Specific IgE to crude cod extracts were present in 36 (20%) of the 179 atopic dogs and in 12 (44%) of the 27 dogs with cod allergy. The allergens in crude cod extracts were analyzed by ELISA, immunoblotting, and liquid chromatography-tandem mass spectrometry. In allergen component analysis, IgE reactivity to tropomyosin and enolase was observed in the sera of dogs with cod allergy. IgE reactivity to parvalbumin, collagen, and tropomyosin was evaluated using the sera of atopic dogs that tested positive for specific IgE to crude cod extracts. Among the 36 dogs with IgE reactivity to crude cod extracts, 9 (25%), 14 (39%), and 18 (50%) dogs tested positive for specific IgE to parvalbumin, collagen, and tropomyosin, respectively. CONCLUSIONS: The IgE reactivity to cod allergens observed in dogs was similar to that in humans, and this finding further supports the use of atopic dogs with fish allergy as a model for fish allergy in humans.

Biology/Disease-Driven Initiative on Protein-Aggregation Diseases of the Human Proteome Project: Goals and Progress to Date.

The Biology/Disease-driven (B/D) working groups of the Human Proteome Project are alliances of research groups aimed at developing or improving proteomic tools to support specific biological or disease-related research areas. Here, we describe the activities and progress to date of the B/D working group focused on protein aggregation diseases (PADs). PADs are characterized by the intra- or extracellular accumulation of aggregated proteins and include devastating diseases such as Parkinson's and Alzheimer's disease and systemic amyloidosis. The PAD B/D working group aims for the development of proteomic assays for the quantification of aggregation-prone proteins involved in PADs to support basic and clinical research on PADs. Because the proteins in PADs undergo aberrant conformational changes, a goal is to quantitatively resolve altered protein structures and aggregation states in complex biological specimens. We have developed protein-extraction protocols and a set of mass spectrometric (MS) methods that enable the detection and quantification of proteins involved in the systemic and localized amyloidosis and the probing of aberrant protein conformational transitions in cell and tissue extracts. In several studies, we have demonstrated the potential of MS-based proteomics approaches for specific and sensitive clinical diagnoses and for the subtyping of PADs. The developed methods have been detailed in both protocol papers and manuscripts describing applications to facilitate implementation by nonspecialized laboratories, and assay coordinates are shared through public repositories and databases. Clinicians actively involved in the PAD working group support the transfer to clinical practice of the developed methods, such as assays to quantify specific disease-related proteins and their fragments in biofluids and multiplexed MS-based methods for the diagnosis and typing of systemic amyloidosis. We believe that the increasing availability of tools to precisely measure proteins involved in PADs will positively impact research on the molecular bases of these diseases and support early disease diagnosis and a more-confident subtyping.

Relationship Between Immunoglobulin Deposition and Early Lesions of Progressive Glomerulonephropathy in Young Common Marmosets.

The authors previously investigated progressive glomerulonephropathy in 2- to 11-year-old common marmosets and characterized age-related changes of the renal glomeruli and development of tubulointerstitial lesions. In this study, immunoglobulin deposition and ultrastructural changes of the glomeruli were investigated in 5 young marmosets from 6 months to 3 years of age with pre-onset or early glomerulonephropathy. In all animals, the foot processes of podocytes were effaced, and IgM was deposited into the glomeruli. In glomeruli without glomerular basement membrane (GBM) alteration, IgM was the only immunoglobulin type deposited in the glomeruli. In cases with more advanced lesions of reticulation and thickening of GBM, IgA and IgG deposits were also observed. Therefore, the authors conclude that IgM may be the primary or earliest immunoglobulin deposited in this nephropathy, whereas IgA and IgG deposition may be connected to the progression of the glomerular lesions. IgM deposition and foot process effacement of podocytes occur early in the life of affected marmosets.

Spontaneous, Experimentally Induced, and Transmissible AA Amyloidosis in Japanese Quail ( Coturnix japonica).

The authors describe a spontaneous case of amyloid A (AA) amyloidosis in an adult female Japanese quail ( Coturnix japonica). The bird developed AA amyloidosis secondary to chronic peritonitis caused by a Gram-negative bacillus infection. Mild amyloid deposition was also identified in the intestinal tract of apparently healthy adult individuals, suggesting that quail may develop intestinal amyloidosis with age. Based on these observations, it was hypothesized that quail can develop AA amyloidosis following inflammatory stimulation with lipopolysaccharide (LPS). Therefore, adult quail were repeatedly injected with LPS and the development of AA amyloidosis was confirmed. The amyloid deposition in this model increased when quail amyloid was intravenously injected as an amyloid-enhancing factor. The experiments were repeated with young quail, but amyloid deposits were not observed following LPS injections. However, AA amyloidosis did develop when quail amyloid was injected in addition to LPS. These results indicated that adult quail develop AA amyloidosis after inflammatory stimulation with LPS. Furthermore, quail AA amyloidosis was shown to have transmissibility regardless of age. Interestingly, the authors found that administration of chicken amyloid fibrils also induced AA amyloidosis in young quail. This is the first report of cross-species transmission of avian AA amyloidosis.

The development of mixed cryoglobulinemia in Capillaria hepatica-infected mice is associated with the capillaria antigen-induced selective proliferation of splenic B-1a cells in response to interleukin-5 stimulation.

Chronic infection by pathogens such as hepatitis C virus induces monoclonal or oligoclonal proliferation of B cells, which produce IgM rheumatoid factor, leading to the development of mixed cryoglobulinemia (MC). Antigen-driven lymphoproliferation is essential to the onset of MC; however, the underlying mechanism is largely unknown. Herein, we show that type II MC is induced by Capillaria hepatica infection through a mechanism in which splenic B-1a cells reacting to C. hepatica-specific antigen selectively proliferate, producing IgM rheumatoid factor under co-stimulation of the specific worm antigen and IL-5. In vitro assays using B-1a cells from infected mice showed that stimulation by C. hepatica soluble fraction promoted the proliferation of B-1a cells and the secretion of IgM, which reacted with the 75-kDa antigen in the soluble fraction. The severity of MC was correlated with the increase in serum IL-5 levels in the infected mice. Furthermore, i.p. injection of the soluble worm fraction caused MC without an inflammatory response in IL-5 transgenic mice, indicating that IL-5 is critical for the development of MC. These results indicate that the selective proliferation of IgM rheumatoid factor-secreting B-1a cells is induced by co-stimulation by the specific pathogen antigen and IL-5 in the development of MC in C. hepatica-infected mice.

SPONTANEOUS LIPOPROTEIN GLOMERULOPATHY-LIKE NEPHROPATHY IN A SQUIRREL (SCIURUS VULGARIS).

Lipoprotein glomerulopathy (LPG) is a rare human glomerular disease caused by abnormal lipid metabolism. Naturally occurring LPG has not been reported in animals. We describe the histopathological characterization of spontaneous LPG-like nephropathy in a captive squirrel ( Sciurus vulgaris ). Macroscopically, swollen glomeruli were distinctively identified as fine white granules in the renal cortex. Histologically, most glomeruli were markedly enlarged with distended capillaries containing faintly eosinophilic and amorphous materials. The amorphous material was negative using the periodic acid-Schiff reaction, periodic acid-methenamine silver stain, or Masson's trichrome stain. Sudan III staining revealed lipid in the materials, and immunohistochemistry demonstrated that the material additionally contained apolipoprotein E. Electron microscopy showed numerous lipid granules and vacuoles of various sizes in the capillary lumina associated with foot process effacement of podocytes. These pathological characteristics bear some resemblance to those of human LPG.

Quantitative Targeted Proteomics of Pancreatic Cancer: Deoxycytidine Kinase Protein Level Correlates to Progression-Free Survival of Patients Receiving Gemcitabine Treatment.

The purpose of the present study is to identify the determinant(s) of gemcitabine (dFdC)-sensitivity in pancreatic cancer tissues of patients treated with dFdC alone and in pancreatic cancer cell lines exposed to dFdC in vitro. Protein expression levels of 12 enzymes and 13 transporters potentially involved in transport and metabolism of dFdC in pancreatic cancer cell lines and tissues were quantified by means of our LC-MS/MS-based quantitative targeted proteomics technology. Protein expression levels of deoxycytidine kinase (dCK), uridine monophosphate-cytidine monophosphate (UMP-CMP) kinase, cytosolic nucleotidase III (cN-III), and equilibrative nucleoside transporter 1 (ENT1) were significantly correlated with IC50 or 1/IC50 in five cell lines with different sensitivities to dFdC (p < 0.05). Expression levels of the selected proteins in pancreatic cancer tissues of 10 patients with different progression-free survival (PFS) (49-955 days) were quantified, and their relationship with PFS was examined. Only the protein expression level of dCK was significantly correlated with PFS (p < 0.05). Multiple regression analysis was also performed, and combinations of ENT1, UMP-CMP kinase, CTPS1, and dCK were highly correlated with PFS. Our results indicate that the protein expression level of dCK in pancreatic cancer tissue is a good predictor of PFS, and thus dCK may be the best biomarker of dFdC sensitivity in pancreatic cancer patients treated with dFdC, although other proteins would also contribute to dFdC-sensitivity at the cellular level in vivo and in vitro.

Clinical and histopathological features resembling those of human focal segmental glomerulosclerosis in a cat with nonimmune-mediated glomerulonephropathy.

BACKGROUND: Nonimmune-mediated glomerulonephropathies are rarely reported in domestic animals with the exception of amyloidosis. Here we describe the pathological features and clinical course of a feline with protein-losing nonimmune-mediated glomerulonephropathy characterized by segmental glomerulosclerosis and severe podocyte injury. CASE PRESENTATION: A castrated male Japanese domestic cat aged 3 years and 8 months had hypertension, persistent proteinuria, and azotemia. Microscopic examination of a renal biopsy revealed many glomeruli with adhesion to the Bowman's capsule and segmental sclerosis. The most characteristic ultrastructural glomerular feature was severe podocyte foot process effacement. No electron-dense deposits were observed. Immunofluorescence revealed no immune deposits, but abnormal expression of nephrin and podocin was detected in the glomeruli. These findings resemble those of human focal segmental glomerulosclerosis. The cat temporarily responded to treatment with angiotensin-converting enzyme inhibitors and prednisolone administration but died of progressive renal failure 32 months after biopsy. CONCLUSIONS: The cat was diagnosed with nonimmune mediated glomerulonephropathy because of the absence of immune deposits and severe podocyte injury. To our knowledge, this is the first report of nonimmune-mediated glomerulonephropathy in a cat resembling human focal segmental glomerulosclerosis.

Validation of uPA/SCID mouse with humanized liver as a human liver model: protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases by LC-MS/MS.

Chimeric mice with humanized liver (PXB mice) have been generated by transplantation of urokinase-type plasminogen activator/severe combined immunodeficiency mice with human hepatocytes. The purpose of the present study was to clarify the protein expression levels of metabolizing enzymes and transporters in humanized liver of PXB mice transplanted with hepatocytes from three different donors, and to compare their protein expressions with those of human livers to validate this human liver model. The protein expression levels of metabolizing enzymes and transporters were quantified in microsomal fraction and plasma membrane fraction, respectively, by means of liquid chromatography-tandem mass spectrometry. Protein expression levels of 12 human P450 enzymes, two human UDP-glucuronosyltransferases, eight human ATP binding cassette (ABC) transporters, and eight human solute carrier transporters were determined. The variances of protein expression levels among samples from mice humanized with hepatocytes from all donors were significantly greater than those from samples obtained from mice derived from each individual donor. Compared with the protein expression levels in human livers, all of the quantified metabolizing enzymes and transporters were within a range of 4-fold difference, except for CYP2A6, CYP4A11, bile salt export pump (BSEP), and multidrug resistance protein 3 (MDR3), which showed 4- to 5-fold differences between PXB mouse and human livers. The present study indicates that humanized liver of PXB mice is a useful model of human liver from the viewpoint of protein expression of metabolizing enzymes and transporters, but the results are influenced by the characteristics of the human hepatocyte donor.