Bulk-Boundary Correspondence in Point-Gap Topological Phases.

A striking feature of non-Hermitian systems is the presence of two different types of topology. One generalizes Hermitian topological phases, and the other is intrinsic to non-Hermitian systems, which are called line-gap topology and point-gap topology, respectively. Whereas the bulk-boundary correspondence is a fundamental principle in the former topology, its role in the latter has not been clear yet. This Letter establishes the bulk-boundary correspondence in the point-gap topology in non-Hermitian systems. After revealing the requirement for point-gap topology in the open boundary conditions, we clarify that the bulk point-gap topology in open boundary conditions can be different from that in periodic boundary conditions. On the basis of real space topological invariants and the K theory, we give a complete classification of the open boundary point-gap topology with symmetry and show that the nontrivial open boundary topology results in robust and exotic surface states.

High expression of autotaxin is associated with poor recurrence-free survival in cholangiocarcinoma.

BACKGROUND AND AIM: Autotaxin (ATX) is an extracellular lysophospholipase D that catalyzes the hydrolysis of lysophosphatidylcholine into lysophosphatidic acid (LPA). Recent accumulating evidence indicates the biological roles of ATX in malignant tumors. However, the expression and clinical implications of ATX in human cholangiocarcinoma (CCA) remain elusive. METHODS: In this study, the expression of ATX in 97 human CCA tissues was evaluated by immunohistochemistry. Serum ATX levels were determined in CCA patients (n = 26) and healthy subjects (n = 8). Autotaxin expression in cell types within the tumor microenvironment was characterized by immunofluorescence staining. RESULTS: High ATX expression in CCA tissue was significantly associated with a higher frequency of lymph node metastasis (p = 0.050). High ATX expression was correlated with shorter overall survival (p = 0.032) and recurrence-free survival (RFS) (p = 0.001) than low ATX expression. In multivariate Cox analysis, high ATX expression (p = 0.019) was an independent factor for shorter RFS. Compared with low ATX expression, high ATX expression was significantly associated with higher Ki-67-positive cell counts (p < 0.001). Serum ATX levels were significantly higher in male CCA patients than in healthy male subjects (p = 0.030). In the tumor microenvironment of CCA, ATX protein was predominantly expressed in tumor cells, cancer-associated fibroblasts, plasma cells, and biliary epithelial cells. CONCLUSIONS: Our study highlights the clinical evidence and independent prognostic value of ATX in human CCA.

The evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reaction.

In regenerative medicine, the tumorigenic potency of cells in cellular therapy products (CTPs) is a major concern for their application to patients. This study presents a method-the soft agar colony formation assay using polymerase chain reaction (PCR)-to evaluate tumorigenicity. MRC-5 cells, contaminated with HeLa cells, were cultured for up to 4 weeks in soft agar medium. Cell-proliferation-related mRNAs, Ki-67 and cyclin B, could be detected in 0.01% of HeLa cells after 5 days of culture, whereas cyclin-dependent kinase 1 (CDK1) could be detected after 2 weeks. On the other hand, CDK2, proliferating cell nuclear antigen (PCNA), and minichromosome maintenance protein 7 (MCM7) were not useful to detect HeLa cells even after 4 weeks of culture. The cancer stem cell (CSC) markers, aldehyde dehydrogenase 1 (ALDH1) and CD133 in 0.01% of HeLa cells, could be detected 2 and 4 weeks after culture, respectively. However, another CSC marker CD44 was not useful because its expression was also detected in MRC-5 cells alone. This study suggests that the application of the PCR method to the soft agar colony formation assay could evaluate not only the tumorigenic potency in the short-term but also characterize the colonies, eventually improving the safety of CTPs.

Characterization and role of collagen gene expressing hepatic cells following partial hepatectomy in mice.

BACKGROUND AND AIMS: The mechanism underlying liver regeneration following partial hepatectomy (PH) is not fully elucidated. We aimed to characterize collagen gene expressing hepatic cells following PH and examine their contribution to liver regeneration. APPROACH AND RESULTS: Col-GFP mice, which express GFP under the control of the collagen gene promoter, were used to detect collagen gene expressing cells following PH. The GFP-expressing cells were analyzed via single-cell RNA sequencing (scRNA-seq). Additionally, Col-ER Cre/RFP and Col-ER Cre/DTA mice were utilized to examine the cell fates and functional roles of collagen gene expressing cells in liver regeneration, respectively. The number of collagen gene expressing cells was found to be increased on day 3 and subsequently decreased on day 7 following PH. ScRNA-seq analysis of sorted collagen gene expressing cells showed that the regenerating liver was characterized by three distinct hepatic stellate cell (HSC) clusters, including one representing classic myofibroblasts. The other HSC clusters included an intermediately activated HSC cluster and a proliferating HSC cluster. Of these, the latter cluster was absent in the CCl 4 -induced liver fibrosis model. Cell fate tracing analysis using Col-ER Cre/RFP mice demonstrated that the collagen gene expressing cells escaped death during regeneration and remained in an inactivated state in the liver. Further, depletion of these cells using Col-ER Cre/DTA mice resulted in impaired liver regeneration. CONCLUSIONS: Heterogeneous HSC clusters, one of which was a unique proliferating cluster, were found to appear in the liver following PH. Collagen gene expressing cells, including HSCs, were found to promote liver regeneration.

Universal Platform of Point-Gap Topological Phases from Topological Materials.

Whereas point-gap topological phases are responsible for exceptional phenomena intrinsic to non-Hermitian systems, their realization in quantum materials is still elusive. Here, we propose a simple and universal platform of point-gap topological phases constructed from Hermitian topological insulators and superconductors. We show that (d-1)-dimensional point-gap topological phases are realized by making a boundary in d-dimensional topological insulators and superconductors dissipative. A crucial observation of the proposal is that adding a decay constant to boundary modes in d-dimensional topological insulators and superconductors is topologically equivalent to attaching a (d-1)-dimensional point-gap topological phase to the boundary. We furthermore establish the proposal from the extended version of the Nielsen-Ninomiya theorem, relating dissipative gapless modes to point-gap topological numbers. From the bulk-boundary correspondence of the point-gap topological phases, the resultant point-gap topological phases exhibit exceptional boundary states or in-gap higher-order non-Hermitian skin effects.

Bile Duct Regeneration with an Artificial Bile Duct Made of Gelatin Hydrogel Nonwoven Fabrics.

Although choledochojejunostomy is the standard technique for biliary reconstruction, there are various associated problems that need to be solved such as reflux cholangitis. Interposition with an artificial bile duct (ABD) to replace the resected bile duct maintains a physiological conduit for bile and may solve this problem. This study investigated the usefulness of an ABD made of gelatin hydrogel nonwoven fabric (GHNF). GHNF was prepared by the solution blow spinning method. The migration and activity of murine fibroblast L929 cells were examined in GHNF sheets. L929 cells migrated into GHNF sheets, where they proliferated and synthesized collagen, suggesting GHNF is a promising scaffold for bile duct regeneration. ABDs made of GHNF were implanted in place of resected bile duct segments in rats. The rats were killed at 2, 6, and 12 weeks postimplantation. The implantation site was histologically evaluated for bile duct regeneration. At postoperative 2 weeks, migrating cells were observed in the ABD pores. The implanted ABD was mostly degraded and replaced by collagen fibers at 6 weeks. Ki67-positive bile duct epithelial cells appeared within the implanted ABD. These were most abundant within the central part of the ABD after 6 weeks. The percentages of Ki67-positive cells were 31.7 ± 9.1% in the experimental group and 0.8 ± 0.6% in the sham operation group at 6 weeks (p < 0.05), indicating that mature biliary epithelial cells at the stump proliferated to regenerate the biliary epithelium. Biliary epithelial cells had almost completely covered the bile duct lumen at 12 weeks (epithelialization ratios: 10.4 ± 6.9% at 2 weeks, 93.1 ± 5.1% at 6 weeks, 99.2 ± 1.6% at 12 weeks). The regenerated epithelium was positive for the bile duct epithelium marker cytokeratin 19. Bile duct regeneration was accompanied by angiogenesis, as evidenced by the appearance of CD31-positive vascular structures. Capillaries were induced 2 weeks after implantation. The number of capillaries reached a maximum at 6 weeks and decreased to the same level as that of normal bile ducts at 12 weeks. These results showed that an ABD of GHNF contributed to successful bile duct regeneration in rats by facilitating the cell migration required for extracellular matrix synthesis, angiogenesis, and epithelialization. Impact Statement Development of an artificial bile duct (ABD) enables physiological biliary reconstruction and may solve clinical problems associated with choledochojejunostomy. In this study, we created ABDs with gelatin hydrogel nonwoven fabric and implanted them in place of resected bile duct in rats. We evaluated the process of bile duct regeneration as well as decomposition of the ABD and demonstrated successful regeneration of resected bile duct, highlighting the possibility of this novel biliary reconstruction method to replace choledochojejunostomy.

Increased Expressions of Programmed Death Ligand 1 and Galectin 9 in Transplant Recipients Who Achieved Tolerance After Immunosuppression Withdrawal.

Programmed death 1 (PD1)/its ligand PD-L1 concomitant with T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3)/its ligand galectin 9 (Gal-9) and the forkhead box P3 (FOXP3) might be involved in tolerance after liver transplantation (LT). Liver biopsies from 38 tolerant, 19 nontolerant (including 16 samples that triggered reintroduction of immunosuppression [IS] and 19 samples after IS reintroduction), and 38 control LT patients were studied. The expressions of PD1, PD-L1, Gal-9, and FOXP3 were determined by immunohistochemical and immunofluorescence (IF) staining. The success period of IS withdrawal was calculated using Kaplan-Meier curve analysis. Tolerant and control patients exhibited higher PD-L1, Gal-9, and FOXP3 levels than nontolerant patients at the moment of triggering IS reintroduction. High expressions of PD-L1 and Gal-9 were associated with prolonged success of tolerance (83.3% versus 36.7% [P < 0.01] and 73.1% versus 42.9% [P = 0.03]). A strong correlation between PD-L1 and Gal-9 expression levels was detected (Spearman r = 0.73; P ≤ 0.001), and IF demonstrated colocalization of PD-L1 and Gal-9 in the cytoplasm of hepatocytes. In conclusion, the present study demonstrated that increased expressions of PD-L1 and Gal-9 were associated with sustained tolerance after IS withdrawal in pediatric liver transplantation.

Structure-activity relationship for the folding intermediate-selective inhibition of DYRK1A.

DYRK1A phosphorylates proteins involved in neurological disorders in an intermolecular manner. Meanwhile, during the protein folding process of DYRK1A, a transitional folding intermediate catalyzes the intramolecular autophosphorylation required for the "one-off" inceptive activation and stabilization. In our previous study, a small molecule termed FINDY (1) was identified, which inhibits the folding intermediate-catalyzed intramolecular autophosphorylation of DYRK1A but not the folded state-catalyzed intermolecular phosphorylation. However, the structural features of FINDY (1) responsible for this intermediate-selective inhibition remain elusive. In this study, structural derivatives of FINDY (1) were designed and synthesized according to its predicted binding mode in the ATP pocket of DYRK1A. Quantitative structure-activity relationship (QSAR) of the derivatives revealed that the selectivity against the folding intermediate is determined by steric hindrance between the bulky hydrophobic moiety of the derivatives and the entrance to the pocket. In addition, a potent derivative 3 was identified, which inhibited the folding intermediate more strongly than FINDY (1); it was designated as dp-FINDY. Although dp-FINDY (3) did not inhibit the folded state, as well as FINDY (1), it inhibited the intramolecular autophosphorylation of DYRK1A in an in vitro cell-free protein synthesis assay. Furthermore, dp-FINDY (3) destabilized endogenous DYRK1A in HEK293 cells. This study provides structural insights into the folding intermediate-selective inhibition of DYRK1A and expands the chemical options for the design of a kinase inhibitor.

13-Weeks subchronic toxicity of isoquercitrin-γ-cyclodextrin (IQC-γCD) molecular inclusion complex in Sprague-Dawley rats.

Flavonoids such as quercetin and its glycoside Isoquercitrin and are abundantly present in the diet and have various pharmacological effects. However, limited data about its potential toxicity is available. In this study, we aim to evaluate the subchronic toxicity of the isoquercitrin-γ-cyclodextrin (IQC-γCD) molecular inclusion complex (SunActive® QCD/EN) in Sprague-Dawley (SD) rats. The IQC-γCD was administrated orally to 40 male and 40 female SD rats at dietary doses up to 5.0 % for 13 consecutive weeks. During the experiment periods, the general clinical signs, mortality, hematological, urinalysis values, biochemical, and histopathological parameters were examined. All animals survived until the scheduled necropsy, and no statistically significant or clinical sign of toxicologically relevant differences including pathology parameters, and histopathological endpoints were observed in any of the IQC-γCD treatment groups, compared with the control group. However, certain observations were noted in the male rats treated with the highest concentration (5.0 %), but these were not seen in female rats. A slight inhibition of weight gain was observed, probably linked to a fall in red blood cells, and hematocrit index in female rats. Statistically significant changes were noted in some clinical measures, such as plasma bilirubin level, alkaline phosphatase total bile acid without evidence of systemic clinical toxicity. The results support no observed adverse effect level (NOAEL) of IQC-γCD of 5.0 % in the diet for males (3338.55 mg/kg/day), and 3.0 % in the diet for females (2177.33 mg/kg/day) SD rats. Therefore, in this 13 weeks repeated-dose SD rat study there were no treatment-related adverse clinical or pathological findings for IQC-γCD of 5.0 % in the diet for males, and 3.0 % in the diet for females SD rats. The results of the present study support the safe use of IQC-γCD as a functional food, food additive, and natural ingredient.

Comparison of biological features between severely immuno-deficient NOD/Shi-scid Il2rgnull and NOD/LtSz-scid Il2rgnull mice.

Biological background data up to 11 weeks of age and tumorigenic susceptibility to xenotransplantation with HeLa cells were compared between severely immuno-deficient NOG and NSG mice. The body weight was lower in NOG mice than in NSG mice. Severe depletion of peripheral blood lymphocytes and lymphoid hypoplasia that are well-known characteristics of these mice were equally observed. No lymphoproliferative lesions developed in any mouse of either strain. The occurrence of ectopic exocrine gland and cyst was a common finding in the thymus of both strains. In addition, minimal spongiotic change was observed in the medulla oblongata and spinal cord in both strains, and its incidence in female NOG mice was a little higher than that in NSG mice. In the adrenal, subcapsular cell hyperplasia that is known as an age-related change in non-genetically modified mice developed earlier and its incidence was higher in NSG mice than in NOG mice. The development of female genital organs of NOG mice was slightly retarded in comparison with that of NSG mice. To evaluate tumorigenic susceptibility to xenotransplantation, female mice were implanted in the dorsal subcutis with 1×103 to 1×106 cells/head of HeLa cells, and were checked up to 16 weeks after implantation. As a result, there was no significant strain difference on tumor formation rate and tumor volume. In conclusion, the present study clearly demonstrated that NOG and NSG mice showed no distinct strain differences in either biological features or biological disadvantages.

Ln(OTf)3-catalysed highly regioselective alcoholysis of 2,3-epoxy alcohols.

Regioselectivity of Ln(OTf)3-catalysed alcoholysis of 2,3- and 3,4-epoxy alcohols was closely investigated to expand the scope of the transformations. The synthetic use was demonstrated by application to the construction of 4-propoxy-5-hydroxy-2,3-pentanedione (C4-propoxy-HPD), which is a potent synthetic mediator in AI-2 quorum sensing.

Extra-gastrointestinal stromal tumor with a large cyst.

Gastrointestinal stromal tumors (GISTs) arising at sites other than the alimentary tract are rare, and they are called extra-GISTs (EGISTs). We report a case of a large EGIST forming a cyst, probably arising in the mesentery of the transverse colon. A 64-year-old Japanese man presented to a hospital with an abdominal tumor forming a large cyst. Intraoperatively, the tumor was neither present in nor in contact with the alimentary tract. It was present in the mesentery of the transverse colon and was attached to the greater omentum and peritoneum, immediately anterior to the body of the pancreas. The tumor was resected with the spleen and a part of the pancreas. Histological examination of the tumor revealed that it belonged to the high-risk category of cystic EGISTs.

[Two Cases of Long-Term Use of TAS-102 for Unresectable Colorectal Cancer].

TAS-102 has been administered to patients with unresectable colorectal cancer. We initiated TAS-102 administration in 2014 and gradually increased the number of indications. In a global, multicenter, randomized, double-blind, phase III study (RECOURSE study), TAS-102 administration improved overall survival by 1.8 months and progression-free survival by 0.3 months compared with those in the placebo group. However, there are limited clinical reports of long-term administration of TAS-102. We encountered 2 cases of continuous long-term use of the TAS-102 for over 2 years. In our cases, although the cancer had been recognized early as exhibiting slow growth during follow-up, the physical symptoms did not appear for an extended period. Although grade 3 neutropenia was pointed out several times during the follow-up term, severe digestive symptoms had not occurred. Therefore, the patients could remain motivated to receive the drug. In our cases, the adequate treatment for neutropenia enabled long-term administration of TAS-102; therefore, TAS-102 would be tolerable for patients of colorectal cancer after receiving chemotherapy for an extended period.

Hepatic epithelioid hemangioendothelioma with metastasis to the mesentery of the small intestine: A case report.

Epithelioid hemangioendothelioma (EHAE) is a vascular tumor which, due to its rarity, is often misdiagnosed as other hepatic tumors based on radiological characteristics. We herein report a case of EHAE in the liver and the mesentery of the small intestine. A 64-year-old asymptomatic woman was admitted to the hospital due to a hepatic tumor identified using computed tomography (CT). An enhanced CT scan revealed multiple tumors in the liver and a tumor in the mesentery. One of the hepatic tumors and the mesenteric tumor were resected and histologically examined. The two tumors exhibited similar histological characteristics and were diagnosed as EHAE. When multiple tumors are found in the liver, EHAE should be included in the differential diagnosis, as the prognosis of EHAE differs from that of carcinoma or benign tumors.

Background data on NOD/Shi-scid IL-2Rγnull mice (NOG mice).

To obtain background data of NOD/Shi-scid IL-2Rγnull (NOG) mice, severely immunedeficient mice, a total of 120 animals were examined at 7, 26 and 52 weeks-old (20 mice/sex/group). The survival rate at 52 weeks-old was 95% (19/20) in both sexes. Clinically, circling behavior in one direction along the cage wall was observed in males after 8 weeks and females after 47 weeks-old, and hunchback position was found in males after 32 weeks-old. Hematologically, lymphocyte count markedly decreased at all ages, while white blood cell count increased in several mice at 52 weeks-old. Blood chemistry results revealed high values of aspartate aminotransferase, lactate dehydrogenase and creatine phosphokinase in some females at 26 weeks-old, without any related histological change. Histologically, lymphoid hypoplasia characterized by severe lymphocyte depletion with poorly developed tissue architectures was observed. In addition, spongiotic change in the nerve tissue was observed in both sexes at 7 and 26 weeks-old, and intracytoplasmic materials known as tubular aggregates in the skeletal muscles were found in males terminated at 26 and 52 weeks-old and in females at 52 weeks-old. Malignant lymphoma was found in one female euthanized at 20 weeks-old. Further, small intestinal adenoma, hepatocellular adenoma, leukemia, cerebral lipomatous hamartoma, Harderian gland adenoma and uterine polyp were also observed, and their incidences were low except for that of uterine polyp. This study provided detailed background data on NOG mice up to 52 weeks-old and provided information on appropriate use of NOG mice in the various research fields.

Sensory incongruence leading to hand disownership modulates somatosensory cortical processing.

The sense of body ownership is based on integration of multimodal sensory information, including tactile sensation, proprioception, and vision. Distorted body ownership contributes to the development of chronic pain syndromes and possibly symptoms of psychiatric disease. However, the effects of disownership on cortical processing of somatosensory information are unknown. In the present study, we created a "disownership" condition in healthy individuals by manipulating the visual information indicating the location of the subject's own left hand using a mirror box and examined the influence of this disownership on cortical responses to electrical stimulation of the left index finger using magnetoencephalography (MEG). The event-related magnetic field in the right primary somatosensory cortex at approximately 50 msec (M50) after stimulus was enhanced under the disownership condition. The present results suggest that M50 reflects a cortical incongruence detection mechanism involving integration of sensory inputs from visual and proprioceptive systems. This signal may be valuable for future studies of the mechanisms underlying sense of body ownership and the role that disrupted sense of ownership has in neurological disease.

Reversible effect of maternal exposure to chlorpyrifos on the intermediate granule cell progenitors in the hippocampal dentate gyrus of rat offspring.

To examine the effects of developmental exposure to chlorpyrifos (CPF) on neurogenesis in the hippocampal dentate gyrus, pregnant rats were treated with 2.8, 14 or 70 ppm CPF in the diet from gestational day 10 to day 21 after delivery. Dams had decreased cholinesterase (ChE) activities in red blood cells (RBC) at intakes of ≥2.8 ppm and in brain at 70 ppm. Offspring on postnatal day (PND) 21 had decreased ChE activities in the RBC and brain at 70 ppm. There were no behavioral abnormalities in the offspring. Immunohistochemical analysis showed decreases in the numbers of cells positive for proliferating cell nuclear antigen and T box brain 2 in the subgranular zone (SGZ) of the dentate gyrus on PND 21 at 70 ppm, while other progenitor cell populations and the apoptotic cell number were unaffected in this zone. However, on PND 77 all changes had disappeared. The distribution of the progenitor cell population expressing nicotinic acetylcholine receptor α7 and lacking expression of postmitotic neuron-specific nuclear protein was unchanged by CPF-exposure, suggesting no effect of cholinergic stimulation on neurogenesis. These results suggest that developmental exposure to CPF directly but transiently affect the proliferation of type-2 progenitor cell populations in the hippocampal neurogenesis. The lowest-observed-adverse-effect level (LOAEL) of CPF was determined to be 2.8 ppm (0.36 mg/kg body weight/day) for dams by the inhibition of ChE activity in the RBC at this dose. As for offspring, no-observed-adverse-effect level (NOAEL) was determined to be 14 ppm (1.86 mg/kg body weight/day) by the decrease of type-2 progenitor cell proliferation in the SGZ and the inhibition of ChE activity in the RBC and brain at 70 ppm. The NOAEL of dams based on the offspring's effects was approximately 2800 times higher than the estimated consumption of CPF through food in the general population and in pregnant women as examined in Japan.

Enhanced liver tumor promotion but not liver initiation activity in rats subjected to combined administration of omeprazole and ß-naphthoflavone.

Omeprazole (OPZ) and ß-naphthoflavone (BNF) are cytochrome P450 (CYP)1A inducers and have liver tumor promoting effects. In this study, we investigated the co-promoting and co-initiating effects of OPZ and BNF in rats. In Experiment 1, male rats were subjected to partial hepatectomy (PH), and given oral doses of 138 or 276 mg/kg OPZ, 0.125% or 0.25% BNF or 138 mg/kg OPZ+0.125% BNF (n = 9~12) for 6 weeks after N-diethylnitrosamine (DEN) initiation. In Experiment 2, male rats were treated with oral doses of 138 or 276 mg/kg OPZ, 0.03% or 0.06% BNF or 138 mg/kg OPZ+0.03% BNF (n = 11~12) for 9 days starting 1 week before initiating treatment. As an initiating treatment, 2-Amino-3,4-dimethylimidazo[4,5-f]quinolone (MeIQx) was orally administered 12 hr after PH. The rats were fed a basal diet for 15 days, followed by a diet containing 0.015% 2-acetylaminofluorene for the next 10 days with a single oral dose of carbon tetrachloride. In Experiment 1, the number and area of glutathione S-transferase placental form-positive foci in the OPZ+BNF group were significantly higher than the average values of the High OPZ or the High BNF group. The expression of cyclooxygenase-2 (Cox-2) and COX-2 protein in the liver significantly increased in the OPZ+BNF group. In Experiment 2, liver initiation activity was not enhanced by the co-administration of OPZ+BNF. The results of our studies suggest that the co-administration of OPZ and BNF results in synergistic effects in the liver tumor promotion probably owing to increased COX-2 expression, but no modifying effect in the liver initiation activity of MeIQx in rats.

Serum alkaline phosphatase isoenzymes in SD rats detected by polyacrylamide-gel disk electrophoresis.

Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. In the present study, we assessed the usefulness of a commercially available polyacrylamide-gel disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co. Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes in rats of the Sprague-Dawley strain (SD rats), which are commonly used in toxicity studies. We also examined age-related changes in serum ALP isoenzymes in SD rats. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine (SI) and serum samples were treated with neuraminidase, antiintestinal ALP antibody, ALP inhibitor levamisole, and/or wheat germ agglutinin. It became clear that pretreatment of serum with neuraminidase is necessary for rat serum ALP isoenzyme analysis. The kit revealed that the main serum ALP isoenzymes in fasted 8-week-old intact rats were bone- and SI-derived and they tended to decrease with age. Serum liver-derived isoenzyme was slightly detected in both sexes of all ages examined, but it greatly increased in cholestasis model rats with bile-duct ligation, and rats of this model also had large molecular ALP detected in the stacking gel, suggesting hepatic damage. High-molecular intestinal ALP isoenzyme was slightly observed at the most cathodal side of the resolving gel. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in SD rats and that concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

Serum alkaline phosphatase isoenzymes in laboratory beagle dogs detected by polyacrylamide-gel disk electrophoresis.

Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. Itoh et al. (2002) reported that a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes was useful for veterinary clinicopathological diagnosis in mongrel dogs. In the present study, based on the report of Itoh et al. (2002), we tried to expand the application range of this kit to laboratory beagle dogs which are commonly used in toxicity studies. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine and serum samples were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The main serum ALP isoenzymes in 5-month-old intact beagle dogs were bone-derived (bone and atypical ALP: corresponding to human variant bone ALP) and they tended to decrease with age. However, liver-derived ALP isoenzyme greatly increased in the serum of cholestasis model dogs. The cholestasis model dogs also had a large molecular ALP detected in the resolving gel. This ALP could be originated from intestinal ALP or corticosteroid-induced ALP (CALP), because the activity remained even after levamisole inhibition. CALP was observed in intact laboratory beagle dogs with individual differences. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in laboratory beagle dogs and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.