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Background: The success of liver resection relies on the ability of the remnant liver to regenerate. Most of the knowledge regarding the pathophysiological basis of liver regeneration comes from rodent studies, and data on humans are scarce. Additionally, there is limited knowledge about the preoperative factors that influence postoperative regeneration., Aim: To quantify postoperative remnant liver volume by the latest volumetric software and investigate perioperative factors that affect posthepatectomy liver regeneration., Methods: A total of 268 patients who received partial hepatectomy were enrolled. Patients were grouped into right hepatectomy/trisegmentectomy (RH/Tri), left hepatectomy (LH), segmentectomy (Seg), and subsegmentectomy/nonanatomical hepatectomy (Sub/Non) groups. The regeneration index (RI) and late regeneration rate were defined as (postoperative liver volume)/[total functional liver volume (TFLV)] × 100 and (RI at 6-months - RI at 3-months)/RI at 6-months, respectively. The lower 25 th percentile of RI and the higher 25 th percentile of late regeneration rate in each group were defined as "low regeneration" and "delayed regeneration". "Restoration to the original size" was defined as regeneration of the liver volume by more than 90% of the TFLV at 12 months postsurgery., Results: The numbers of patients in the RH/Tri, LH, Seg, and Sub/Non groups were 41, 53, 99 and 75, respectively. The RI plateaued at 3 months in the LH, Seg, and Sub/Non groups, whereas the RI increased until 12 months in the RH/Tri group. According to our multivariate analysis, the preoperative albumin-bilirubin (ALBI) score was an independent factor for low regeneration at 3 months [odds ratio (OR) 95%CI = 2.80 (1.17-6.69), P = 0.02; per 1.0 up] and 12 months [OR = 2.27 (1.01-5.09), P = 0.04; per 1.0 up]. Multivariate analysis revealed that only liver resection percentage [OR = 1.03 (1.00-1.05), P = 0.04] was associated with delayed regeneration. Furthermore, multivariate analysis demonstrated that the preoperative ALBI score [OR = 2.63 (1.00-1.05), P = 0.02; per 1.0 up] and liver resection percentage [OR = 1.02 (1.00-1.05), P = 0.04; per 1.0 up] were found to be independent risk factors associated with volume restoration failure., Conclusion: Liver regeneration posthepatectomy was determined by the resection percentage and preoperative ALBI score. This knowledge helps surgeons decide the timing and type of rehepatectomy for recurrent cases., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Although liver-humanized animals are desirable tools for drug development and expansion of human hepatocytes in large quantities, their development is restricted to mice. In animals larger than mice, a precondition for efficient liver humanization remains preliminary because of different xeno-repopulation kinetics in livers of larger sizes. Since rats are ten times larger than mice and widely used in pharmacological studies, liver-humanized rats are more preferable. Here, Fah -/- Rag2 -/- IL2rg -/- (FRG) rats are generated by CRISPR/Cas9, showing accelerated liver failure and lagged liver xeno-repopulation compared to FRG mice. A survival-assured liver injury preconditioning (SALIC) protocol, which consists of retrorsine pretreatment and cycling 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) administration by defined concentrations and time intervals, is developed to reduce the mortality of FRG rats and induce a regenerative microenvironment for xeno-repopulation. Human hepatocyte repopulation is boosted to 31 ± 4% in rat livers at 7 months after transplantation, equivalent to approximately a 1200-fold expansion. Human liver features of transcriptome and zonation are reproduced in humanized rats. Remarkably, they provide sufficient samples for the pharmacokinetic profiling of human-specific metabolites. This model is thus preferred for pharmacological studies and human hepatocyte production. SALIC may also be informative to hepatocyte transplantation in other large-sized species., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Dispersions of platelet γ-Fe2O3 particles 30-50nm in size were intravenously administered to mice and the amount of particles accumulated in each tissue was obtained by magnetization measurement using a vibrating sample magnetometer. Background noise was greatly reduced by measuring dried tissues under a magnetic field of 500 Oe so that the effect of diamagnetism was slight. Remarkable particle accumulation was observed in the liver and spleen. Considerable particle accumulation was observed in the lung when a large quantity of γ-Fe2 O3 particles was administered. There was no significant particle accumulation in the kidney and heart.

Hypothermia is used in the clinic for protection of organs such as the brain against ischemic injury during aortic/complex congenital cardiac surgery or post-resuscitation encephalopathy. The principal mechanism of hypothermic protection is suppression of metabolism, however, the pleiotropic effects of cooling are incompletely understood. Here, we used a rat model system to evaluate metabolic changes induced by deep hypothermia. The hypothermia-induced changes were identified using fluorescence-based two-dimensional (2-D) difference gel electrophoresis (DIGE) and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry. Rats were randomly assigned to a normothermic control group (37°C, n=6) or hypothermia group (23°C, n=6) that received surface cooling for 3h. Liver tissue was excised for assessment. Functional profiling of differently expressed proteins was performed as an enrichment analysis of Gene Ontology (GO) terms and pathways. We found that the livers of anesthetized rats with deep hypothermia showed significant downregulation of proteins in the endoplasmic reticulum and mitochondria, and of those involved in ATP binding, amino acid metabolism and urea cycle, response to oxidative stress, anti-apoptosis, negative regulation of apoptosis. The changes in the proteome of the hypothermic rats showed similarities, except with regard to endoplasmic reticulum chaperones, to those identified elsewhere in mammals undergoing hibernation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Liver ischemia-reperfusion (I/R) injury is one of the most serious complications of hepatic surgery. In I/R, activated Kupffer cells cause platelet adhesion to sinusoidal endothelium as well as neutrophils and cause liver dysfunction. The aim of this study was to evaluate platelet dynamics in the hepatic microcirculation after I/R by intravital microscopy (IVM) and to clarify the relationship between platelet adhesion and neutrophil activation. Male Sprague-Dawley (SD) rats were divided into two groups: the control (administration of saline) group and the sivelestat group in which neutrophil activation was suppressed by sivelestat before I/R. The number of adherent platelets in sinusoid was observed up to 120 minutes after I/R by IVM. Samples of liver tissue and blood were taken for examination of histological findings, liver enzymes and inflammatory cytokines. The number of adherent platelets was significantly increased after I/R in both groups. Compared with the control group, the number of adherent platelets significantly decreased after hepatic I/R in the sivelestat group. Moreover, sivelestat improved changes of histological findings and elevation of liver enzymes. However, there was no significant difference in inflammatory cytokines of TNF-alpha, IL-1beta or IL-6. Platelet adhesion in the sinusoid is associated with liver dysfunction after I/R as well as neutrophils. Activated neutrophils induce platelet adhesion in the sinusoid of the liver.

We have examined assay methods and conditions for human liver alanine:glyoxylate aminotransferase (AGT). This enzyme shows not only the AGT activity but also serine:pyruvate and serine: glyoxylate aminotransferase (SPT and SGT) activities. In the assay of AGT activity using crude enzyme preparations, there is the complication that glutamate:glyoxylate aminotransferase (GGT) also contributes to AGT activity, but at present no other enzyme is known to catalyze transamination between L-serine and glyoxylate or pyruvate. Therefore, an assay for AGT using its SGT activity was investigated in which hydroxypyruvate formed from L-serine in the enzymic reaction with glyoxylate was determined by lactate dehydrogenase (LDH) in the presence of tris(hydroxym ethyl)aminomethane (Tris) at pH 8.4. A possible obstacle to this assay is that pyruvate formed from L-serine by serine dehydratase (SDH) interferes with SGT assay as an additional substrate of LDH and AGT. However, the SDH activity in human liver is very low, and by performing the SGT reaction in the presence and absence of glyoxylate the SGT activity was represented as the glyoxylate-dependent hydroxypyruvate formation from L-serine. There was a combined good correlation between the AGT, SGT and SPT activities, and the activity ratio, AGT : SGT : SPT was about 1.0 : 0.17 : 0.13.

Previously, we identified a peroxisome-specific isoform of Lon protease using subcellular proteomics. In the present study, we investigated changes in the level of the Lon protease in peroxisomes during recovery from peroxisomal proliferation induced by di-(2-ethylhexyl)phthalate (DEHP) to elucidate the function of peroxisomal Lon protease (PSLP). Following a 2-week treatment with DEHP, the level of PSLP was monitored for 15 days. The amount of protease was greatly increased after the 2-week treatment, followed by a further increase 3 days after cessation of the treatment. Afterward, it decreased and reached the control level on day 15. On the other hand, level peroxisomal beta-oxidation enzymes induced to express by DEHP started to decrease soon after discontinuation of treatment. The results suggest that PSLP functions to degrade beta-oxidation enzymes induced by DEHP during recovery from perxisomal proliferation.

Serine:pyruvate aminotransferase (SPT; EC 2.6.1.51) of rat liver is a unique enzyme in that it is located in both mitochondria and peroxisomes. To analyze a peroxisomal targeting signal (PTS) of SPT, we constructed in this study various peroxisomal SPT clones having mutations at the C-terminal 20-amino acid region in which a putative PTS is located, and we examined subcellular localization of mutated products expressed in transfected COS-1 cells. When the mutant SPTs were unstable in transfected COS-1 cells, their translocation into peroxisomes was examined using an in vitro peroxisomal import system. Deletion of the C-terminal tripeptide, NKL, and amino acid substitution of K2 (the second lysine from the C-terminus), K4, or E15 abolished or impaired the peroxisomal import of the translated product, resulting in cytosolic accumulation in the cell. In the cases of mutation of R18G, D19A, or K2Q and the conversion to proline of L9, L13, V17, or A20, no products were detected in transfected cells. However, the results of an in vitro peroxisomal import experiment showed that the mutation of L9P, L13P, V17P, and A20P caused loss of the PTS function. When serine was introduced instead of N3 to generate a typical PTS1, the SKL motif, at the C-terminus, all of the proteins having mutations at P5, E11, R12, or E15 showed extensive localization in peroxisomes. These results suggest that the putative C-terminal PTS of SPT is not equivalent to the typical PTS1 shown in acyl-CoA oxidase and urate oxidase, because the PTS of SPT is not restricted to the C-terminal tripeptide. The results also suggest that the alpha-helical structure of the C-terminal region of SPT is important for the stable conformation of the enzyme and the peroxisomal targeting function of its PTS.

Our earlier electron microscopic observations revealed that prolonged exposure of glutaraldehyde-fixed rat liver sections to buffer solutions induced focal membrane disruptions of peroxisomes with catalase diffusion as shown cytochemically. Recently, it was suggested that 15-lipoxygenase (15-LOX) might be involved in natural degradation of membrane-bound organelles in reticulocytes by integrating into and permeabilizing the organelle membranes, leading to the release of matrix proteins. We have now investigated the localization of 15-LOX and its role in degradation of peroxisomal membranes in rat liver. Aldehyde-fixed liver slices were incubated in a medium that conserved the 15-LOX activity, consisting of 50 mM HEPES-KOH buffer (pH 7.4), 5 mM mercaptoethanol, 1 mM MgCl(2), 15 mM NaN(3), and 0.2 M sucrose, in presence or absence of 0.5-0.05 mM propyl gallate or esculetin, two inhibitors of 15-LOX. The exposure of aldehyde-fixed liver sections to this medium induced focal disruptions of peroxisome membranes and catalase diffusion around some but not all peroxisomes. This was significantly reduced by both 15-LOX inhibitors, propyl gallate and esculetin, with the latter being more effective. Double immunofluorescent staining for 15-LOX and catalase revealed that 15-LOX was co-localized with catalase in some but not all peroxisomes in rat hepatocytes. By postembedding immunoelectron microscopy, gold labeling was localized on membranes of some peroxisomes. These observations suggest that 15-LOX is involved in degradation of peroxisomal membranes and might have a physiological role in programmed degradation and turnover of peroxisomes in hepatocytes. (J Histochem Cytochem 49:613-621, 2001)

Primary hyperoxaluria Type 1 (PH1) is caused by a functional deficiency of a liver enzyme, serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), which catalyzes transamination between L-serine or l-alanine as an amino acid substrate and glyoxylate or pyruvate as an alpha-keto acid substrate. A high affinity for glyoxylate is a notable feature of this enzyme, suggesting a role in glyoxylate metabolism in vivo. Another conspicuous feature of SPT/AGT is its species-specific and food habit-dependent subcellular distribution. Thus, the enzyme is located in peroxisomes in herbivores and man, largely in mitochondria in carnivores, and in both the organelles in rodents. The mechanism of the species-specific dual organelle localization of SPT/AGT is either transcription of the gene from two different start sites or loss of the upstream translation initiation ATG codon by mutations. It appears that the mitochondrial versus peroxisomal distribution of SPT/AGT in different animal species is indispensable in meeting the metabolic needs caused by their respective food habits. As for the peroxisomal localization, glycolate is contained in plants much more than in animal tissues, and when ingested, it is converted to glyoxylate, an immediate precursor of oxalate, in liver peroxisomes. Therefore, peroxisomal localization of SPT/AGT may be indispensable for herbivores to convert the glyoxylate formed in peroxisomes into glycine in situ rather than forming oxalate. On the other hand, our recent studies showed that SPT/AGT contributed substantially to serine metabolism in rabbit, human, and dog livers; i.e., irrespective of its mitochondrial or peroxisomal localization. Thus, the mitochondrial localization of SPT/AGT was not a prerequisite for the metabolism of L-serine. Another source of glyoxylate is the metabolism of L-hydroxyproline, and in this case, the enzyme responsible for the glyoxylate formation has been reported to be a mitochondrial matrix enzyme. Collagen accounts for about 30% of total animal proteins and contains about 13% (w/w) hydroxyproline. It is therefore possible that both mitochondrial and peroxisomal SPT/AGT contribute to the metabolism of glyoxylate and serine, but the subcellular site for glyoxylate metabolism is different in herbivores and carnivores.

Serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, SPTp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.

L-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by L-serine dehydratase (SDH), another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagon-treated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mM L-serine and 0.25 mM pyruvate) was about (1)/(10) of that through SDH. Flux through GCS accounted for only several percent of the amount of L-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from L-serine were evaluated in vivo based on the principle that 3H at the 3 position of L-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10-20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of L-serine in rat liver.

The effects of ligustrazine on hepatic oxygenation in the isolated rat liver were investigated during prolonged perfusion and following the injection of norepinephrine. After injection of erythrocytes into the perfusate, the hemoglobin spectra in the liver were measured by Erlangen microlightguide spectroscopy, and the hemoglobin oxygenation (HbO2) in the liver was calculated on the basis of the Kubelka-Munk theory. During artificial perfusion, the HbO2 value was decreased from 59.3 +/- 6.4% (after one hour's perfusion) to 25.5 +/- 19.5% (n = 441; after six hours' perfusion). However, when ligustrazine was injected into the perfusate after six hours' perfusion, the HbO2 values recovered to 56.4 +/- 9.7% (n = 441). After injection of norepinephrine, HbO2 in the liver decreased from 48.8 +/- 10.4% to 25.2 +/- 18.4% (n = 961), while subsequent administration of ligustrazine caused a recovery to 62.9 +/- 6.0% (n = 961). Our results suggested that ligustrazine is a powerful hepatic vasodilator for improving hepatic oxygenation.

In rat liver, a single serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) gene is transcribed from two transcription initiation sites. Transcription from the upstream site generates the mRNA encoding the precursor for mitochondrial SPT (pSPTm) and is markedly enhanced by the administration of glucagon or cAMP. In this report we show the increase in the downstream transcript, the peroxisomal SPT (SPTp) mRNA, caused by peroxisome proliferators and triiodothyronine (T3). In the case of T3, the pSPTm mRNA was also increased 72 h after a single administration of the hormone in addition to an earlier increase in SPTp mRNA.

To understand the targeting mechanisms of peroxisomal serine:pyruvate aminotransferase, in vitro import experiments were carried out using this 43 kDa peroxisomal enzyme, which was synthesized in a coupled transcription/translation system. Being different from other peroxisomal enzymes, such as acyl-CoA oxidase and urate oxidase used in previous in vitro import experiment, the soluble form of peroxisomal serine:pyruvate aminotransferase was fairly resistant to proteinase K digestion. However, the enzyme recovered in the peroxisomal fraction was proteinase K sensitive. This indicates that the association of peroxisomal serine:pyruvate aminotransferase with the peroxisomal membrane causes a marked conformational change in the structure of this protein.

Effects of skim milk and its fermented product by Lactobacillus acidophilus on plasma and liver triglyceride and cholesterol levels were examined in diet-induced hypertriglyceridemic rats. Male Sprague-Dawley rats at 4 weeks of age were fed a hypertriglyceridemic diet that contained 20% coconut oil, 17.5% fructose, and 17.5% sucrose for 14 days. The test diet was supplemented with either 20% skim milk powder or 20% powder of skim milk fermented by L. acidophilus SBT 2062. Hypertriglyceridemia was observed in the control group, but plasma cholesterol levels were not increased. Skim milk suppressed the elevation of plasma triglyceride levels, while its fermented product had no significant effect. Both dairy products prevented the elevation of liver triglyceride and cholesterol levels, but had no effect on plasma cholesterol levels.

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26 degrees C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C-terminal 10 amino acid residues of acyl-CoA oxidase (the C-terminal tripeptide is Ser-Lys-Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C-terminal Ser-Lys-Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C-terminal Ser-Arg-Leu (SRL) sequence was deleted or mutated to Ser-Glu-Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C-terminus the import activity was retained. These results show that urate oxidase is important into peroxisomes via a common pathway with acyl-CoA oxidase, and that the C-terminal SRL sequence functions as a peroxisomal-targeting signal.

Effects of dietary oat, barley, and guar gums on serum and liver triglyceride or cholesterol concentrations were examined in diet-induced hypertriglyceridemic rats. Male Sprague-Dawley rats were fed a hypertriglyceridemic diet that contained 20% coconut oil, 17.5% fructose, 17.5% sucrose, and 5% cellulose at 4 weeks of age for 14 days. In the gum-supplemented diets, 2% cellulose was replaced by oat gum, barley gum, or guar gum. Hypertriglyceridemia was observed in the control group, whereas serum cholesterol concentration was not increased. All of the gums lowered serum and liver cholesterol concentrations except barley gum which had no significant effect on liver cholesterol. Both oat and barley gums suppressed the elevation of serum and liver triglyceride concentrations but guar gum had no effect.

The effects of glucagon on serine: pyruvate/alanine: glyoxylate aminotransferase (SPT/AGT) gene expression were studied in primary cultured rat hepatocytes. When hepatocytes had been precultured for 16-18 h under serum- and hormone-free conditions, the addition of glucagon caused (after a lag period of about 2 h) a remarkable increase in the cellular level of SPT/AGT mRNA by 4 h in a time- and dose-dependent manner. The induced mRNA was that for mitochondrial SPT/AGT, as judged by ribonuclease protection analysis. A nuclear run-on assay revealed that activation of transcription is responsible for the increase in mitochondrial SPT/AGT mRNA and that the maximal rate of transcription occurs 1.5 h after glucagon addition. The effect of glucagon was mimicked by 8-bromo-cAMP and suppressed by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cAMP-dependent protein kinase (protein kinase A), while both 12-O-tetradecanoylphorbol-13-acetate and A23187 were without effect in elevating the SPT/AGT mRNA level, suggesting that the cAMP/protein kinase A system is involved in the regulation of SPT/AGT gene expression. In hepatocytes precultured for 16-18 h under serum- and hormone-free conditions, the glucagon-induced transcription was severely inhibited by cycloheximide. When the preculture was for 2 h, on the other hand, the activation of transcription by glucagon was more rapid, and the inhibition by cycloheximide was less than that observed with cells precultured for 16-18 h, suggesting that a short-lived protein factor is involved in the hormonal regulation. The glucagon-induced expression of the SPT/AGT gene was also turned off by dexamethasone.

Effects of soluble and insoluble fiber preparations isolated from defatted oat, barley, and wheat on liver cholesterol accumulation were examined in cholesterol-fed rats. The soluble and insoluble fiber preparations were isolated by a modification of the procedure of Asp et al. Male Sprague-Dawley rats, aged 5 weeks, were fed a control diet containing 5% cellulose or diets containing soluble and insoluble fiber preparations for 9 days. Soluble fiber preparations from oat, barley, and wheat were added to diets corresponding to 1.9, 2.8, and 0.6% fiber, respectively. Insoluble fiber preparations from oat, barley, and wheat were added to diets corresponding to 3.1, 2.2, and 4.4% fiber, respectively. All of the soluble fiber preparations suppressed liver cholesterol accumulation, but they did not suppress the elevation of plasma cholesterol concentrations. A significant inverse relationship between the quantity of ingested soluble fiber and liver cholesterol accumulation was observed; n = 28, r = -0.943, p < 0.0001. None of the insoluble fiber preparations had any significant effect on liver and plasma cholesterol concentrations. These results indicate that the water-soluble fractions of oat, barley, and wheat are the active components that suppressed liver cholesterol accumulation, and that these effects are related to the quantity of ingested soluble fiber.

To determine whether natural human interferon administered under the usual therapeutic dosing scheme would inhibit the hepatic drug metabolism, we performed an antipyrine test in eight patients with chronic B or non-A, non-B hepatitis before and after a subchronic interferon therapy (6 megaunits/day for 17 +/- 4 days, mean +/- SD). Six patients received interferon-beta and 2 received interferon-alpha. To circumvent a possible influence of interferon-induced fever on the hepatic drug metabolism, the antipyrine test during the interferon therapy was performed at least 14 days after the interferon-induced fever disappeared. The kinetic parameters of antipyrine were obtained from seven saliva samples over 32 hours postdose. There were no significant differences in any kinetic parameters of antipyrine observed before and during the interferon therapy. With the sample size of the study, there was only a 20% chance (i.e., beta-power = 0.8 at alpha = 0.05) that we might have missed a 17% reduction in antipyrine clearance by the interferon therapy (type II error). On the other hand, the subchronic interferon therapy lowered serum aminotransferases and DNA polymerase activity significantly (P less than .05) compared with the respective baseline values. Our results suggest that the subchronic therapeutic dosing scheme of interferon as conducted in the present study does not cause the inhibitory effect on the oxidative drug metabolism to a statistically significant or clinically relevant degree in patients with chronic hepatitis, while it improves their liver function. Further studies are required for determining if different types of interferons administered under the different dosing schemes would alter the hepatic drug metabolism and the inhibitory effect would be time-dependent.

In rat liver there are two types of serine:pyruvate aminotransferase (SPT) whose natures are indistinguishable but whose subcellular localization are different. One is a mitochondrial enzyme (SPTm) and the other a peroxisomal enzyme (SPTp). We compared, in this study, the structure of mRNAs encoding SPTm and SPTp by comparison of the sizes after removal of poly(A) tail by ribonuclease H and by means of RNA blot analysis and S1 nuclease protection assay. No differences were detected between these two mRNAs other than that about 100 nucleotides of the 5'-terminal sequence of SPTm mRNA are lacking in SPTp mRNA, and the length of the poly(A) tail is different. Southern blot analysis of rat genomic DNA showed that the SPT gene is single. Primer extension and S1 nuclease mapping analyses, using a DNA fragment of a genomic clone, revealed that the SPTm and SPTp mRNAs are transcribed from different initiation sites, about 70 nucleotides apart, in the same exon, exon 1. Ribonuclease protection assay performed with RNA hybridization probe corresponding to 5'-terminal portion of SPTm mRNA also showed that the 5'-terminal sequence of SPTp mRNA is about 70 nucleotides shorter than that of hormone-responsive SPTm mRNA. These results indicate that the different organelle distribution of SPTm and SPTp, the products of the same SPT gene, arises from transcription from different initiation sites, conferring N-terminal extension peptide, the mitochondrial targeting signal, only on the translation product of SPTm mRNA.

In order to clarify morphological changes of the liver in the uremic state, 16 uremic patients treated with chronic hemodialysis were studied. Biopsy was performed in 14 cases under laparoscopic observation and in two on the occasion of renal transplantation. One uremic patients not being treated with dialysis was also studied for comparison. All biopsy specimens were examined by light and electron microscopy. The liver usually appeared mildly or moderately swollen under laparoscopic observations, which was considered at least partially due to the enlargement of the hepatocytes. All patients had hepatocytes with an Orcein-negative "ground glass" appearance, in which marked proliferation of smooth endoplasmic reticulum (SER) was found by electron microscopy. Since the patient not being on dialysis also had such hepatocytes, this finding may be characteristic of uremia. With electron microscopy, in addition to proliferation of SER, alteration of mitochondria and rough endoplasmic reticulum (RER) and an increase in cytoplasmic lipid droplets were observed. Hypertrophy of the Golgi apparatus containing electron-dense particles (VLDL) was often found in patients associated with hypertriglyceridemia. Amorphous electron-dense inclusions in microbodies were occasionally observed. Siderosis was observed in nine patients including three having parenchymal siderosis. With electron microscopy, various siderosomes were seen in the cytoplasm of hepatocytes in patients with parencymal siderosis. Conclusively, these histological and ultrastructral features of hepatocytes are rather associated with several metabolic abnormalities in uremia.

Immunocytochemical localization of serine:pyruvate aminotransferase (SPT) in rat hepatocytes was studied using a protein A-gold technique. Rat liver was fixed by perfusion. Vibratome sections (100 micron thick) of the liver were embedded in Epon or Lowicryl K4M. Ultrathin sections were incubated with antiSPT, followed by protein A-gold complex. Gold particles representing the antigenic sites for SPT were seen in three subcellular compartments, peroxisomes, mitochondria, and cytoplasm. In the control experiments the specificity of the immunolabelling was confirmed. Quantitative analysis of the labelling density showed that main subcellular compartments containing SPT are mitochondria and peroxisomes. In addition, the gold particles distributing in the cytoplasm were 16%-29% of the total labelling. The result indicated that the cytoplasm also contains SPT with a low density.

Nascent peptides on free and bound ribosomes prepared from rat liver were labeled and released with [3H]puromycin, and the amounts of the nascent peptides of two secretory proteins, serum albumin and transferrin, were determined by immunoprecipitation with specific antibodies. An appreciable amount of serum albumin nascent peptides was associated with free ribosomes, although most of them were carried by bound ribosomes. Serum albumin nascent peptides associated with free ribosomes were smaller in size than those carried by bound ribosomes. When the products of cell-free translation programmed by free and bound polysomes were immunoprecipitated with antibodies, the percentages of serum albumin to the total translation products were in good agreement with those obtained in the nascent peptides experiment. Cell-free translation of free polysomes produced serum albumin with a similar molecular weight to the authentic one. Although nascent peptides of transferrin were detected only on bound ribosomes, and not on free ribosomes, the transferrin antibody immunoprecipitated 0.2% of total translation products of free polysomes. These observations indicate that free polysomes have complete messenger RNA's and shorter growing nascent peptides of serum albumin and transferrin.

An electron-microscopic study was carried out using chimpanzees' livers infected with experimental hepatitis B for the elucidation of intracellular development of HBV-associated ultrastructures and extracellular release of HBV. Core particles were first detected in the nucleus of liver cells at around the time of the first seropositiveness for HBsAg, and then in the cytoplasm. Subsequently, their budding into endoplasmic reticular cisterna was seen together with other core particles in the surrounding cytoplasm. Dane-like particles were seen in the cisterna, and also extracellularly nearby a liver cell with a marked proliferation of microvilli at the onset of liver cell injury. Thereafter, core-like particles were seen within electrondense amorphous material at the site of the contact between liver cell and lymphocyte. The above sequence of features suggested us the assembly of core particles and surface envelope at the cisternal membrane of endoplasmic reticulum, and a reversed pinocytosis whereby Dane or HBV particles were released extracellularly. The filamentous structures within endoplasmic reticular cisternae, which were thought to be HBsAg, were never detected.

A 32-yr-old female who suffered from typical Weber-Christian disease developed hepatosplenomegaly and hepatic dysfunction. A liver biopsy specimen displayed many fat droplets and alcoholic hyalins (Mallory bodies). Electron microscopic hepatic injury was indicated by many fat droplets, alcoholic hyalins, marked deformities of the rough endoplasmic reticulum, and by some deformed nuclei and nuclear bodies. There has been no other report describing alcoholic hyalins in the hepatic changes of Weber-Christian disease. Additionally, based on electron microscopic observation, we assume that fatty changes were due to a relative reduction in lipoprotein synthesis in the deformed rough endoplasmic reticulum.

The alterations of the alkaline phosphatase (ALP) activity in the rat liver following bile duct ligation were investigated by electron microscopical techniques. Serum ALP activity reached the maximum at 24 hours after ligation and two isozymes of ALP, high molecular and low molecular one, appeared in the serum. Bile canaliculi became dilated at 48 hours after ligation and the microvilli were destructed and diminished in number. ALP activity was observed almost only on the bile canalicular membrane of the liver cells in the control. On the other hand, in the bile duct-ligated rat, the ALP activity on the canalicular membrane was markedly increased initially, then it appeared on the lateral membrane, and finally on the sinusoidal membrane also. It was not stainable on the canalicular membranes which lacked microvilli. The proposed pathway through which hepatic ALP enters the blood stream in bile duct-ligated rats is as follows: ALP, being synthesized in the microsomes of hepatocytes, is initially transferred to the bile canalicular membrane and diffused to lateral membrane through tight junction, reaches to sinusoidal membrane then released into the blood stream.

Cloned cDNAs for rat liver serine: pyruvate aminotransferase were obtained by screening of a cDNA expression bank of rat liver with an antibody against the enzyme. Nineteen clones were isolated from 33 000 transformants and most of them had common fragments of cDNA on analysis by digestion with some restriction enzymes. These clones were identified as those containing cDNA for serine:pyruvate aminotransferase by the following criteria. (a) At the nucleic acid level, a 500-base-pair fragment of cDNA prepared by digestion of cDNAs with EcoRI and PstI hybridized with the mRNA coding for serine:pyruvate aminotransferase as judged by hybrid-selected and hybrid-arrested translations. (b) Specific proteins were detected in nine bacterial clones, a 40-kDa protein in one clone and a 39-kDa protein in eight clones. Among them only the 40-kDa protein was found to be solubilized from the cell by sonication, and this protein was immunoprecipitated with an antibody against serine:pyruvate aminotransferase of rat liver. (c) High activity of serine:pyruvate aminotransferase was expressed both in whole cell suspension and sonicated extract prepared from the transformant producing the 40-kDa protein, and 99% of the activity was immunoreactive with the antibody. Two types of mRNA for serine:pyruvate aminotransferase were detected on the RNA blot analysis by using cloned cDNA fragment as a probe. The larger mRNA (approximately 1600 nucleotides) was glucagon-inducible while the smaller one (approximately 1500 nucleotides) was not affected by the hormone.

The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.

The methylation state of cellular oncogenes (c-oncs) and epidermal growth factor (EGF) receptor gene from human liver tissues was examined by means of restriction endonuclease analysis. c-myc and EGF receptor gene from hepatocellular carcinoma and fetal liver were substantially hypomethylated in comparison with those genes from normal liver, while the extents of methylation of c-mos and c-Ki-ras genes were the same among these tissues. It can be speculated that the specific hypomethylation of c-myc and EGF receptor genes may be associated with the development of hepatocellular carcinoma.

Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.

A cDNA expression library constructed in a plasmid pUC8 from poly(A)+ RNA of rat liver was screened immunologically, using an antibody against arginase of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.35 kilobase pairs in length. In the bacterial clone, we detected a specific protein of Mr = about 43,000 that is slightly larger than the purified arginase (Mr = about 40,000) and a high activity of arginase was expressed. The arginase mRNA species of about 1600 bases long was detected in the liver, but not in the small intestine, kidney, spleen and heart of the rats.

Between December 1973 and September 1987, 21 patients with primary liver cancer and 41 patients with metastatic liver cancer were treated with external irradiation, intra-arterial infusion chemotherapy and/or transarterial embolization (TAE) at the National Medical Center Hospital, the National South Kyushu Central Hospital and the National Kure Hospital. Of the patients with primary liver cancer, 13 cases were treated with intra-arterial infusion chemotherapy (30-40 mg adriamycin or 10 mg mitomycin C) and hepatic irradiation. Eight cases were treated by TAE and hepatic irradiation. In the Child A group, the survival period of the chemotherapy + hepatic irradiation cases (mean: 608 days) was longer than that of the TAE + hepatic irradiation cases (mean: 216 days). The median survival period of all the cases was 7.0 months (mean: 10.9 months). For 16 of the 21 patients (who had absorbed over 40 Gy), the median survival period was 11.9 months (mean: 11.7 months). For 5 of the 21 patients (who had absorbed below 40 Gy), the median survival period was 4.3 months (mean: 7.9 months). Of the patients with metastatic liver cancer, the median survival period was 7.2 months (mean: 8.0 months). For 22 of the 41 patients (who had absorbed over 40 Gy), the median survival was 7.9 months (mean: 12.6 months). For 19 of the 41 patients (who had absorbed below 40 Gy), the median survival period was 1.7 months (mean: 2.6 months). The pretreatment serum GOT (glutamate oxaloacetate transaminase) levels and the pretreatment Karnofsky performance status index were the factors governing the prognosis of the cases with metastatic liver cancer, while toxicity was generally mild.

A cDNA expression library constructed from poly(A)+ RNA of rat liver was screened immunologically using an antibody against argininosuccinate lyase (EC 4.3.2.1), a urea cycle enzyme, of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.5 kilobase pairs in length. In the bacterial clone, a specific protein of Mr = about 25,000 was expressed. The argininosuccinate lyase mRNA of about 2.1 kilobases long was detected in the liver and in a lesser amount in the kidney and spleen, but not in the small intestine and heart of the rats.

Free ribosomes and membrane-bound ribosomes were prepared from rat livers, and the contributions of these two types of ribosomes to the synthesis of aldolase B were studied by the immunoprecipitation of [3H]puromycin-labeled nascent peptides with a rabbit antibody to this enzyme. Although rat liver aldolase was recovered in both cytosolic and microsomal fractions by the fractionation of liver homogenate, the microsomal aldolase was immunologically identical with its cytosolic counterpart as confirmed by Ouchterlony immunodiffusion test. We examined the nascent peptide fractions prepared from free and bound ribosomes, and found that the nascent peptides of aldolase were mainly localized in free ribosomes. About 0.5% of the total nascent peptides of free ribosomes and 0.08% of those of bound ribosomes was aldolase. The site of synthesis of serum albumin was also examined as a reference standard by the immunoprecipitation of labeled nascent peptides, and the nascent peptides of this secretory protein were mainly associated with bound ribosomes, as reported by other workers. These observations confirm that aldolase B is mainly synthesized by free ribosomes in rat liver cells.

Ultrastructural studies on liver cell necrosis and the interaction of lymphocyte and liver cell were carried out in experimental hepatitis B in chimpanzees. Two types of liver cell necrosis were identified. One was a lytic necrosis, and the other was a coagulation necrosis. Both types of liver cell necrosis were closely associated with the apposition of lymphocytes. The interaction (or close contact) of lymphocyte and non-necrotic liver cell infected with HBV was also detected. There were two distinct patterns of the contact. One was the direct contact, and the other was the contact with the intervention of electron-dense fuzzy material containing 20 to 22 mu spherical particles and 51 to 55 mu double-layered spherical particles. The ultrastructural characteristics of lymphocytes in each pattern of the contact were different. The results suggested that the pathogenesis of liver cell necrosis in hepatitis B was closely associated with the action of lymphocytes, and two modes of lymphocytic reaction were conceivable.

Six hours after oral administration in rat liver, diisopropyl 1,3-dithiol-2-ylidenemalonate (malotilate) decreased AMP concentrations by 60% with unchanged total adenine nucleotide concentrations and significantly increased adenylate energy charges of 0.87 in comparison to those of 0.81 in vehicle administered control animals. Twelve hours after incubation in primary cultured rat hepatocytes, malotilate decreased AMP concentrations by 18% and increased ATP and GTP concentrations by 13 and 18% respectively with significantly increased adenylate energy charges of 0.89 in comparison to 0.87 in vehicle control. Increased adenylate energy charges by malotilate coincided with the initiation of increases in protein and de novo purine synthesis in vivo, and are associated with the increased rate of de novo purine synthesis in vitro.