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C57BL/6J (BL6J) and C57BL/6N (BL6N) inbred substrains are most widely used to understand the pathological roles of target molecules in a variety of diseases, including non-alcoholic steatohepatitis (NASH), based on transgenic mouse technologies. There are notable differences in the metabolic phenotypes, including glucose tolerance, between the BL6J and BL6N substrains, but the phenotypic differences in NASH are still unknown. We performed a comparative analysis of the two mouse substrains to identify the pathological phenotypic differences in NASH models. In the CCl 4 -induced NASH model, the BL6J mice exhibited a more severe degree of oxidative stress and fibrosis in the liver than the BL6N mice. In contrast, in the high-fat diet-induced NASH model, more accumulation of hepatic triglycerides but less weight gain and liver injury were noted in the BL6J mice than in the BL6N mice. Our findings strongly suggest caution be exercised with the use of unmatched mixed genetic background C57BL6 mice for studies related to NASH, especially when generating conditional knockout C57BL6 mice.

Backfat thickness is one of the most important traits of commercially raised pigs. Meishan pigs are renowned for having thicker backfat than Landrace pigs. To examine the genetic factors responsible for the differences, we first produced female crossbred pig lines by mating Landrace (L) × Large White (W) × Duroc (D) females (LWD) with Landrace (L) or Meishan (M) boars (i.e., LWD × L = LWDL for Landrace offspring and LWD × M = LWDM for the Meishan offspring). We confirmed that LWDM pigs indeed had a thicker backfat than LWDL pigs. Next, we performed gene expression microarray analysis in both genetic lines to examine differentially expressed genes (DEGs) in energy metabolism-related tissues, subcutaneous adipose (fat), liver, and longissimus dorsi muscle tissues. We analyzed the annotation of DEGs (2-fold cutoff) to functionally categorize them by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. The number of DEGs in muscle tissues of both lines was much less than that in fat and liver tissues, indicating that DEGs in muscle tissues may not contribute much to differences in backfat thickness. In contrast, several genes related to muscle (in fat tissue) and lipid metabolism (in liver tissue) were more upregulated in LWDM pigs than LWDL pigs, indicating that those DEGs might be responsible for differences in backfat thickness. The different genome-wide gene expression profiles in the fat, liver, and muscle tissues between genetic lines can provide useful information for pig breeders., Competing Interests: The authors have declared that no competing interests exist.

Background: Islet transplantation is an attractive treatment for patients with insulin-dependent diabetes mellitus, and currently, the liver is the favored transplantation site. However, an alternative site is desirable because of the low efficiency of hepatic transplantation, requiring 2 to 3 donors for a single recipient, and because the transplanted islets cannot be accessed or retrieved., Methods: We developed a novel procedure of islet transplantation to the inguinal subcutaneous white adipose tissue (ISWAT) of mice and described functional and morphological characteristics of transplanted syngeneic islets. Also, it was determined whether islet allograft rejection in the ISWAT can be prevented by immunosuppressive agents. Furthermore, it was examined whether human islets function when grafted in this particular site of immune-deficient mice., Results: In this site, transplanted islets are engrafted as clusters and function to reverse streptozotocin-induced diabetes in mice. Importantly, transplanted islets can be visualized by computed tomography and are easily retrievable, and allograft rejection is preventable by blockade of costimulatory signals. Of much importance, the efficiency of islet transplantation in this site is superior to the liver, in which hyperglycemia of diabetic recipient mice is ameliorated after transplantation of 200 syngeneic islets (the islet number yielded from 1 mouse pancreas) to the ISWAT but not to the liver. Furthermore, human islets transplanted in this particular site function to reverse diabetes in immune-deficient mice., Conclusions: Thus, the ISWAT is superior to the liver as the site of islet transplantation, which may lead to improved outcome of clinical islet transplantation.

Purpose: The options for improving the chemotherapeutic regimen consisting of bolus plus infusion of 5-fluorouracil (5-FU) include omitting the 5-FU bolus injection. We examined the effects of a 5-FU bolus injection on the activity of dihydropyrimidine dehydrogenase (DPD), which is the first and rate-limiting enzyme of 5-FU catabolism, in rats., Methods: The rats were divided into three groups, and then continuous infusion (50 mg/m(2)/h) for 4 h was started with a bolus injection of saline, 20 mg/kg 5-FU, or 60 mg/kg 5-FU. Plasma 5-FU, uracil (Ura), dihydrouracil (UH2) levels, and hepatic DPD activity were determined after administration of 5-FU., Results: The half-life after the end of the infusion (t 1/2, 4-8 h) of 5-FU in the rats given the bolus injection was significantly longer than in those that had been given saline, and it increased with increasing 5-FU bolus injection dosage (r = 0.801, p < 0.01). The plasma UH2/Ura ratio, an indirect biomarker of hepatic DPD activity, tended to be lower in the rats that had received a 5-FU bolus injection than in those that had not, and it remained low after infusion ended. The hepatic DPD activity in rats that had received a 5-FU bolus injection was significantly lower than in those that had not. Negative correlation was observed between DPD activity and bolus injection dosage (r = -0.691, p < 0.05)., Conclusions: A bolus injection suppresses hepatic DPD activity and its effects are dependent on dosage, resulting in slower elimination of 5-FU from the blood and contributing to long-term systemic exposure to 5-FU.

Objectives: Significant inverse association between coffee intake and the levels of liver enzymes has been reported. We demonstrated higher prevalence of metabolic syndrome in Korean immigrants (KIs) than in indigenous Japanese (IJs). The aim of this study was to investigate whether the association between coffee intake and liver enzyme levels was different between the 2 ethnic groups., Methods: This study is a cross-sectional study including a total of 966 subjects comprising KIs and IJs. The association between the quintiles of coffee intake and dichotomous values of liver enzymes was evaluated by logistic regression analysis in KIs, IJs, a high-risk group (current smokers or alcohol drinkers ≥45 g/day), and a low-risk group (non-smokers and alcohol drinkers <45 g/day)., Results: In KIs, a significant inverse association between coffee intake and serum aspartate aminotransferase (AST) levels was observed. In the IJs, a significant inverse association between coffee intake and serum alanine aminotransferase levels was observed. In the high-risk group, a significant inverse association between coffee intake and serum AST and gamma-glutamyltransferase levels was observed., Conclusion: No difference was observed between KIs and IJs regarding the association between coffee and liver enzymes. Coffee might inhibit hepatic damage by alcohol drinking and smoking., (© 2016 S. Karger AG, Basel.)

Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule.

In living-donor liver transplantation with a left lobe graft, which can reduce the burden on the donor compared to right lobe graft, the main problem is small-for-size (SFS) syndrome. SFS syndrome is a multifactorial disease that includes aspects related to the graft size, graft quality, recipient factors and even technical issues. The main pathophysiology of SFS syndrome is the sinusoidal microcirculatory disturbance induced by shear stress, which is caused by excessive portal inflow into the smaller graft. The donor age, the presence of steatosis of the graft and a poor recipient status are all risk factors for SFS syndrome. To resolve SFS syndrome, portal inflow modulation, splenectomy, splenic artery modulation and outflow modulation have been developed. It is important to establish strict criteria for managing SFS syndrome. Using pharmacological interventions and/or therapeutic approaches that promote liver regeneration could increase the adequate outcomes in SFS liver transplantation. Left lobe liver transplantation could be adopted in Western countries to help resolve the organ shortage.

Aim: To evaluate the clinical utility of hepatic clearance (HC) measured with technetium-99m-diethylenetriaminepenta-acetic acid-galactosyl human serum albumin ((99m)Tc-GSA) single-photon emission computed tomography (SPECT) to estimate the degree of liver fibrosis., Methods: Seventy-eight consecutive patients who underwent initial hepatectomy due to hepatocellular carcinoma were enrolled in this study. Indocyanine green clearance (ICG R15), quantitative indices estimated by (99m)Tc-GSA [the receptor index (LHL15 and HH15) and HC via SPECT analysis], and conventional liver function tests were performed before hepatectomy. Correlations among the quantitative indices for liver functional reserve, conventional liver function tests, and the degree of liver fibrosis were evaluated., Results: The degree of liver fibrosis was correlated with ICG R15, HH15, LHL15, and HC. HC showed the best correlation with conventional liver function tests. According to multivariate analysis, HC and LHL15 were significant independent predictors of severe fibrosis. HC was the most valuable index for predicting severe fibrosis., Conclusion: HC measured with (99m)Tc-GSA SPECT is a reliable index for assessing liver fibrosis before hepatectomy.

Background: The mortality rates associated with hepatectomy are still not zero. Our aim was to define the risk factors for complications and to evaluate our perioperative management., Study Design: Between 2001 and 2008, 793 consecutive patients (547 men and 246 women; mean age ± SD, 56.1 ± 14.9 years) underwent hepatectomy without gastrointestinal resection and choledocojejunostomy at our center. Of these patients, 354 (44.6%) were positive for the hepatitis B virus surface antigen and/or the hepatitis C virus antibody. We categorized 783 (98.7%) patients as Child-Pugh class A. Major resection (sectionectomy, hemihepatectomy, and extended hemihepatectomy), was performed in 535 patients (67.5%) and re-resection in 81 patients (10.2%)., Results: The median operative time was 345.5 minutes and median blood loss was 360 mL. The rate of red blood cell transfusion was 6.8%. The morbidity rate was 15.6%. Reoperations were performed in 19 patients (2.4%). The mean postoperative hospital stay was 18.4 ± 10.4 days. The in-hospital mortality rate was 0.1% (1 of 793 patients; caused by hepatic failure). The independent relative risk for morbidity was influenced by an operative time of more than 360 minutes, blood loss of more than 400 mL, and serum albumin levels of less than 3.5 g/dL, as determined using multivariate logistic regression analysis., Conclusions: Shorter operative times and reduced blood loss were obtained by improving the surgical technique and using new surgical devices and intraoperative management, including anesthesia. Additionally, decision making using our algorithm and perioperative management according to CDC guidelines reduced the morbidity and mortality associated with hepatectomy., (Copyright © 2010 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Because hepatic vasculatures exhibit variations, a preoperative evaluation of the vascular anatomy and an estimation of the volume of the liver graft are essential for successful adult living donor liver transplantation. Using 3-dimensional (3D) computed tomography (CT), we analyzed the volumetric and anatomical relationship of the hepatic vasculatures of liver grafts. The livers of 223 potential donors were analyzed by 3D CT. Volumetric analysis was performed for each hepatic vein and its tributaries. The anatomy of the portal vein and hepatic artery was assessed along with the biliary system via intraoperative cholangiography in 110 recipients. On the basis of the anatomical presentation of the inferior right hepatic vein (IRHV), the hepatic veins were classified as follows: in type I, the IRHV was absent; in type II, the IRHV was smaller than the right hepatic vein (RHV); and in type III, the IRHV was greater than or equal to the RHV in size. The drainage volume of the middle hepatic vein (MHV) and especially its tributaries in the right lobe increased with the size of the IRHV (P < 0.001). In type III hepatic veins with a large IRHV (17% of the donors), the MHV tributaries had the largest drainage volume in the right lobe (41.2% +/- 11.8%). Furthermore, type III hepatic veins typically exhibited biliary variations in 75% of the donors. No correlation was observed between variations in the hepatic artery and portal vein. In conclusion, a right lobe graft with a large IRHV is accompanied by a large drainage volume via the MHV and by bile duct variations in 17% of livers. Therefore, anatomical and volumetric analysis is important for preoperative evaluations., ((c) 2010 AASLD.)

The cholesterol-lowering action of soybean protein was studied with rats from the aspect of sulfur-containing amino acids using casein as a counterpart. Weanling rats were fed for 3 wk on a soybean protein isolate (SPI) or casein diet. Serum cholesterol levels did not differ between the two diet groups, but were lowered by supplementing methionine to a 10% SPI diet or cystine to an amino acid mixture diet, equivalent to a 10% SPI or to a 20% SPI diet. By adding methionine or cystine to a 10% SPI diet, cholesterol 7alpha-hydroxylase activity was elevated concomitantly with elevated hepatic glutathione (GSH) level, while hydroxyl methyl-glutalyl coenzyme A reductase activity was reduced by methionine, regardless of GSH levels. Excretion of fecal steroid was not significantly changed by addition of either amino acid, as expressed per body weight. These results indicate that the relative amount of methionine and cystine in a diet affected cholesterol metabolizing enzyme activity in a way not parallel to GSH concentration.

In order to obviate a small-for-size graft syndrome (SFSGS), a portacaval (PC) shunt had been considered in a case of adult-to-adult living donor liver transplantation (AA-LDLT). In a recent AA-LDLT case, we adopted the PC shunt to resolve SFSGS; however, graft atrophy was observed in the late period of LDLT, thereby resulting in liver dysfunction. Due to the surgical closure of the PC shunt at 11 months post-LDLT, the graft regenerated gradually and resulted in the recovery of the liver function. This experience indicates that the portacaval shunt would overcome SFSGS in the early period of LDLT, while it would cause the graft atrophy and the graft dysfunction in the late period of LDLT.

Background: Adenosine is known to exert protective roles in hepatic ischemia and reperfusion injury, while all adenosine receptors do not play the cytoprotective roles. We have tested our hypothesis that blockage of adenosine binding to A(1) receptor by its antagonist, KW3902 [8-(noradamantan-3-yl)-1,3-dipropylxanthine] attenuates hepatic ischemia-reperfusion injury., Methods: Adult female beagle dogs underwent a 2 h total hepatic vascular exclusion (THVE) with a venovenous bypass. Nontreated animals that underwent THVE with a venovenous bypass alone were used as the control (Group CT, n=6). KW3902 was given to the animals by continuous intraportal infusion for 60 min before ischemia at a dose of 1 microg/kg/min (Group KW, n=6). Two wk survival, hemodynamics, hepatic tissue blood flow (HTBF), liver function, energy metabolism, cAMP concentration, and histopathological findings were studied., Results: Two wk animal survival was significantly improved in group KW compared with that in group CT (group CT: 16.7% versus group KW: 83.3%). HTBF, liver function, and hepatic adenine nucleotide concentration were remarkably better in group KW than group CT. In addition, cAMP concentration in group KW was maintained significantly higher than group CT. Histopathological examination revealed preservation of hepatic architecture and suppression of neutrophil infiltration into hepatic tissue in group KW., Conclusion: Administration of adenosine A(1) receptor antagonist before ischemia attenuates hepatic ischemia-reperfusion injury. To elicit the beneficial effect of adenosine against ischemia and reperfusion injury of the liver, it is important to oppose adenosine A1 receptor activation.

In right lobe living donor liver transplantation (RL-LDLT), venous reconstruction of large middle hepatic vein (MHV) tributaries is often necessary. However the standard for reconstruction is unclear. In two recent RL-LDLT cases, we reconstructed a MHV tributary after estimating the donor's liver volume by three-dimensional images using helical computed tomography (3D-CT). 3D-CT demonstrated that the expected donor right lobe volume was 437 ml, which represented 46.2% of graft volume/recipient's standard volume ratio (GV/SV ratio) in case 1, and 600 ml (46.4%) in case 2. Moreover, 3D-CT revealed a large MHV tributary, 8 mm in diameter, drained 190 ml in volume of segment 8 in case 1, and, 7 mm in diameter, drained 138 ml in case 2. In these cases, when the area drained by the MHV tributary was excluded, GV/SV ratio were 26.1% and 35.7%, respectively. Based on this data, we reconstructed a large MHV tributary in both cases.

A role of natural killer T (NKT) cells in transplant rejection remains unknown. Here, we determined whether NKT cells participate in rejection of islet allografts, using NKT cell-deficient mice. Survival of islet allografts in streptozotocin-induced diabetic CD1d(-/-) mice or Valpha14 NKT cell(-/-) mice was significantly prolonged without immunosuppression when grafted into the liver, but not beneath the kidney capsule, compared with wild-type mice. Acceptance of intrahepatic islet allografts was achieved in CD1d(-/-) mice by a subtherapeutic dose of rapamycin, which was abrogated in conjunction with the transfer of hepatic mononuclear cells from wild-type, but not from CD1d(-/-), mice at islet transplantation. The second islet grafts from a donor-specific, but not from a third-party, strain in CD1d(-/-) mice bearing functional islet allografts were accepted without immunosuppression at 120 days after the initial transplantation. These findings demonstrate that NKT cells play a significant role in rejection of islet allografts in the liver of mice, but that NKT cells are not essential for induction of donor-specific unresponsiveness in this model. The current study indicates that NKT cells might be considered as a target for intervention to prevent islet allograft rejection when the liver is the site of transplantation.

We recently reported an involvement of ANG II and the ANG II type 1 (AT1) receptor in the hepatic expression of IL-1beta induced in dehydrated rats by LPS. Here, we first confirmed that ANG II and AT1 receptors contribute to the LPS-induced increase in the splenic concentration of IL-1beta in dehydrated rats. We then investigated whether ANG II contributes to IL-1 production through a modulating effect on the activation of proinflammatory transcription factors (NF-kappaB and AP-1) that is induced in the dehydrated rat's liver and spleen by intravenous injection of LPS. Surprisingly, LPS markedly increased the hepatic activation of NF-kappaB, an effect that was significantly enhanced (rather than reduced) by pretreatment with an ANG-converting-enzyme (ACE) inhibitor or AT1-receptor antagonist. Furthermore, the same ACE inhibitor and AT1-receptor antagonist each increased the resting NF-kappaB activity in the liver and spleen, although they had no effect on the LPS-induced splenic expression of NF-kappaB. Both hepatic and splenic AP-1 expressions were enhanced by LPS. This response was significantly augmented by pretreatment with the AT1-receptor antagonist (but not with the ACE inhibitor) in the spleen, while in the liver, neither drug had any effect. These results suggest that the endogenous ANG II or AT1 receptor suppresses the activation of hepatic or splenic transcription factors in dehydrated rats given LPS. Our results seem not to support the idea that NF-kappaB and AP-1 play key roles in the ANG II-induced enhancement of the production of proinflammatory cytokines that is induced by LPS in dehydrated rats.

Gamma interferon (IFN-gamma)-regulated chemokines of the CXC family have been implicated as key regulators of a variety of T-cell-dependent inflammatory processes. However, the cellular source(s) of IFN-gamma that regulates their early expression has rarely been defined. Here, we have directly addressed this question in mice after Leishmania donovani infection. Comparison of CXCL10 mRNA accumulation in normal and IFN-gamma-deficient mice confirmed an absolute requirement for IFN-gamma for sustained (24 h) expression of CXCL10 mRNA accumulation in this model. In normal mice, IFN-gamma was produced by both CD3int NK1.1+ NKT cells and CD3- NK1.1+ NK cells, as detected by intracellular flow cytometry. Strikingly, B6.Jalpha281-/- mice lacking NKT cells that express the invariant Valpha14Jalpha18 T-cell-receptor alpha chain, although retaining a significant population of IFN-gamma-producing NK cells and NKT cells, were unable to sustain CXCL10 mRNA accumulation. These data indicate that invariant NKT cells are indispensable for the regulation of hepatic CXCL10 gene expression during L. donovani infection.

Natural killer-T (NKT) cells are rich in the liver. However, their involvement in liver injury is not fully understood. We developed here a new murine model of NKT-cell-activation-associated liver injury, and investigated a role of tumor necrosis factor alpha (TNF-alpha) and Fas in pathogenesis. We injected intraperitoneally alpha-galactosylceramide (alpha-GalCer), an NKT-cell stimulant, into D-galactosamine (GalN)-sensitized mice. Survival rate, pathological changes of the liver, and plasma concentrations of cytokines were studied. Alpha-GalCer/GalN administration gave a lethal effect within 7 h, making pathological changes such as massive parenchymal hemorrhage, hepatocyte apoptosis, sinusoidal endothelial cell injury, and close apposition of lymphocytes to apoptotic hepatocytes. Anti-NK1.1 mAb-pretreated mice and Valpha14NKT knock out (KO) mice did not develop liver injury. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were elevated at 4 h in the plasma. These cytokines were produced by hepatic lymphocytes as demonstrated by in vitro stimulation with alpha-GalCer. The lethal effect was suppressed in TNF-alpha KO mice, TNF receptor-1 KO mice, and lpr/lpr (Fas deficient) mice, whereas it was not in IFN-gamma KO mice. These results indicate that the present liver injury is characterized by parenchymal hemorrhage and hepatocyte apoptosis, and mediated by TNF-alpha secretion and direct cytotoxicity of alpha-GalCer-activated NKT cells.

Prolonged hepatic warm ischemia has been incriminated in oxidative stress after reperfusion. However, the magnitude of oxidative stress during ischemia has been controversial. The aims of the present study were to elucidate whether lipid peroxidation progressed during ischemia and to clarify whether oxidative stress during ischemia aggravated the oxidative damage after reperfusion. Rats were subjected to 30 to 120 min of 70% warm ischemia alone or followed by reperfusion for 60 min. Lipid peroxidation (LPO) was evaluated by amounts of phosphatidylcholine hydroperoxide (PC-OOH) and phosphatidylethanolamine hydroperoxide (PE-OOH) as primary LPO products. Total amounts of malondialdehyde and 4-hydroxy-2-nonenal (MDA + 4-HNE), degraded from hydroperoxides, were also determined. PC-OOH and PE-OOH significantly increased at 60 and 120 min ischemia with concomitant increase of oxidized glutathione. These hydroperoxides did not increase at 60 min reperfusion after 60 min ischemia, whereas they did increase at 60 min reperfusion after 120 min ischemia with deactivation of phospholipid hydroperoxide glutathione peroxidase and superoxide dismutase. The amount of MDA + 4-HNE exhibited similar changes, but the velocity of production dropped with ischemic time longer than 60 min. In conclusion, oxidative stress progressed during ischemia and triggered the oxidative injury after reperfusion. Secondary LPO products are less sensitive, especially during ischemia, which may cause possible underestimation and discrepancy.

Background: Adenosine, a metabolite of adenosine triphosphate degradation during ischemia, is reported to attenuate ischemia and reperfusion injury in several tissues. Dipyridamole is a nucleoside transport inhibitor that augments endogenous adenosine. In this study, we tested whether dipyridamole would attenuate hepatic I/R injury. For this purpose, dipyridamole was applied to a 2-hour total hepatic vascular exclusion model in dogs., Study Design: Dipyridamole (DYP) was given by continuous intravenous infusion for 1 hour before ischemia at a dose of 0.25 mg/kg (high-DYP, n = 6), 0.1 mg/kg (medium-DYP, n = 6), or 0.05 mg/kg (low-DYP, n = 6). Nontreated animals were used as ischemic controls (CT, n = 12). Two-week survival, systemic and hepatic hemodynamics, liver function tests, energy metabolism, adenosine 3', 5'-cyclic monophosphate (cyclic AMP) levels, platelet numbers, arachidonic acid metabolites, and histopathology were analyzed., Results: Two-week animal survival was 25% in CT, 17% in high-DYP, 100% in medium-DYP, and 17% in low-DYP. Dipyridamole significantly improved postreperfusion hepatic blood flow and energy metabolism, attenuated liver enzyme release and purine catabolite production, and augmented cyclic AMP levels. The medium dose of dipyridamole lessened platelet aggregation, thromboxane B2 production, and polymorphonuclear neutrophil infiltration, and improved survival., Conclusions: We demonstrated marked hepatoprotective effects of dipyridamole against severe ischemia and reperfusion injury in canine livers. Dipyridamole is a promising agent for liver surgery and transplantation.

The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear. In this paper we characterized the Valpha14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection determined an early expansion of Valpha14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30. However, an NK1.1(+) Valpha14 NKT population preferentially producing IFN-gamma predominated at an early stage (day 8), which was substituted by an NK1.1(-) population preferentially producing IL-4 at later stages (day 30). Despite the fact that Valpha14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs. Additionally, while control mice developed organized small granulomas, those in Valpha14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that Valpha14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology. In agreement, we found an increased spontaneous production and mRNA expression of TNF-alpha in liver and lungs of Valpha14 NKT-deficient mice, whose neutralization in vivo by anti-TNF-alpha mAbs consistently reduced the number of granulomas in liver and lungs. Together, our results support a regulatory role for Valpha14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology.

The reductive-oxidative status of tissues regulates the expression of many inflammatory genes that are induced during gram-negative bacterial infections. The cytokine gamma interferon (IFN-gamma) is a potent stimulus for host inflammatory gene expression, and oxidative stress has been shown to inhibit its production in mice challenged with Escherichia coli bacteria. The objective of the present study was to characterize the cells that produced IFN-gamma in a mouse bacterial peritonitis model and determine the effects of oxidative stress on their activation. The liver contained large numbers of IFN-gamma-expressing lymphocytes following challenge with viable E. coli bacteria. The surface phenotypes of IFN-gamma-expressing hepatic lymphocytes were those of natural killer (NK) cells (NK1.1(+) CD3(-)), conventional T cells (NK1.1(-) CD3(+)), and NK T cells (NK1.1(+) CD3(+)). Treating mice with diethyl maleate to deplete tissue thiols significantly impaired IFN-gamma production by NK cells, conventional T cells, and CD1d-restricted NK T cells in response to E. coli challenge. However, IFN-gamma expression by a subset of NK T cells, which did not bind alpha-galactosylceramide-CD1d tetramers, was resistant to the inhibitory effects of tissue oxidative stress. Stress-resistant IFN-gamma-expressing cells were also predominantly CD8(+) and bore gamma delta T-cell antigen receptors. The residual IFN-gamma response by NK T cells may explain previous reports of hepatic gene expression following gram-negative bacterial challenge in thiol-depleted mice. The finding also demonstrates that innate immune cells differ significantly in their responses to altered tissue redox status.

Background: Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). The effect of parenteral nutrition (PN) on hepatic DPD activity and metabolism of 5-FU remains unknown., Materials and Methods: Rats were divided into two groups: a sham-operated oral feeding group (FED) and a PN group. After 7-day PN infusion, hepatic DPD activity, serum 5-FU levels and thymidylate synthase (TS) levels in the jejunum and tumor were measured., Results: PN administration significantly decreased hepatic DPD activities. After infusion of 5-FU (40 mg/kg body), the serum 5-FU concentration and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP)-bound TS levels in the jejunum were significantly higher in the PN group than the FED group (156.8 +/- 51.9 vs 100.5 +/- 51.9 ng/ml, p < 0.001 and 38.55 +/- 7.61 vs 22.89 +/- 4.46 pmol/g of tissue, p < 0.01, respectively). In Yoshida sarcoma-bearing rats, the FdUMP-bound TS level in the tumor did not differ significantly between the PN and FED rats., Conclusion: PN decreases hepatic DPD activity, which may lead to increased toxicity of 5-FU.

Background: In a series of canine liver ischemia experiments, we have shown that amelioration of hepatic injury is achievable by the inhibition of vasoconstriction, cytokine production, platelet aggregation, and neutrophil infiltration. Cyclic adenosine diphosphate (cAMP) was considered to be involved in most of these events. In our study, we tested our hypothesis that augmentation of endogenous cAMP by phosphodiesterase (PDE) 3 inhibitor, amrinone (AM), or adenylate cyclase stimulator, NKH477 (NKH), could attenuate ischemia and reperfusion injury of the liver., Methods: Thirty-six beagle dogs were used. They were divided into group CT (untreated control), group AM, group NKH, and group CB (treated by both agents). AM or NKH were administered i.v. 1 hr before ischemia (group preAM and group preNKH) or 15 min before reperfusion (pos-AM and postNKH). Combination group animals were treated only before ischemia. Animal survival, hepatic tissue blood flow, liver enzymes, platelet counts, energy metabolism, hepatic cAMP and cyclic guanosine 3',5'-cyclic monophosphate levels, and histopathology were analyzed., Results: Two-week animal survival was significantly improved by pre- or posttreatment with either agent. After reperfusion, hepatic tissue blood flow, liver enzyme release, platelet counts, energy metabolism, tissue cAMP levels, and histological architecture were also ameliorated markedly. Combination of both agents induced severe liver damage and lethal hypotension. AM treatment exhibited more protective effects than NKH, particularly when it was given before ischemia. Interestingly, not only cyclic guanosine 3',5'-cyclic monophosphate, were also restored at higher levels after reperfusion by preischemia treatment., Conclusions: Administration of amrinone or NKH477 maintained hepatic tissue concentrations of cyclic nucleotides, and attenuated ischemia and reperfusion injury of the liver. Thus, regulation of hepatic tissue cyclic nucleotides is an important alternative for prevention of hepatic damage in liver preservation and surgery.

GBP28 (gelatin-binding protein of 28 kDa)/adiponectin is an adipocyte-producing plasma protein proposed to interact with the extracellular matrix. To examine the production of GBP28 in non-adipose tissues, we herein analyzed its expression and localization in mouse livers before and after CCl(4) treatment. In immunohistochemical analyses, the boundary of hepatocytes provided positive signals for GBP28 after 3-6 h and their cytoplasm was intensely stained after 18 h of CCl(4) treatment. Quantitative RT-PCR and in situ hybridization revealed that GBP28 mRNA expression was markedly elevated in CCl(4)-treated mouse livers. These results suggest that the circulating GBP28 binds the extracellular matrices of hepatocytes during the initial stage of CCl(4)-induced hepatic injury and the damaged hepatocytes themselves started to produce GBP28 thereafter. The induced expression of GBP28 was also observed in human hepatoma HepG2 cells after treatment with IL-6. Thus, GBP28 is also produced by the liver, where it undergoes tissue damage-induced transcriptional regulation., (Copyright 2001 Academic Press.)

Background: Membrane phospholipid breakdown, caused by ischemia and reperfusion (I/R) of the liver, releases free fatty acids including arachidonic acids and lysophospholipids, which serve as precursors of various inflammatory lipid derivatives. Phospholipase A2 (PLA2) is a key enzyme that initiates this reaction. In this study, we tested our hypothesis that a type II PLA2 inhibitor, LY329722, could attenuate hepatic I/R injury caused by a 2-hr total hepatic vascular exclusion (THVE) in dogs., Methods: Eighteen beagle dogs, subjected to a 2-hr THVE, were divided into three groups. Group 1 (n=6) was untreated and served as a control group. LY329722 was administered to animals in group 2 (n=6) intravenously (0.2 mg x kg(-1) x hr(-1)) for 60 min before ischemia, and to animals in group 3 (n=6) for 60 min starting 15 min before reperfusion (0.2 mg x kg(-1) x hr(-1)). Animal survival, systemic and splanchnic hemodynamics, hepatic tissue blood flow, liver functions, energy metabolism, hepatic venous thromboxane B2 and endothelin-1 levels, phospholipid levels and tumor necrosis factor-a mRNA expression in liver tissue, and histopathologic findings were evaluated., Results: Two-week animal survival was 33% (two of six) in group 1, and 100% (six of six) in groups 2 and 3. LY329722 improved systemic and splanchnic hemodynamics, hepatic tissue blood flow, and energy metabolism, reduced liver enzyme, thromboxane B2, and endothelin-1 release, prevented hepatic phospholipid degradation and tumor necrosis factor-alpha mRNA expression, and lessened histopathologic damage and the number of neutrophil infiltrating into the liver tissue., Conclusion: The present study demonstrated that a type II PLA2 inhibitor, LY329722, attenuated hepatic I/R injury caused by a 2-hr THVE model in dogs.

Background: Microcirculatory disturbance has been shown to play a critical role in hepatic ischemia and reperfusion (I/R) injury. Angiotensin II (AngII) is one of the most potent endogenous vasoconstrictors. Angiotensin II type I (AT1) receptor antagonist has been reported to have protective effects on I/R injury of the heart and kidney. However, effect on hepatic I/R injury has not been determined. In this study, we investigate our hypothesis that AT1 receptor antagonist, CV-11974, attenuates hepatic I/R injury., Methods: Twelve beagle dogs underwent a 2-hr total hepatic vascular exclusion with veno-venous bypass. CV-11974 was given to animals at a dose of 0.002 mg/ kg/min for 5 min followed by 0.001 mg/kg/min for 25 min via portal vein before ischemia (group II, n=6). Nontreated animals were used as the control (group I, n=6). Animal survival, hemodynamics, hepatic tissue blood flow (HTBF), liver function, platelet count, renin activity, and AngII concentration of hepatic vein, energy metabolism, and histopathology were analyzed., Results: Two-week survival was 33% in group I, in contrast, 100% in group II. Mean arterial blood pressure during early reperfusion was maintained, and HTBF after reperfusion was significantly higher in group II. Treatment attenuated liver enzyme release and decrease of platelet count, increased renin and AngII, suppressed ATP degradation during ischemia and enhanced ATP resynthesis after reperfusion. Neutrophil infiltration and histopathological damages were lessened in group II., Conclusions: Our data demonstrated that the local renin-angiotensin system might play a role in hepatic microcirculation. AT1 receptor blockade with CV-11974 attenuated hepatic microcirculatory disturbance and ameliorated I/R injury.

The alpha1,6 fucosyltransferase (alpha1,6 FucT) catalyzes the transfer of a fucose from GDP-fucose to the innermost GlcNAc residue of N-linked glycans via an alpha1,6 linkage. alpha1,6 FucT was overexpressed in transgenic mice under the control of a combined cytomegalovirus and chicken beta-actin promoter. Histologically numerous small vacuoles, in which lipid droplets had accumulated, were observed in hepatocytes and proximal renal tubular cells. Electron microscopic studies showed that the lipid droplets were membrane-bound and apparently localized within the lysosomes. Cholesterol esters and triglycerides were significantly increased in liver and kidney of the transgenic mice. Liver lysosomal acid lipase (LAL) activity was significantly lower in the transgenic mice compared to the wild mice, whereas LAL protein level, which was detected immunochemically, was increased, indicating that the specific activity of LAL was much lower in the transgenic mice. In all of the transgenic and nontransgenic mice examined, the activity of liver LAL was negatively correlated with the level of alpha1,6 FucT activity. As evidenced by lectin and immunoblot analysis, LAL was found to be more fucosylated in the transgenic mice, suggesting that the aberrant fucosylation of LAL causes an accumulation of inactive LAL in the lysosomes. Such an accumulation of inactive LAL could be a likely cause for a steatosis in the lysosomes of the liver and kidney in the case of the alpha1,6 FucT transgenic mice.

Effects of dietary protein and S-containing amino acids on oxidative damage were investigated in rat liver. After feeding rats for 3 weeks from weaning, lower GSH levels and higher metallothionein (MT) levels were found in the liver of rats fed on a 10% soyabean-protein-isolate (SPI)-based diet than those fed on a 10% casein-based diet. After injection of N-nitrosodimethylamine (NDMA) at 20 mg/kg body weight, increases in lipid peroxide, determined as thiobarbituric-acid reactive substances (TBARS), and gamma-glutamyltransferase (GGT) activity in the liver were observed in the 10% SPI diet group. By supplementing the 10% SPI diet with 0.3% cystine or methionine, GSH levels were increased, while MT levels were decreased, and elevation in TBARS levels after NDMA injection was diminished. On the other hand, increase in GGT activity could be prevented only by methionine supplement. On a 20% SPI or casein diet, TBARS concentration and GGT activity were not altered after NDMA injection with concomitant increase in GSH levels and decrease in MT levels. These results indicate that sufficient amounts of methionine and cystine in a diet are important to protect the liver from oxidative damage after NDMA administration, and GSH plays a primary role in the cellular protective function when compared with MT.

Pancreatic islet transplantation represents a potential treatment for insulin-dependent diabetes mellitus. However, the precise cellular and molecular mechanisms of the immune reactions against allogeneic and xenogeneic transplanted islets remain unclear. Here, we demonstrate that CD4(+) Valpha14 natural killer T (NKT) cells, a recently identified lymphoid cell lineage, are required for the acceptance of intrahepatic rat islet xenografts. An anti-CD4 mAb, administrated after transplantation, allowed islet xenografts to be accepted by C57BL/6 mice, with no need for immunosuppressive drugs. The dose of anti-CD4 mAb was critical, and the beneficial effect appeared to be associated with the reappearance of CD4(+) NKT cells at around 14 days after transplantation. Interestingly, rat islet xenografts were rejected, despite the anti-CD4 mAb treatment, in Valpha14 NKT cell-deficient mice, which exhibit the normal complement of conventional lymphoid cells; adoptive transfer of Valpha14 NKT cells into Valpha14 NKT cell-deficient mice restored the acceptance of rat islet xenografts. In addition, rat islet xenografts were accepted by Valpha14 NKT mice having only Valpha14 NKT cells and no other lymphoid cells. These results indicate that Valpha14 NKT cells play a crucial role in the acceptance of rat islet xenografts in mice treated with anti-CD4 antibody, probably by serving as immunosuppressive regulatory cells.

To investigate the roles of intrahepatic T cells in liver injury after Salmonella infection, we examined serum alanine transaminase (ALT), histopathology, and bacterial numbers in liver after infection with Salmonella choleraesuis strain 31N-1 in mice genetically lacking TCRalpha beta+, CD4(+), CD8(+), or NK1.1(+)T cells with C57BL/6 background. In control (+/+) mice, serum ALT reached a peak level by day 7 after an intraperitoneal inoculation of 2 x 10(6) CFU Salmonella choleraesuis 31N-1. In TCR-beta-/- mice, liver injury, as assessed by serum ALT level and histological examination, was significantly suppressed on day 7 after Salmonella infection but the numbers of bacteria in liver did not differ from those in normal mice, suggesting that alpha beta T cells are responsible for liver injury induced by Salmonella infection. To further determine which subsets in alpha beta T cells are important for the liver injury, we compared serum ALT level in mice genetically lacking CD4, CD8, beta2-microglobulin (beta2m, IAbeta, or Jalpha281 after Salmonella infection. In CD4(-/-) mice, serum ALT was significantly lower in comparison with control mice, but there was no difference in serum ALT levels in CD8(-/-) and IAbeta-/- mice from that in control mice. Notably, serum ALT levels and pathological lesions in liver were significantly decreased in beta2m-/- or Jalpha281(-/-) mice, which lacked in NK1.1(+) T cells bearing TCR Valpha14-Jalpha281 specific for beta2m-associated CD1d, following Salmonella infection. Taken together, it is suggested that alpha beta T cells bearing NK1.1 and CD4 may be main effector cells for liver injury after Salmonella infection.

To investigate oxidative effects of N-nitrosodimethylamine (NDMA) on the liver, rats were challenged by the reagent with a dose range of 10 to 40 mg/kg. With lower dose levels, protective responses were prominent, such as elevation of the hepatic glutathione and metallothionein (MT) levels. Increased activities were also evident of gamma-glutamylcysteine synthetase, glucose-6-phosphate dehydrogenase (G6PD), and malic enzyme. In the high dose range, however, toxic responses, such as increases in lipid peroxide levels in liver and serum, and glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and ketone bodies in serum became marked. Some of the protective responses became less marked at the highest dose. Catalase and glutathione peroxidase activities in the liver were also inhibited by NDMA treatment. On the other hand, when NDMA was injected as a series of doses (10 mg/kg on four separate occasions), the effects were less marked, and the hepatic levels of MT and lipid peroxide remained unchanged even after the 4th injection. Only the increase in G6PD activity was more marked after four times repeated injection than after a single injection. These results suggest that oxidative and hepatotoxic effects of NDMA are more moderate when given in repeated doses than in a single dose. In contrast to the liver, elevation of MT levels was the only detectable change in the kidney.

The regulatory mechanism of albumin gene transcription was examined using male Donryu rats (7 wk old) starved for 1 or 3 d. At the designated times, the rats were sacrificed to harvest the liver and to measure the serum albumin level. Neither the serum albumin nor the albumin messenger RNA (mRNA) level showed a significant change for these starvation periods. Among nuclear factors binding to the D site of albumin gene promoter, the CCAAT/enhancer binding protein alpha (C/EBP alpha) mRNA level showed a decrease and the D site binding protein (DBP) mRNA level tended to decrease after 3 d of starvation. In contrast, the C/EBP beta mRNA level showed a significant increase at day 1. As a B site binding nuclear factor, the hepatocyte nuclear factor 1 (HNF-1) mRNA level significantly increased at day 1. Gel mobility-shift analysis combined with Western immunoblotting confirmed the presence of D site binding proteins composed of DBP and C/EBP alpha and beta in both groups subjected to oral feeding and to 3-d starvation, though quantitative analysis could not be done. In conclusion, the nuclear transcription factors binding to the albumin gene promoter undergo regulatory changes during 3 d of starvation, whereas there is no significant decrease in the albumin mRNA level.

UK-2A and UK-3A are structural relatives of antimycins, which were isolated as antifungal antibiotics with little cytotoxicity that demonstrated inhibition of respiratory activity. They halve the cellular respiration of yeast within 4-5 minutes and the intracellular ATP content within 2-5 minutes. Moreover, they inhibited the yeast mitochondrial respiration using beta-hydroxybutyrate and succinate as a respiratory substrate, but no inhibition was observed using ascorbate-reduced tetramethyl p-phenylenediamine as the substrate. The site of respiratory inhibition of UK-2A and UK-3A was thought to be the cytochrome bc1 complex in the mitochondrial electron transport chain of yeast cells. They also inhibited the mitochondrial respiration of rat liver. It has been suggested that intact animal cells might have some system to defend themselves from the actions of UK-2A and UK-3A.

The aim of this study was to investigate the changes in the circadian rhythm of the expression of liver-specific genes caused by different schedules of parenteral nutrition (PN). Rats received PN continuously throughout the day or intermittently during the night or day for 7 days. They were examined for gene expression of D-site binding protein (DBP), albumin, and cytochrome P450 cholesterol 7alpha-hydroxylase (CYP7) in the liver. The nocturnal PN group showed circadian expression of DBP messenger RNA (mRNA) and protein with a peak at 10 PM, in the same manner as the control rats receiving normal chow feeding. However, the diurnal PN group showed inverted expression of DBP mRNA and protein with a peak at 10 AM. CYP7 mRNA levels exhibited good synchronization with the levels of DBP mRNA in all groups, whereas albumin mRNA levels did not show such synchronization. Gel mobility-shift assay disclosed that the binding activity of the nuclear extracts to the CYP7 gene promoter was changed by the PN schedule in accordance with the expression of CYP7 mRNA. The PN schedule modulates the circadian rhythm of DBP expression and may have an effect on hepatic bile acid formation through transcriptional regulation of the CYP7 gene.

We found that a homogenous CD4-/CD8- T cell bearing an invariant TCR encoded by V alpha 14J alpha 281 with a one-base N-region is highly dominant in the periphery (2-3% in spleen). Surprisingly, the high expression of this invariant V alpha 14 TCR is a general phenomenon in all laboratory strains irrespective of MHC haplotype and in some wild mouse subspecies. The majority of V alpha 14+ TCR is associated with J alpha other than J alpha 281 during the neonatal stage, after which the frequency of invariant V alpha 14J alpha 281 TCR expression increases with time reaching a maximum at around 5-8 weeks after birth. The dominant expression of V alpha 14J alpha 281 TCR is found in both euthymic and athymic mice. These results indicate that homogenous V alpha 14J alpha 281 T cells are positively selected in the periphery without thymic influence and that their VJ junction is important for positive selection. We also demonstrate that V alpha 14+ TCR gene rearrangements take place at extrathymic sites, such as bone marrow, liver, and intestine, since frequent nonproductive V alpha 14 TCR products and V alpha 14-J alpha 281 gene mediated signal sequences in circular DNA are detected as a result of TCR rearrangements in extrathymic tissues rather than in the thymus. This indicates the extrathymic development of V alpha 14J alpha 281 T cells. Furthermore, the biological roles of homogenous T cells bearing V alpha 14J alpha 281 TCR and the human counterpart of this invariant TCR are also discussed.

The authors present a patient with massive liver sarcoidosis, showing only slight hepatomegaly on precontrast computed tomogram and multiple low-density intrahepatic septa on postcontrast computed tomogram. Sarcoidosis frequently involves the liver, but rarely shows abnormal findings other than hepatomegaly on computed tomograms. Only a few cases have demonstrated a low density intrahepatic area on computed tomograms. In this report, we present a patient with massive liver and pulmonary sarcoidosis, showing low density intrahepatic septa and pulmonary fibrosis on computed tomograms.

Metallothionein contains about 30% cysteine and is a major protein in newborn rat liver. This protein and glutathione constitute two major intracellular cysteine pools in newborn rat liver. When pregnant rats were fed on a soya-bean-protein diet, low in sulphur amino acids, the hepatic glutathione levels of the dams were decreased. However, this did not affect the levels of glutathione or metallothionein in the pups. The activity of the glutathione-degrading enzyme gamma-glutamyltransferase (EC 2.3.2.2) in the livers of pups was maximum at birth and gradually decreased with age when the hepatic glutathione was transported to the kidney. In the pups born from dams fed on soya-bean-protein diet the decline in the hepatic enzyme activity was delayed, suggesting a continued degradation of glutathione in the liver. These results suggest that even with a maternal nutritional deficiency of sulphur amino acids, the transfer of cysteine to the fetus is not impaired. However, the hepatic intra-organ degradation of glutathione is continued in these pups for a prolonged period after birth compared with pups born from control mothers. The increased degradation of glutathione in the liver may be essential to meet the requirement of cysteine in pups born from dams fed on the soya-bean-protein diet.

Changes in the level of glutathione (GSH), the turnover rate, and gamma-glutamyltransferase (GGT) activity were examined in newborn, weanling, and adult male Wistar rats, the objective being to elucidate the mechanisms which control the hepatic GSH level during maturation as well as under conditions of different degrees of protein ingestion. The hepatic GGT activity in the newborn rats was high at birth, decreased within a few days to 1 to 2% of the initial level, and remained unchanged thereafter, when these rats were fed a normal diet after 3 weeks of age. In contrast, the hepatic GSH level increased 3-4-fold while total GGT activity in the kidney increased 6-8-fold. When weanling rats were fed a low protein diet (containing 10% soy protein) for 3 weeks, the hepatic GSH level decreased markedly while the GGT activity increased 5-6-fold. The turnover rate of hepatic GSH also increased, as determined by the use of buthionine sulfoximine, a specific inhibitor of GSH synthesis; a value of 2.1 h was obtained in comparison with 3.5 h for that of rats fed the normal laboratory chow (CRF-1). On the other hand, feeding adult rats on the low protein diet resulted in a marked decrease in hepatic GSH level with no effect on either hepatic or renal GGT activity. These results together with other observations may suggest that GSH translocated out of liver cells in the newborn rats is degraded mainly by these cells, while the tripeptide secreted by hepatocytes of adult rats is metabolized predominantly in extrahepatic tissues, such as the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Transaminative metabolism of L-cysteine was investigated using homogenates of guinea pig liver and kidney. L-Cysteine was transaminated in the presence of 2-oxoglutarate and the homogenate of either liver or kidney. S-(2-Hydroxy-2-carboxyethylthio)cysteine (HCETC) (3-mercaptolactate-cysteine disulfide) was formed by liver homogenate, but the amount was very small. On the other hand, a relatively large amount of HCETC was formed in the presence of kidney homogenate. Transamination between 3-mercaptopyruvate and certain amino acids was catalyzed actively by both liver and kidney homogenates in the presence of L-glutamate. However, more half-cysteine was formed by liver than kidney, and more HCETC was produced by kidney than liver. L-Glutamate was the most potent amino donor, and L-aspartate strongly inhibited the reaction. Results indicate that L-cysteine can be transaminated both in liver and kidney of the guinea pig, and that kidney is more active than liver. 2-Oxoglutarate is the most active 2-oxo acid for cysteine transamination. Oxaloacetate (and aspartate in the reverse reaction) is inhibitory to the reaction. These results are in agreement with the previous conclusion that cysteine aminotransferase is identical with aspartate aminotransferase.

Glutathione reductase has been purified 5,500-fold from rat liver mitochondrial matrix in a yield of 30%. The mitochondrial enzyme was immunochemically indistinguishable from that of the cytosol and the subunit molecular weight was apparently similar to that of the cytosolic enzyme, that is, 50,000 daltons. The optimum pH and kinetic properties investigated were not significantly different from those of the cytosolic enzyme. When rats were fed a riboflavin-deficient diet, the enzyme activity in the mitochondria decreased to a greater extent than that in the cytosol, and greater accumulation of apo-enzyme in the former than that in the latter was confirmed by the amount of immunoprecipitable protein, activation by FAD addition in vitro, and the enzyme activity recovery after injection of riboflavin, into riboflavin-deficient rats.

Weanling male rats were fed a riboflavin- or selenium-deficient diet for 5 weeks, and the glutathione content and its relating enzyme activities in the livers were examined. The glutathione content and glutathione reductase activity were decreased by deficiency of riboflavin, but not by that of selenium. Glutathione peroxidase activity was increased by addition of selenium to the diet, but without its addition, the activity was higher in the riboflavin-deficient rats than in the riboflavin-sufficient rats, in spite of the increase of lipid peroxides in the former rats. Glucose 6-phosphate dehydrogenase activity was decreased significantly by riboflavin deficiency. In another experiment, riboflavin was intraperitoneally injected into rats fed the diet to which neither riboflavin nor selenium had been added. The glutathione content and activities of glutathione reductase and glucose 6-phosphate dehydrogenase returned to the control level of riboflavin-sufficient rats in 24 h, the lipid peroxide level in 48 h, and the glutathione peroxidase activity, being higher than that in the control rats, in 72 h after the injection, respectively. These findings indicate that the increase of lipid peroxides in the livers of riboflavin-deficient rats is caused by the decrease in the glutathione content as well as glutathione reductase activity rather than by that in the selenium-dependent glutathione peroxidase activity.

Discordant hepatic uptake between Tc-99m HIDA and Tc-99m colloid occurred in a 63-year-old female with segmental biliary obstruction due to cholangioma. Radiographic CT and a percutaneous transhepatic cholangiogram revealed the obstructed right hepatic duct as well as the dilated intrahepatic duct in the right lobe. At surgery this was confirmed, and a 2 cm mass encasing the right hepatic duct was identified. It should be included in the gamut of discordant hepatic uptake of Tc-99m IDA and Tc-99m colloid.