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Histidine decarboxylase (HDC), histamine synthase, is expressed in hematopoietic stem cells and in lineage-committed progenitors in the bone marrow (BM). However, the role of histamine in hematopoiesis is not well described. To evaluate the role of histamine in hematopoiesis, we analyzed the changes in HDC expression at hematopoietic sites, the BM, spleen, and liver of 2-, 3-, and 6-week-old wild-type mice. We also performed morphological analyses of the hematopoietic sites using HDC-deficient (HDC-KO) mice. In wild-type adults, HDC expression in the BM was higher than that in the spleen and liver and showed an age-dependent increase. Histological analysis showed no significant change in the adult BM and spleen of HDC-KO mice compared to wild-type mice. In the liver, HDC expression was temporarily increased at 3 weeks and decreased at 6 weeks of age. Morphological analysis of the liver revealed more numerous hematopoietic colonies and megakaryocytes in HDC-KO mice compared to wild-type mice at 2 and 3 weeks of age, whereas no changes were observed in adults. Most of these hematopoietic colonies consisted of B220-positive B-lymphocytes and TER119-positive erythroblasts and were positive for the cell proliferation marker PCNA. Notably, these hematopoietic colonies declined in HDC-KO mice upon N-acetyl histamine treatment. A significant increase in the expression of hematopoiesis-related cytokines, Il3, Il7, Epo, Gcsf, and Cxcl12 mRNA was observed in the liver of 3-week-old HDC-KO mice compared to wild-type mice. These results suggest that histamine-deficiency may maintain an microenvironment suitable for hematopoiesis by regulating hematopoiesis-related cytokine expression in the liver of postnatal mice., (© 2021 American Association for Anatomy.)

Background: Chemotherapy after hepatectomy for colorectal liver metastasis has not been established, due to the toxic side effects, which are likely related to impaired drug clearance during liver regeneration. We investigated the pharmacokinetic and toxicodynamic evaluation of 5-fluorouracil (5-FU) during liver regeneration after major hepatectomy in a rat model., Methods: Thirty-six male Wistar rats were divided into control (C), control with chemotherapy (CC), hepatectomy (H), and hepatectomy with chemotherapy (HC) groups. The CC and HC groups were administered 5-FU for 4 days. Plasma 5-FU, liver weight, and liver dihydropyrimidine dehydrogenase (DPD) were measured. The ileal villous height was measured to determine adverse effects., Results: The area under the curve and maximum plasma concentration of 5-FU increased by up to 51% and 32%, respectively, in the HC group compared to the CC group. The liver regeneration rate was significantly lower in the HC group than in the H group (67.3 ± 7.4 vs 33.0 ± 5.7%, p < 0.001). The HC group had a significantly lower liver DPD than the CC group (4.4 ± 1.1 mg vs 6.9 ± 1.1 mg, p < 0.01). The HC group had a significantly lower ileal villous height than the CC group (253 ± 40 μm vs. 318 ± 36 μm, p < 0.05)., Conclusions: Reduction of the total liver DPD following major hepatectomy caused increased plasma 5-FU levels and 5-FU-associated toxicity.

Systemic platelet behaviors in experimental animals are often assessed by infusion of isotope-labeled platelets and measuring them under anesthesia. However, such procedures alter, therefore may not reveal, real-life platelet behaviors. 5-Hydroxytryptamine (5HT or serotonin) is present within limited cell-types, including platelets. In our studies, by measuring 5HT as a platelet-marker in non-anesthetized mice, we identified stimulation- and time-dependent accumulations in liver, lung, and/or spleen as important systemic platelet behaviors. For example, intravenous, intraperitoneal, or intragingival injection of lipopolysaccharide (LPS, a cell-wall component of Gram-negative bacteria), interleukin (IL)-1, or tumor necrosis factor (TNF)-α induced hepatic platelet accumulation (HPA) and platelet translocation into the sinusoidal and perisinusoidal spaces or hepatocytes themselves. These events occurred "within a few hours" of the injection, caused hypoglycemia, and exhibited protective or causal effects on hepatitis. Intravenous injection of larger doses of LPS into normal mice, or intravenous antigen-challenge to sensitized mice, induced pulmonary platelet accumulation (PPA), as well as HPA. These reactions occurred "within a few min" of the LPS injection or antigen challenge and resulted in shock. Intravenous injection of 5HT or a catecholamine induced a rapid PPA "within 6 s." Intravenous LPS injection, within a minute, increased the pulmonary catecholamines that mediate the LPS-induced PPA. Macrophage-depletion from liver and spleen induced "day-scale" splenic platelet accumulation, suggesting the spleen is involved in clearing senescent platelets. These findings indicate the usefulness of 5HT as a marker of platelet behaviors, and provide a basis for a discussion of the roles of platelets as both "defenders" and "guardians."

Transcription from chromosomes is regulated by posttranslational modifications to histones, such as methylation and ubiquitination. Monoubiquitination of histones H2A and H2B influences H3 methylation to reinforce the activation or repression of gene expression. Here, we provide evidence that H3 polyubiquitination represses transcription of fetal and cell-cycle genes in postnatal mouse liver by crosstalk with H3K9 methylation. We found that the CRL4 ubiquitin ligase targets H3 for polyubiquitination at K79 via the DCAF8 substrate receptor in hepatocytes. Genetic inactivation of DCAF8 and overexpression of an H3K79 mutant in cells or inducible deletion of CRL4 in mouse liver abrogates H3 ubiquitination, reactivates the expression of fetal liver and cell-cycle genes by interfering with methylated H3K9 occupancy, and leads to cell senescence. Restoring CRL4 DCAF8 expression in cells with decreased H3 ubiquitination reinstates the epigenetic gene silencing. Our results suggest that progressive H3 ubiquitination plays an important role in postnatal liver maturation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Reverse cholesterol transport (RCT) is pivotal in the return of excess cholesterol from peripheral tissues to the liver for excretion in bile and eventually feces. RCT from macrophages is a critical anti-atherogenicity mechanism of HDL. As the cholesterol absorption inhibitor ezetimibe promoted RCT in mice, which lack cholesterol ester transfer protein (CETP), we investigated its effects in hamsters, which have CETP. A high-cholesterol diet (HC) increased cholesterol levels throughout lipoprotein fractions and ezetimibe markedly reduced VLDL/LDL cholesterol levels under both normal chow (NC) and HC. However, ezetimibe did not affect and reduced HDL-cholesterol levels under NC and HC, respectively. Intraperitoneal injection of (3)H-cholesterol pre-labeled macrophages in an in vivo RCT assay increased tracer accumulation in the liver but reduced it in bile under HC, and these changes were completely cancelled by ezetimibe. Under both NC and HC, ezetimibe reduced tracer levels in the liver but increased them in feces, indicating promotion of RCT in vivo. We performed a RCT assay using hamsters subjected to bile duct ligation (BDL) to clarify whether a transintestinal cholesterol efflux (TICE) pathway contributes to ezetimibe's enhancement of RCT. BDL markedly inhibited macrophage-derived (3)H-cholesterol excretion to feces and cancelled ezetimibe's stimulatory effect on RCT, suggesting that biliary cholesterol excretion is a major contributor in RCT promotion by ezetimibe but the contribution of the TICE pathway is minimal. In conclusions, ezetimibe exerts an additive anti-atherogenic property by enhancing RCT in hamsters. Our findings suggest that this property is independent of the TICE pathway., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Oxaliplatin-based chemotherapy is widely used for advanced colorectal cancer treatment, but it occasionally induces liver injury that is characterized histologically by sinusoidal dilatation, hepatic plate atrophy and/or venular obstruction. Most of the patients do not reveal apparent radiological abnormalities, however. Here, we report the case of a 47-year-old man with a radiologically detectable mass-forming oxaliplatin-induced sinusoidal injury that mimicked multiple liver tumors. These mass lesions were found on computed tomography images after the administration of six cycles of folinic acid, fluorouracil and oxaliplatin therapy as adjuvant chemotherapy for Stage III rectal cancer. The patient had to undergo liver resection because imaging studies could not exclude metastases. The histological examination revealed that a resected mass lesion was composed of severe sinusoidal dilatation. Milder dilatation was also seen in the surrounding parenchyma. We diagnosed the patient as having an oxaliplatin-induced sinusoidal injury with severe deviation. As oxaliplatin is a standard agent in colorectal cancer therapy today, all clinicians and pathologists should be aware of such non-neoplastic lesions as one of the rare differential diagnoses of metastatic liver tumor, to prevent overtreatment.

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.

Platelets are reportedly causal in hepatitis. We previously showed that in mice, lipopolysaccharide (LPS) induces a reversible and macrophage-dependent hepatic platelet accumulation (HPA), including translocation of platelets into Disse spaces and their entry into hepatocytes. Concanavalin A (ConA), which induces hepatitis in mice via both T cells and macrophages, also induces HPA. Here, we examined the relationship between HPA and ConA-hepatitis. ConA-hepatitis and HPA were evaluated by serum transaminases, hepatic 5-hydroxytryptamine, and/or electron microscopy. Unlike LPS-induced HPA, ConA-induced HPA was only moderately dependent on phagocytic macrophages. Against expectations, platelet-depletion significantly exacerbated ConA-hepatitis, and anti-P-selectin antibody and P-selectin receptor blockade reduced both ConA-induced HPA and hepatitis. Prior induction of HPA by pretreatment with low-dose LPS powerfully reduced ConA-hepatitis. Such protection by LPS-pretreatment was not effective in mice depleted of phagocytic macrophages. In platelet-depleted mice, LPS-pretreatment severely exacerbated ConA-hepatitis. In mice depleted of both macrophages and platelets, neither ConA nor LPS-pretreatment+ConA induced hepatitis. In mice deficient in IL-1α and IL-1β (but not in TNFα), ConA-induced hepatitis was mild, and a protective effect of LPS was not detected. These results suggest that (i) there are causal and protective types of HPA, (ii) the causal type involves hepatic aggregation of platelets, which may be induced by platelet stimulants leaked from injured hepatocytes, (iii) the protective type is inducible by administration of prior low-dose LPS in a manner dependent on phagocytic (or F4/80-positive) macrophages, and (iv) IL-1 is involved in both the causal and protective types., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Etiologic risk factors for hepatocellular carcinoma can be involved in the transformation process by directly targeting intracellular signaling pathways or by indirectly stimulating chronic cycles of hepatocyte destruction and regeneration. However, the contribution of these two routes to hepatocarcinogenesis has not been determined, partly because of the difficulty in distinguishing damaged and regenerated hepatocytes. Here we report that induced deletion of the damaged DNA binding protein 1 (DDB1) abrogates the self-renewing capacity of hepatocytes, resulting in compensatory proliferation of DDB1-expressing hepatocytes. Constitutive stimulation of this regeneration process leads to development of hepatocellular carcinoma, which surprisingly contains no disruption of the DDB1 gene, indicating a cell-nonautonomous role of DDB1 inactivation in tumor initiation. Our results suggest that viruses and hepatoxins may cause liver tumors by simply driving hepatocyte turnover without directly targeting cancer cells.

Background: Adiponectin (APN), which is an adipose tissue-derived hormone, is known as an anti-inflammatory cytokine. The effects of APN on the production of inflammatory mediators and hepatic injury during polymicrobial sepsis were evaluated using APN-knockout (KO) mice that had undergone a cecal ligation and puncture (CLP) and rosiglitazone, a selective peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, which increases the plasma APN concentration., Materials and Methods: Wild type (WT) and APN-KO mice were underwent CLP. The plasma and hepatic levels of inflammatory mediators, including tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), were measured before, at 24, and 48 h after CLP. A histological analysis of the liver and the plasma alanine aminotransferase (ALT) levels were examined to evaluate hepatic injury. The plasma levels of inflammatory mediators after CLP with pretreatment of rosiglitazone were compared with those without rosiglitazone., Results: APN deficiency resulted in significant increases in the plasma levels of TNF-alpha, IL-6, and MCP-1 at 24 h after CLP. Hepatic MCP-1 and plasma AST levels in APN-KO mice were significantly higher than those in WT mice at 48 h after CLP. A steatosis change and MCP-1 expressions in hepatocytes were induced in APN-KO mice during sepsis. The administration of rosiglitazone significantly lowered the plasma levels of inflammatory mediators, including TNF-alpha, IL-6, and MCP-1, in WT mice but not in APN-KO mice during sepsis., Conclusion: These results suggest that an APN deficiency induces an excessive systemic inflammatory status and exacerbates hepatic injury during polymicrobial sepsis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Recent studies indicated that periodontitis induces systemic low-grade inflammation. The increase in systemic low-grade inflammation induced by periodontitis may alter the effects of obesity on the production of inflammatory molecules, including C-reactive protein (CRP), interleukin (IL)-6, and tumor necrosis factor-alpha (TNF-alpha), in the liver and white adipose tissue (WAT). The purpose of the present study is to investigate the effects of periodontitis on the expression of proinflammatory cytokines in the liver and WAT in obese Zucker rats., Methods: Obese Zucker rats and their lean litter mates were divided into four groups of six rats each: lean Zucker rats without periodontitis (control group), lean Zucker rats with periodontitis (periodontitis group), obese Zucker rats without periodontitis (obesity group), and obese Zucker rats with periodontitis (combination group). Periodontitis was ligature induced for 4 weeks in the periodontitis and combination groups, whereas the other groups were left unligated., Results: At 4 weeks, the gene expression for CRP, IL-6, and TNF-alpha in the liver and CRP and IL-6 in the WAT of combination groups was significantly higher than in each of the three groups. Serum TNF-alpha in the periodontitis and obesity groups was significantly higher than in the control group. Serum CRP and TNF-alpha in the combination group was significantly higher than in each of the three groups., Conclusion: Systemic low-grade inflammation after experimental periodontitis was associated with increased gene expression for hepatic levels of TNF-alpha and CRP and adipose tissue levels of IL-6 and CRP in the obese-rat model.

Platelets are reported to be causally involved in experimental hepatitis. Jo2, an agonistic anti-Fas antibody, induces hepatitis in mice. We examined the in vivo behaviors of platelets in mice injected with this antibody (analyzed by measuring 5-hydroxytryptamine, a constituent of platelets). We found that Jo2 induces platelet accumulation predominantly in the liver, and that this hepatic platelet accumulation (HPA) precedes the increases in hepatitis markers (alanine- and asparagine-aminotransferases [ALT and AST]). By electron microscopy, we detected entry of platelets into hepatocytes, and also evidence of apoptosis among hepatocytes. A caspases-3/6/7/8/10 inhibitor prevented the Jo2-induced HPA and hepatitis. In platelet-depleted mice, contrary to our expectations, the Jo2-induced hepatitis was not reduced, and actually the increase in AST was significantly augmented, although the survival time of mice given a lethal dose of Jo2 was significantly increased (nearly doubled). Interestingly, prior induction of HPA by a low dose of lipopolysaccharide markedly reduced Jo2-induced hepatitis. Jo2 also induced HPA and hepatitis in mice deficient in both IL-1 and TNFalpha, although Jo2 increased the blood level of TNFalpha in wild-type mice. These results suggest that in Jo2-induced hepatitis: (i) platelets accumulate predominantly in the liver as a result of hepatic lesions, and that this precedes the release of transaminases from hepatocytes, and (ii) IL-1 and TNFalpha are not essential for Jo2-hepatitis. We hypothesize that platelet accumulation in the liver may, contrary to our expectations, be protective when the hepatitis is local or not severe, but harmful when hepatitis is severe.

Objective: We investigated effects of Japanese torreya (Torreya nucifera) seed oil containing non-methylene-interrupted polyunsaturated fatty acid of all-cis-5,11,14-eicosatrienoic acid (sciadonic acid) on rat lipid metabolism., Methods: Male Sprague-Dawley rats were fed the experimental diets based on AIN-93 containing 10% corn, soybean, or torreya oil for 4 wk. Blood and tissues were recovered from each rat, and concentrations of triacylglycerol, cholesterol, and phospholipid in plasma and liver were determined by enzymatic assays. Moreover, fatty acid composition was analyzed for triacylglycerol, cholesterol ester, and phospholipid isolated from plasma and liver lipids by gas liquid chromatography., Results: Plasma triacylglycerol level in rats fed torreya oil was lower than that in rats fed corn or soybean oil, although there were no significant differences in plasma cholesterol and phospholipid levels in all rats. Liver triacylglycerol level was also lower in rats fed torreya oil, whereas liver cholesterol and phospholipid levers were same for all rats. omega-3 Polyunsaturated fatty acids such as 22:6 (omega-3) were lower in plasma and liver lipids of torreya and corn oil groups, whereas omega-6 polyunsaturated fatty acids such as 22:4 (omega-6) and 22:5 (omega-6) were higher. Considerable amounts of sciadonic acid were detected in cholesterol ester, triacylglycerol, and phospholipid in plasma and liver of rats fed torreya oil., Conclusion: These observations suggest that torreya seed oil can modify lipid metabolism, resulting in lower triacylglycerol levels in plasma and liver of rats.

Intravenous injection of Klebsiella O3 lipopolysaccharide (LPS) into BALB/c mice induces an anaphylaxis-like shock within minutes. Using 5-hydroxytryptamine as a marker for platelets, we previously suggested that a rapid platelet accumulation in the liver and lung precedes the shock, and that a complement-dependent platelet-degradation is involved in the shock. Here, we examined (i) the effect of platelet-depletion (using an anti-platelet monoclonal antibody) on the shock and (ii) the contribution of macrophages to the platelet-accumulation in those organs. LPS-induced platelet-accumulations in the liver and lung were confirmed by immunostaining. In platelet-depleted mice, the shock was largely prevented. The number of F4/80-positive macrophages was much greater in liver than in lung, and the hepatic macrophages were largely lost in mice given clodronate-encapsulated liposomes. In mice treated with such liposomes, both the LPS-induced accumulation of platelets in the liver (but not in the lung) and the shock were largely prevented, and repopulation of hepatic macrophages restored these LPS-induced responses. These results suggest that (i) platelets are indeed involved in the shock, (ii) Kupffer cells mediate the hepatic platelet accumulation, and (iii) preventing this hepatic accumulation can largely prevent rapid shock being induced by LPS (at the dose used here).

There have been many reports about the severity of hepatic necrosis caused by fulminant hepatitis; however, the relation between proliferated bile ductules and osteopontin (OPN) expression in inflamed areas in each of the clinical forms of fulminant hepatitis has not been described. To analyze the mechanism in the onset of fulminant hepatitis, we classified not only 16 autopsy cases of fulminant hepatitis into two clinical forms--acute and subacute--but also 3 autopsy cases of late-onset hepatic failure (LOHF) associated with fulminant hepatitis, and examined liver specimens by light microscopy and immunohistochemistry and also serum transaminase levels. Histopathologic study revealed that some of the proliferated bile ductules were associated directly with deteriorating hepatocytes and that bile plugs were present in the proliferated bile ductules. The value of the proliferative cell nuclear antigen labeling index (PCNA-L I) for proliferated bile ductules was very high during the acute form of fulminant hepatitis. Immunohistochemical analysis revealed that OPN expression was higher in the proliferated bile ductules of acute-form fulminant hepatitis than in cirrhotic and normal liver bile ducts. Transaminase levels in acute-form fulminant hepatitis were significantly elevated in comparison with levels in the other forms of the disease. Comparison of acute form fulminant hepatitis with the subacute form and LOHF showed OPN expression in proliferated bile ductules and serum aspartate aminotransferase (ALT)max to be decreased in the subacute form of fulminant hepatitis. OPN expression is an important marker of the degree of liver inflammation, and its regulation mechanism is very important to understanding the pathophysiology of fulminant hepatitis.

Since O(2) is the bare necessity for multicellular organisms, they develop multiple protective mechanisms against hypoxia. Mammals will adapt to hypoxia in short and long terms. The short-term responses include enhancement of the respiratory and cardiac functions, adrenaline secretion from adrenal medullary cells, and pulmonary vasoconstriction, whereas the long-term response is the increase in erythropoietin production with the consequent increase in red blood cells. Although much work has been done to elucidate molecular mechanisms for O(2)-sensing for the last ten years, the majority of the mechanisms remain unclear. We will review mechanisms proposed for hypoxia detection in carotid body type I cells, pulmonary artery smooth muscle, adrenal medullary cells, and liver cells, with the special focus on adrenal medullary cells.

To gain a comprehensive understanding of the molecular mechanisms underlying the effects of dietary protein on alternations in physiologic and pathologic status, the GeneChip microarray system was used to investigate the consequences of two different states of protein malnutrition on gene expression in rat liver. Expressions of 281 genes were increased or decreased by twofold or more by treatment with a protein-free diet for 1 wk compared with control rats fed a casein diet. Similarly, 111 genes were affected in rats fed a wheat gluten diet compared with those fed the casein diet. Although some of the genes identified were known to respond to protein nutrition, a majority were newly identified as responders to protein nutritional status. Interesting findings included the drastic changes in the levels of genes for Id (inhibitor of DNA binding) proteins, which are involved in the regulation of multiple genes, and of a set of genes in the pathway of cholesterol biosynthesis and disposal. This study represents a step toward a more global understanding of gene expression changes in states of protein malnutrition.

We report on a case of metastatic adenocarcinoma of liver that was removed and examined histochemically after microwave coagulation therapy (MCT). The patient was a 65-year-old woman who had a metastatic tumor in the liver (S3) after high anterior resection due to a rectal adenocarcinoma and received MCT against the tumor. One month after MCT, multiple metastatic tumors were detected by abdominal computed tomography (CT) scan. As it was difficult to control them by MCT alone, we performed lateral segmentectomy. To assess the effects of microwave ablation on cellular viability of metastatic tumor, we used enzyme histochemistry for acid phosphatase (AcP), which is positive in macrophages infiltrating in the tumor. In a part of the ablated area of resected liver, there was remaining neoplastic tissue of which the morphology was maintained in H&E staining. This was found to be microwave-fixed non-viable tissue because no enzyme activity of AcP was detected in the infiltrating macrophages. This case report suggests that enzyme histochemistry was useful to assess the effect of MCT, enabling us to distinguish fixed cells from viable cells.

1. Mast cells and basophils are believed to trigger allergic reactions and anaphylaxis. They rapidly release histamine (H), a typical mediator of inflammation, in response to antigens. In the mouse, platelets contain much 5-hydroxytryptamine (5HT), an additional inflammatory mediator, while human platelets contain both H and 5HT. Here, we examined the response of platelets in sensitized mice to antigen challenge. 2. Platelets accumulated in the lung and liver almost immediately after intravenous injection of ovalbumin (OVA), in mice sensitized to it, and platelet degranulation occurred during these reactions. 3. These responses of platelets preceded H release from mast cells and/or basophils, occurred at doses of OVA lower than those inducing H release, and contributed to the signs of shock. 4. We reported previously that intravenous injection into mice of LPS (a membrane constituent of gram-negative bacteria) induces a similar platelet response (accumulation of platelets in the lung and liver) and shock. 5. Blood that has passed through the body (other than the digestive tract) passes first to the lungs before being recirculated by the heart, and blood that has passed through the digestive tract passes next to the liver. Thus, our findings suggest that in addition to their role in haemostasis, platelets, tiny anuclear cytoplasts, may be important in both innate and acquired immunity, and that the lung and liver may be the fronts at which platelets wage war on pathogens.

A second female-predominant murine CYP3A, CYP3A44, was isolated from liver and its mRNA expression was compared with that of the previously described CYP3A41. The expression of CYP3A44 was relatively constant after birth in females, whereas it gradually declined in males after 5 weeks of age. The expression of CYP3A41 increased with age in females after 3 weeks of age, whereas it gradually declined in males after 5 weeks of age. Hypophysectomy and growth hormone replacement indicated that expression of both CYP3A mRNAs in females was dependent on the feminine plasma growth hormone profile. Estradiol induced the expression of both mRNAs and the effect was dependent on the presence of the pituitary gland. These observations suggest that endocrine control of expression might be similar, but not identical, for two female-predominant CYP3A mRNAs., (Copyright 2002 Elsevier Science (USA).)

We investigated the relationship between the motility of hepatic Ito cells and their myofibroblastic transformation in cultures. Ito cells were freshly isolated from rat liver and cultured in a 10% FBS-supplemented medium. On day 2 after beginning the culture, transmission electron microscopy and oil red O staining showed that Ito cells possessed numerous lipid droplets but not actin filaments in the cytoplasm. On day 8, actin filaments were abundantly found in the cytoplasm, whereas lipid droplets dramatically decreased in number. Western blot analysis also demonstrated that protein levels of alpha-smooth muscle actin in the cell markedly increased with time, but no obvious change was detected in those of desmin and tubulin beta. In Boyden's chamber assay, the migration of Ito cells, which was induced by platelet-derived growth factor-BB (PDGF-BB), was activated in a time-dependent manner. This migration of transformed Ito cells was independent of the degree of their adhesion to various substances of the extracellular matrix. Among these molecules, laminin showed the highest effect upon the migratory activity. The migration of transformed Ito cells on laminin was completely inhibited by cytochalasin D, colchicine, or taxol. Furthermore, their adhesion to the matrix was also decreased by cytochalasin D or colchicine, but not by taxol. These findings indicate that the motility of Ito cells is acquired in conjunction with their myofibroblastic transformation, which is accompanied by morphological changes with a new organization of actin filaments. The results also suggest that microtubules as well as the extracellular matrix are deeply associated with the motility of Ito cells.

The biosynthetic properties of wax esters in the liver were compared between two types of myctophid fishes having different body lipid composition, i.e., three triglyceride-rich species (Lampanyctus jordani, Diaphus theta, and Symbolophorus californiensis) and three wax ester-rich species (L. regalis, Stenobracius nannochir, and Stenobracius leucopsarus). n-Heptadecanol (17:0-ALC) and/or 10-cis-heptadecenoic acid (17:1-ACID) was incubated with liver homogenate of the six myctophid fishes and with co-factors such as NADPH and ATP for 2 to 5 h. Considerable amounts of wax esters with odd-numbered fatty acids and/or alcohols were produced in the liver homogenate of the wax ester-rich species. Stenobracius nannochir and L. regalis, which exclusively contained wax esters as neutral lipids, showed the highest activity of wax ester synthesis, followed by S. leucopsarus, which contained triglyceride as the minor constituent. Only trace amounts at most of odd-numbered fatty acids and alcohols were incorporated into the wax esters after incubation with the liver homogenates of the triglyceride-rich fishes. Active interchange between the fatty acids and the alcohols occurred during wax ester biosynthesis in the wax ester-rich fishes. The chain elongation and shortening of acyl moieties were also observed during incubation. These results suggested that the deposition of lipids in myctophid fishes is mainly due to their biosynthetic activities.

Treatment of 30S-5SRNP with 1 M Cs(2)SO(4) at 2 degrees C overnight followed by sucrose density-gradient centrifugation yielded particles smaller than 30S-5SRNP, designated as CsS-particles. CsCl density-gradient centrifugation of CsS-particles showed the homogeneity of the particles containing about half the amount of proteins in 30S-5SRNP particles. The particles contained 18SrRNA, 5SRNP and about half the number of proteins in 30S-5SRNP. The ATPase activity of freshly prepared CsS-particles was about half the original 30S-5SRNP level although it was unstable even at 2 degrees C. Poly(U) slightly enhanced the activity, and phe-tRNA(phe) stimulated it concentration-dependently. EF-1a alone enhanced it, and in combination with poly(U) and phe-tRNA(phe) stimulated it markedly. EF-2 alone markedly increased it. The activity with the full components for elongation described above became very high, being comparable to that of the original 30S-5SRNP and twice that of 40S subunits. A two-dimensional electrophoretogram of the protein in CsS-particles revealed 9 small subunit protein species, in addition to L5, which included proteins interacting with mRNA and two elongation factors. Taken together with the results of our preceding study indicating the participation of ATPase of 80S ribosomes in peptide elongation, the present results indicate CsS-particles may be a part of the ATPase centre of 80S ribosomes.

Expression of a female-specific CYP3A in the adult mouse liver was observed on immunoblotting analysis. To characterize this cytochrome P450, we determined the primary structure of its cDNA and examined its expression profile. This cytochrome P450 consisted of 504 amino acids and showed 92, 68, 88, and 69% amino acid sequence identity with mouse CYP3A11, 3A13, 3A16, and 3A25, respectively, and was designated as CYP3A41, a new mouse CYP3A gene. In the female liver, levels of CYP3A41 mRNA expression were comparable to those of CYP3A11, the major CYP3A enzyme in the adult mouse liver. Expression of CYP3A41 mRNA was detected immediately after birth in the livers of animals of both sexes, but increased with age in females, whereas it was gradually reduced in males, resulting in predominantly female-specific expression in livers. Lesser amounts of CYP3A41 mRNA were detected in the kidneys of female mice, with traces in the stomach, ovary, and heart of female mice and in the testis of male mice. Gonadectomy and sex hormone treatment indicated that estradiol and testosterone were able to induce and suppress the expression of CYP3A41 mRNA in the liver, respectively. Among the classical CYP3A inducers, dexamethasone, rifampicin, and 3-methylcholanthrene did not affect the level of CYP3A41 mRNA in the liver of either sex. On the other hand, pregnenolone 16alpha-carbonitrile and phenobarbital suppressed CYP3A41 level to half that of untreated female mice. These observations indicated that CYP3A41 is a female-specific CYP3A and one of the major CYP3A forms in the female mouse liver., (Copyright 2000 Academic Press.)

Effects of raw and gelatinized sago and tapioca starches on serum and liver lipid concentrations were compared in rats fed a cholesterol-enriched diet. Total serum cholesterol and the atherogenic index in the rats fed raw sago starch showed a statistically significant decrease and tendency to decrease, respectively, when compared with those of rats fed raw tapioca starch. Both the serum total cholesterol and atherogenic index in the rats fed gelatinized sago starch showed a tendency to decrease when compared with those of rats fed gelatinized tapioca starch. Serum triacylglycerol concentrations in the rats fed raw sago starch were significantly lower than those in the rats fed raw tapioca starch. The apparent digestibility of raw sago starch was lower than that of tapioca starch. The oval shapes of undigested raw sago starch granules collected from feces remained almost unchanged in shape, but had some holes and scrapes on their surfaces. The shape surfaces of undigested raw tapioca starch collapsed, thereby not retaining their original granule shapes. On the other hand, the amylose contents in the raw and gelatinized sago starches were higher than those in the raw and gelatinized tapioca starches. These results suggested that a decrease or a tendency to decrease in serum total cholesterol levels and atherogenic indexes in raw or gelatinized sago starch-fed rats compared to those of raw or gelatinized tapioca starch-fed rats, and a decrease in serum triacylglycerol concentrations in raw sago starch-fed rats compared to those of raw tapioca starch-fed rats, were due to the lower digestibility and higher amylose content of sago starch.

The properties and role in peptide elongation of ATPase intrinsic to rat liver ribosomes were investigated. (i) Rat liver 80S ribosomes showed high ATPase and GTPase activities, whereas the GTPase activity of EF-1alpha and EF-2 was very low. mRNA, aminoacyl-tRNA, and elongation factors alone enhanced ribosomal ATPase activity and in combination stimulated it additively or synergistically. The results suggest that these translational components induce positive conformational changes of 80S ribosomes by binding to different regions of ribosomes. Translation inhibitors, tetracyclin and fusidic acid, inhibited ribosomal ATPase with or without elongational components. (ii) Two ATPase inhibitors, AMP-P(NH)P and vanadate, did not inhibit GTPase activities of EF-1alpha and EF-2 assayed as uncoupled GTPase, but they did inhibit poly(U)-dependent polyphe synthesis of 80S ribosomes. (iii) Effects of AMP-P(NH)P and ATP on poly(U)-dependent polyphe synthesis at various concentrations of GTP were examined. ATP enhanced the activity of polyphe synthesis even at high concentrations of GTP, suggesting a specific role of ATP. At low concentrations of GTP, the extent of inhibition by AMP-P(NH)P was very low, probably owing to the prevention of the reduction of the GTP concentration. (iv) Vanadate inhibited the translocation reaction by high KCl-washed polysomes. These findings together indicate that ribosomal ATPase participates in peptide translation by inducing positive conformational changes of mammalian ribosomes, in addition to its role of chasing tRNA from the E site.

We found unique behaviors among platelets within a few minutes of the intravenous injection of lipopolysaccharide (LPS) into mice. Platelets accumulated primarily in the liver at lower doses of LPS, but at higher doses they accumulated largely in the lungs. When the platelets accumulated in these organs were degraded, there was a rapid anaphylactoid shock. The platelet response depended on the strain of mouse and on the source of LPS. Of various LPSs tested, the LPS from the smooth type of Klebsiella O3 (KO3-S LPS) was the most potent at inducing the platelet response and shock. K-76 monocarboxylic acid, an inhibitor of complement C5, effectively prevented the KO3-S LPS-induced degradation (but not accumulation) of platelets and the ensuing rapid shock in BALB/c mice. Moreover, in DBA/2 mice (which are deficient in complement C5), platelets accumulated in the lungs and liver in response to KO3-S LPS but soon returned to the circulation without degradation, and there was no rapid shock. The LPS from the rough type of KO3 induced an accumulation of platelets in the liver and lungs but not a degradation of platelets. On the basis of these results and those reported by other investigators, we propose that in the platelet response to LPS, the lectin pathway to form C3 convertase from C4 and C2 is involved in the rapid accumulation of platelets in the liver and lungs and that the pathway from C5 to C9 is involved in the destruction of platelets and the consequent anaphylactoid shock.

1. D-Galactosamine (GalN) depletes UTP primarily in the liver, resulting in decreased RNA synthesis in hepatocytes. Co-injection of GalN and lipopolysaccharide (LPS) into mice produces fulminant hepatitis with severe hepatic congestion, resulting in rapid death. Although the underlying mechanism is uncertain, GalN enhances the sensitivity to tumour necrosis factor (TNF). Administration of uridine (a precursor of UTP) prior injection of either LPS itself or interleukin-1 (IL-1) reduces the lethality of GalN+LPS. The present study focused on the effects of these agents on TNF production. 2. Intraperitoneal injection of GalN+LPS into mice greatly elevated serum TNF. Although large doses of LPS alone also greatly elevated serum TNF, LPS itself induced neither hepatic congestion nor rapid death. Administration of a macrophage depletor, liposomes encapsulated with dichloromethylene bisphosphonate, reduced both the TNF production and mortality induced by GalN+LPS. 3. Uridine, when injected 0.5 h after the injection of GalN+LPS, reduced the production of TNF. Prior injection of LPS, but not of IL-1, also reduced this TNF production. 4. Serum from LPS-injected mice reduced the TNF production induced by GalN+LPS, but it was less effective at reducing the lethality. Its ability to reduce TNF production was abolished by heat-treatment. 5. We hypothesize that a factor inhibiting TNF production by macrophages is produced by hepatocytes in response to LPS. Possibly, production of this hepatocyte-derived TNF-down-regulator (TNF-DRh) may be: (i) inhibited by GalN, causing over-production of TNF by macrophages and (ii) stimulated by LPS-pretreatment (and restored by uridine), causing reduced TNF production.

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 microM led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [alpha-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3'-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5'- or 3'-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule.

Background/aims: Injection into mice of a small dose of either a lipopolysaccharide or interleukin-1 induces a slowly developing accumulation of 5-hydroxytryptamine, predominantly in the liver. We have established that this 5-hydroxytryptamine accumulation is the result of the translocation of platelets to hepatic sinusoidal spaces and, further, into Disse spaces, and that the platelets make direct contact with hepatocytes. In the present study, we report our recent findings on this phenomenon., Methods: Platelets contain a large amount of 5-hydroxytryptamine, but the 5-hydroxytryptamine content of the liver is normally very small. Therefore, the translocation of platelets to the liver was assessed by measuring 5-hydroxytryptamine as in previous studies, and it was also analysed by electron microscopy., Results: Anti-platelet agents, such as heparin and inhibitors of prostaglandin synthesis, were ineffective in preventing the lipopolysaccharide-induced accumulation of 5-hydroxytryptamine in the liver. Of the various cytokines tested, only interleukin-1 and tumour necrosis factor induced such an accumulation of 5-hydroxytryptamine. Intravenous injection of liposomes encapsulating dichloromethylene bisphosphonate resulted in an almost complete depletion of macrophages from the liver. The lipopolysaccharide- and cytokine-induced hepatic accumulations of 5-hydroxytryptamine were abolished almost completely in such macrophage-depleted mice. Electron microscopy revealed no accumulation of platelets in the liver after injection of lipopolysaccharide into the macrophage-depleted mice. Surprisingly, in normal mice injected with lipopolysaccharide, several platelets were found inside some hepatocytes, even though there was no visible damage to these hepatocytes. In fact, there were many polysomes around the degranulated platelets within the hepatocytes, suggesting an enhanced protein synthesis., Conclusion: These results suggest that, in response to lipopolysaccharide, interleukin-1 or tumour necrosis factor, platelets translocate into the liver in a way that is different from aggregation, and that some, at least, enter hepatocytes. During these processes, hepatic macrophages play an essential role.

1. Injection of lipopolysaccharides (LPS) or endotoxin into mice and rats induces a prolonged increase in serotonin (5-hydroxytryptamine: 5HT), predominantly in the liver. 2. The 5HT increase reflects the accumulation of platelets in the sinusoidal and perisinusoidal Disse spaces (spaces between endothelial cells and hepatocytes) in the liver. 3. Most of the platelets which accumulated in these spaces still retained their intact structure and a large amount of 5HT. 4. Interleukin-1 and/or tumor necrosis factor also induce the platelet response. 5. Kupffer's cells play a key role in this platelet response. 6. Anti-platelet drugs currently used, except for anti-inflammatory steroids, were ineffective in preventing the platelet response. 7. This platelet response is different from the well known platelet aggregation. 8. The possible involvement of this platelet response in insulin-independent hypoglycaemia, disseminated intravascular coagulation, septic shock, hepatitis, Shwartzman type reactions or self-defense mechanisms is discussed.

We investigated the synergistic efficacy of CDDP and UFT, an oral anticancer derivative of 5-fluorouracil, on DNA synthesis of 3'-MeDAB-induced hepatocarcinomas in rats. Chronic treatment with CDDP slightly reduced tissue activities of thymidylate synthetase and thymidine kinase in the hepatocarcinomas, and, moreover, simultaneous administration of a low dose of UFT with CDDP markedly suppressed both enzyme activities, i.e., UFT and CDDP showed a potent synergism on DNA synthesis in hepatocarcinomas.

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.

The cDNA clone encoding HMG 2a of chick liver was isolated from a lambda gt11 expression library using polyclonal antibodies. DNA sequence analysis revealed an open reading frame of 201 amino acids. Comparison of the nucleotide sequences of cDNA coding for chick liver HMG 2a with pig thymus HMG 2 and human monocytic leukemia cell HMG 2 showed 70% homology. 2.0 kb and 1.2 kb mRNAs were found in newly hatched chick liver and decreased during postnatal development of chicks.

1. Injection of lipopolysaccharide (LPS; 0.5-500 microgram kg-1) into mice induced a dose-dependent, slowly developing increase in hepatic content of 5-hydroxytryptamine (5-HT). This sustained increase could not be attributed to an LPS-induced alteration of the pharmacokinetic handling of 5-HT by stimulation of its uptake or inhibition of its degradation. 2. Regional differences were apparent in the tissue content of histamine and 5-HT between mast cell-deficient (W/Wv) and normal (+/+) mice. LPS administration (0.5 mg kg-1) gave comparable increases in the hepatic level of 5-HT in mast cell-deficient and normal mice. 3. Reserpine pretreatment (1 mg kg-1) selectively reduced 5-HT levels in the blood, spleen, liver, brain and lung of normal mice. Prior treatment with this agent also abolished the LPS (0.5 mg kg-1)-induced hepatic accumulation of 5-HT. 4. Accumulation of 5-HT in the liver by LPS (0.1 mg kg-1) was temporally associated with both a fall in the levels of circulating platelets, and a reduction in the concentration of 5-HT in the blood. The LPS dose-dependent (0.5-500 micrograms kg-1) increase in hepatic 5-HT content was associated with a similar dose-dependent reduction in the circulating levels of 5-HT. 5. Interleukin-1, alpha and beta (10 micrograms kg-1) and tumour necrosis factor alpha (TNF alpha) (1 mg kg-1) significantly enhanced the accumulation of 5-HT within the liver. Administration of TNF alpha (10 micrograms kg-1) potentiated the increase in hepatic 5-HT content seen with IL-1 beta (10 micrograms kg-1). 6. Electron microscopy revealed numerous platelets in the sinusoidal and perisinusoidal Disse spaces within the liver, in animals pretreated with LPS (0.1 mg kg '). The platelets retained their intact structure and showed no evidence of degranulation. 7. These data suggest that the LPS and cytokine-induced mobilization of 5-HT in the liver is associated with the hepatic translocation of platelets. This migration appears to be independent of platelet aggregation.

By means of a highly sensitive and quantitative assay for specific detection of metastasized tumor cells in chick embryonic organs using the polymerase chain reaction (PCR), we have examined the experimental metastatic ability of individual clones of NIH 3T3 cells, transformed with oncogenes: v-Ki-ras, v-Ha-ras, v-src, v-fos, and v-abl. Such a transformed clone had different metastatic abilities in different embryonic organs. Among them, two clones of NIH 3T3 cells transformed with ras-oncogenes (v-Ki-ras or v-Ha-ras) metastasized to liver and lungs of chick embryo, and grew there more rapidly than the other clones. The parental NIH 3T3 cells were detected as slight bands of PCR products after iv injection, indicating some cells were trapped in chick embryonic organs, but did not grow. These findings indicate that the transformed cells are able to invade the organ tissues and grow in embryonic chick organs, but non-metastatic cells such as the untransformed-NIH 3T3 cells are not able to grow in the secondary sites. These experiments clearly demonstrate the usefulness of this assay system to study genes involved in malignant transformation.

Administration of tryptophan or hydrocortisone to rats caused a several-fold increase in tryptophan-2,3-dioxygenase activity in the liver. Highly purified lysosomes were isolated from livers of tryptophan- or hydrocortisone-treated animals as well as the control rats. Immunoblotting of lysosomal proteins with anti-tryptophan-2,3-dioxygenase showed 48 kDa band, corresponding to the subunit molecular weight of the enzyme. The relative amount of the immuno-reactive substance in the lysosomes from hydrocortisone-treated rats was 3 times higher than the control while the value in the lysosomes from tryptophan-treated rats was essentially the same as in the control. These results indicate that administration of tryptophan renders cytosolic tryptophan-2,3-dioxygenase less vulnerable to lysosomal uptake and causes an accumulation of the enzyme in the cytosol.

Endotoxins or lipopolysaccharides (LPS), when injected into mice, increase serotonin (5-hydroxytryptamine; 5HT) in the liver and produce hypoglycaemia. In the present study, it was found that the cytokines produced by macrophages in response to LPS, interleukin-1 (IL-1, 0.1 microgram/kg or more) and tumour necrosis factor (TNF alpha, 100 micrograms/kg or more), can also induce an increase in liver serotonin and produce hypoglycaemia. The two responses correspond well with each other in terms of time course and dose dependency. These results suggest that the two responses induced by LPS may be mediated by IL-1 and/or TNF alpha and that the phenomenon of accumulation of 5HT in the liver may be relevant to hypoglycaemia. The hypoglycaemic response to IL-1 was moderate at a wide dose range but was induced by smaller amounts than with insulin.

Glucocorticoid-binding activities of the granuloma cytosol were compared with those of the liver cytosol and of the serum in vitro. The granuloma cytosol bound cortisol (HC) about 4-fold higher than dexamethasone (DX) and triamcinolone acetonide (TA); the liver cytosol bound these two synthetic agonists more than HC. The kinetic parameters of the glucocorticoid-binding components of the granuloma and the liver cytosols were studied by the Scatchard method. The binding components of the granuloma cytosol had a single class of binding sites with high affinity for these three steroids, whereas the binding site of the liver cytosol had negative cooperativity or consisted of two distinct classes, because its Scatchard plot showed a hyperbolic curve. The granuloma glucocorticoid-binding components will be protein since their binding was prevented by a trypsin treatment and completely lost by heating at 60 C for 5 min. Heating at 25 or 37 degrees C for 30 min did not affect the HC-binding activity of the granuloma cytosol, regardless of prelabeling with the steroid. The binding activity for DX and TA were heat-labile and completely lost by heating the cytosol at 37 degrees C for 30 min without the respective steroid. The results of thermal inactivation and ammonium sulfate fractionation show the granuloma HC-binding protein closely resembles corticosteroid-binding globulin (CBG). From enzymatic determination of hemoglobin in tissue cytosols, attribution of the contaminating blood to the HC-binding activity of the cytosol is considered to be negligible.

It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced by such azo dye carcinogens as 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). Thymidine kinase (TK) catalyzes the formation of deoxythymidine monophosphate (dTMP) by the phosphorylation of thymidine via the salvage pathway. In the present study, we investigated serum alpha-fetoprotein (AFP) and TK levels, and tissue TK and its isozyme activities in the liver of rats treated with 3'-MeDAB. Serum TK activities rose abruptly directly after the onset of 3'-MeDAB treatment, peaking after 1 week and then gradually decreasing. At 3 weeks, though serum TK was decreasing, serum AFP and tissue TK began to increase, and oval cells appeared in the liver. At 5 weeks, though serum TK reached a nadir, serum AFP and tissue TK formed transient peaks, and oval cells occupied a major part of the hepatic lobules with hyperplastic nodules. Thereafter, serum TK continued to increase, and serum AFP and tissue TK, after transiently decreasing, re-increased; at 20 weeks, each value was at high level, and mixed type hepatocarcinoma was observed. The liver TK isozymes were separated into three types by DEAE-cellulose column chromatography. A 3'-MeDAB diet induced a remarkable increase in activity of cytosolic and fetal type isozyme in non-tumorous regions of livers at 5 weeks and tumorous regions at 20 weeks. These results indicate that early biochemical changes in 3'-MeDAB-induced hepatocarcinoma in rats may serve as a useful model and provide a valuable insight in hepatocarcinogensis.

168 HBAg seropositive and 105 HBAg seronegative liver biopsies were studied for correlations between anti-HBc titers (indirect immunofluorescence method) and tissue expression of HBsAg and HBcAg (immunofluorescence), Dane particles in blood (immune electron microscopy) and inflammatory reaction. 98.8% of the HBAg seropositive patients were positive for anti-HBc. The mean titers showed statistically significant differences mainly between chronic aggressive hepatitis (1:2(11.3)) versus lobular hepatitis (1:2(10.1)), chronic persistent hepatitis (1:2(9.9)) and nonspecific reactive hepatitis (1:2(7.6)). Due to the considerable deviation of titers within the histological groups, however, titers below 1:2(11) are of low diagnostic relevance, whereas titers above 1:2(12) are mainly indicative of chronic aggressive hepatitis, although acute lobular hepatitis with signs of possible transition to chronicity or chronic persistent hepatitis with strong inflammatory activity may occur. Among HBAg seronegative patients 20% were positive for anti-HBc (mean titer = 1:2(7.7)). Among 78 patients also tested for anti-HBs, 10.2% were positive for both anti-HBc and anti-HBs. In an additional 12.8%, anti-HBc was the only marker of past hepatitis B virus infection. Anti-HBs was the only marker in a further 33%. In none of the HBAg seronegative patients and in only 59% of all HBAg seropositive patients, there was an association of anti-HBc with complete virus synthesis as measured by the demonstration of HBcAg in tissue or Dane particles in blood. It is concluded that anti-HBc is not a criterion of infectiosity but a specific, although non-characteristic, marker for HBAg seropositive acute and chronic hepatitis as well as for terminated HBV infection of all possible inflammatory and HBAg expression types.

When rat liver was pulse-labeled with [3H]valine in vivo, the nascent peptide on membrane-bound polysomes was found to be more highly labeled than that on free polysomes. Nascent peptides were purified from both classes of polysomes and, after hydrolysis, the amino acids were reacted with 14C-labeled 1-fluoro-2,4-dinitrobenzene. The specific activity of [3H]valine was determined from the [14C]-dinitrophenyl-[3H]valine after purification by two-dimensional thin layer chromatography. With this approach we found that the specific activity of [3H]valine in the nascent peptide of membrane-bound polysomes was more than twice that of free polysomes. Moreover, when rats were pretreated with a lysosomal protease inhibitor, the differences between the specific activities of valine in nascent peptides of the two classes of polysomes tended to decrease. Our results indicate the existence of two distinct pools for valine used for protein synthesis in liver cells; one serves as a precursor for the synthesis of secretory proteins on membrane-bound polysomes and the other as a precursor for the synthesis of intracellular proteins on free polysomes.

After the injection into mice of culture medium of P388D1 cells, a murine macrophage cell line, 5-hydroxytryptamine accumulated in the liver and blood glucose declined. The factors capable of inducing these responses were purified by gel filtration and chromatofocusing. With these procedures, the activity to induce the increase in 5-hydroxytryptamine in the liver accompanied the activity to induce hypoglycaemia. Moreover, through the purification, the factors were found in the fraction of interleukin 1, a lymphocyte-activating factor. These results suggest that the factors capable of inducing the increase in 5-hydroxytryptamine and hypoglycaemia are likely to be interleukin 1 molecules or molecules closely related to interleukin 1. The present and previous findings together with those in the literature support the idea that the increase in 5-hydroxytryptamine in the liver might be a cause of hypoglycaemia. These findings may provide new and important information about the roles of macrophages in inflammation or in immune responses.

A simple method for the assay of bradykinin (BK)-degrading enzymes was investigated. The procedure of the method includes enzymatic degradation of BK, separation of the residual BK on a small P-cellulose column (0.6 X 3 cm), and its fluorometrical determination based on the reaction with fluorescamine. BK was separated completely from its fragments produced during enzymatic reaction by the column chromatography. The recovery rate of BK was 96 +/- 3%. Quantitative determinations could be carried out on 0.2 nmol of BK, at least in the fluorometry. This method was available for the assay of the enzymes in tissue homogenates as well as in purified preparations, and its usefulness for the study of the enzymes is presented.

Mitochondrial ornithine aminotransferase was synthesized in a rabbit reticulocyte cell-free system using polysomal mRNA from rat liver. Upon analyzing the primary translation product by electrophoresis and fluorography, we found that ornithine aminotransferase is synthesized not as a large precursor but as one with its authentic size. Free ribosomes were found to be the main site of synthesis of ornithine aminotransferase.

Various mitogenic substances (concanavalin A, pokeweed mitogen, polyI : polyC and a phorbol diester), as well as lipopolysaccharides (LPS or endotoxins), produced hypoglycaemia after being injected into mice. However, non-mitogenic immuno-stimulants (zymosan, carrageenan, an adjuvant peptide and interferon) did not induce hypoglycaemia. All of the mitogenic substances also induced an increase in 5-hydroxytryptamine (5-HT) in liver, but the non-mitogenic substances did not have this effect. The time course of the development of hypoglycaemia was similar to that of the increase in liver 5-HT. The dose-dependence of the hypoglycaemia induced by LPS was similar to that of the increase in liver 5-HT. In C3H/HeJ mice, the macrophages and/or lymphocytes of the mice are known to be less responsive to LPS, and both the LPS-induced hypoglycaemia and increase in 5-HT were less in these mice than in control mice (C3H/He and ddI mice). These results suggest that macrophages and/or lymphocytes may participate in the induction of hypoglycaemia and the increase in 5-HT induced by mitogenic substances and LPS. A possible correlation between hypoglycaemia and the increase in hepatic 5-HT is discussed, although the relationship is not substantiated.

The administration of ethionine to female rats causes breakdown of hepatic polysomes. The state of mRNA and monomeric ribosomes after the polysome dissociation was studied. The mRNA was selectively labeled with [14C] orotate after a low dose of actinomycin D. Sucrose density gradient centrifugation of Triton X-100-treated cytoplasm revealed an accumulation of heterodisperse radioactive material with very large S values. This material was converted to smaller S values with deoxycholate treatment and was extremely sensitive to mild ribonuclease treatment. Since this material was banded at around 1.43 g/cm3 in CsCl gradient centrifugation and contained RNA with a distribution of S values characteristic of polysomal mRNA, this material was identified as mRNA-containing ribonucleoprotein particles. The monomeric ribosomes were shown to be dissociated into subunits in the presence of 0.5 M KCl, indicating that these lacked nascent polypeptide chains. When the animals were recovered from the ethionine treatment by subsequent administration of adenine and methionine, the heterodisperse ribonucleoprotein particles and monomeric ribosomes appeared to be utilized for the reformation of polysomes.

The enzyme catalyzing the deacetylaction of N-acetylhistamine was partially purified about 160-fold from rat liver extract and its properties were investigated. The purification procedure included DEAE-cellulose chromatography, precipitation with ammonium sulfate and DEAE-cellulose rechromatography. The enzyme contains a labile -SH group that is essential for its activity. Mn2+ and Co2+ enhanced the deacetylation reaction at low concentration. The molecular weight of the deacetylase was estimated to be about 70 000 from gel-filtration. Among various acetyl derivatives tested so far, N-acetylhistamine and to a lesser extent N-acetyltyramine served as the substrates. The Km value was 0.3 mM at the optimum pH 8.0 for N-acetylhistamine. Diphenhysramine, an antihistaminergic agent, inhibited the deacetylation remarkably.

Experimental granuloma pouches were induced in the dorsum of Sprague-Dawley rats by the subcutaneous injection of either carrageenin or agar. In the former inflammation model, the hepatic activities of aminopyrine N-demethylase and aniline hydroxylase and the contents of cytochromes P-450 and b5 were reduced significantly from control and pentobarbital sleeping time was prolonged. Hydrocortisone inhibited significantly the increased vascular permeability, exudation and proliferation of the carrageenin-induced granuloma in the daily, oral dose of 10 mg/kg for 3 days. On the other hand, the animals with agar granuloma pouch did not show any decline of the hepatic drug metabolism and a same dosage level of hydrocortisone showed substantially no inhibitory effect on the agar granuloma. Consequently, it is suggested that impairment of the hepatic drug metabolism may be responsible for the increased potency of hydrocortisone in the carrageenin-induced granuloma.