A useful ELISA system for human liver-type arginase, and its utility in diagnosis of liver diseases.

OBJECTIVES: To develop a new ELISA system for liver-type arginase using monoclonal antibodies against the enzyme, and to verify the utility of the arginase in diagnosis of hepatic disorders. DESIGN AND METHODS: We have developed an enzyme-linked immunosorbent assay (ELISA), using two kinds of monoclonal antibodies (Mo6G3 and Mo9C5) for human liver-type arginase as the first and second antibodies respectively. We have also developed a new method to eliminate the influence of erythrocyte-derived arginase contamination in hemolytic samples. This ELISA was applied to specimens received from patients with acute and chronic hepatic disease and also patients who had undergone partial hepatectomy. RESULTS: This assay is sensitive and reproducible for the measurement of liver-type arginase in the sera of patients with liver dysfunction, and enabled us to detect enzyme concentrations as low as 27 pmol/L without any processing of the samples. The assay showed within-run coefficients of variation (CV) ranging from 1.9 to 4.1% and between-day CV from 3.6 to 5.1% for arginase concentrations varying from 57.1 to 1200 pmol/L. The recovery was 113% (mean) with a range of 96 to 129%. These antibodies reacted strongly with both recombinant and native liver-type arginases, while, to some extent, with erythrocyte-derived arginase. Correction for erythrocyte-derived arginase contamination in hemolytic samples was, however, easily made by assaying peroxidase-like activity of hemoglobin. From the view of a limited localization of arginase in the liver, the marked increase of the enzyme in serum reflects initiation of liver injury, while the rapid decrease reflects termination of the damage. Such quick normalization in circulating liver-type arginase indicated another merit of the enzyme in diagnosis of liver diseases. CONCLUSIONS: The changes in circulating liver-type arginase level could be helpful not only in the diagnosis of liver diseases but also subsequent treatment of the patients with liver damage.

[Clinical significance of anti-liver-type arginase autoantibody in blood of recipients after partial liver transplantation].

Autoantibody against human liver-type arginase was detected in blood of patients treated with partial liver transplantation and consisted of all subclasses of IgG, i.e., IgG1, IgG2, IgG3 and IgG4, and IgM. We newly constructed an ELISA system for the antibodies by the aid of arginase protein immunopurified from extracts of human liver tissues. Addition of 2.0 mol/l urea in 0.1 mol/l citrate buffer(pH 4.5) was effective for elimination of immunoglobulins, such as IgG and IgM, and rheumatoid factors adsorped non-specifically to liver-type arginase-autoantibody complexes on the plate. We found that, during a short period of about two months after operation, in successful cases, liver-type arginase increased, remarkably and repeatedly, in blood of recipients followed by elevation of IgM level within a week and also IgG2 level two or three weeks later. Thus the change in IgG2 level seemed to depend on those of the arginase and/or IgM. However, in unsuccessful cases, such fluctuation was not so clear as the successful cases. To be noteworthy was production of autoantibodies directed to liver-type arginase in blood of patients with liver injury although the arginase, as well as AST and ALT, is an enzyme which leaks out of liver tissue. Appearance of the autoantibodies in blood supports occurrence of liver injury, in part, in graft liver because the enzyme exists exclusively in the liver. Among immunoglobulins to liver-type arginase, IgG2 seemed to be the most helpful index to know rightly postoperative conditions of recipients of liver transplantation, and its measurement could be useful for long-term follow-up of the patients.

Origin of anomalous lattice expansion in oxide nanoparticles

Anomalous lattice expansions have been measured for the first time in monodisperse CeO2-x nanoparticles and in BaTiO3 single nanoparticles by electron diffraction. X-ray photoelectron spectroscopy studies on CeO2-x nanoparticles and ab initio computer simulation on BaTiO3 clusters show that the origin of expansion is the decrease of electrostatic force caused by valence reduction of Ce ions and the increase in ionicity of Ti ions, respectively. The lattice constant change of oxide (ionic) nanoparticles with the increase in ionicity would depend on the structure of the particles. Hence, first-principles calculations of large ionic clusters are indispensable.

Altered expression level of a systemic nuclear autoantigen determines the fate of immune response to self.

One of the hallmarks of systemic autoimmune diseases is immune responses to systemic nuclear autoantigens. We have examined the fate of the immune response against a nuclear autoantigen using human U1 small nuclear ribonucleoprotein-A protein (HuA) transgenic (Tg) mice by adoptive transfer of autoreactive lymphocytes. We obtained two Tg lines that have different expression levels of the transgene. After spleen cells from HuA-immunized wild-type mice were transferred to Tg mice and their non-Tg littermates, these recipients were injected with HuA/IFA to induce a recall memory response. HAB69, which expressed a lower amount of HuA, exhibited a vigorous increase in the autoantibody level and glomerulonephritis. Moreover, the autoreactivity spread to 70K autoantigen. Alternatively, in HAB64, which expressed a higher amount of HuA, the production of autoantibody was markedly suppressed. The immune response to HuA autoantigen was impaired as demonstrated in a both delayed-type hypersensitivity response and proliferation assay. This inhibition was Ag-specific and was mediated by T cells. These data suggest that the expression level of systemic autoantigens influences the outcome of the immune response to self.

Far-Infrared Rotational Spectrum of ArD+

Rotational transition frequencies of ArD+ were measured with an accuracy of a few hundred kilohertz in the 0.95-4.7 THz region, using a tunable far-infrared radiation source. Effective rotational parameters B, D, and H were determined for the ground vibrational state. The observed frequencies were combined with the previous data on all isotopic species of protonated argon ion to improve the mass-independent Dunham parameters Ukl, DeltaArkl, and DeltaHkl. Copyright 1999 Academic Press.

Analysis of Rotational Transitions in Excited Vibrational States of Propionitrile (C2H5CN).

We have observed rotational transitions of propionitrile (C2H5CN) in the 8-200 GHz region and assigned 208 transitions with J

Tunable Far-Infrared Spectroscopy of 82KrD+, 84KrD+, 86KrD+, and 82KrH+

Pure rotational spectra of isotopic species of protonated krypton 82KrD+, 84KrD+, 86KrD+, and 82KrH+ were observed in the 0.75-3.5 THz region, using a tunable far-infrared radiation source. Rotational parameters B, D, and H of these molecular ions were determined. By analyzing the observed frequencies with the previous data on all the isotopic species, the mass independent Dunham parameters Ukl, DeltaKrkl, and DeltaHkl have been improved. Copyright 1998 Academic Press.

Liver-type arginase in serum during and after liver transplantation: a novel index in monitoring conditions of the liver graft and its clinical significance.

We quantified liver-type arginase in sera of 47 patients undergoing partial liver transplantation with use of an ELISA method. The level of liver-type arginase fluctuated slightly beyond the normal range in successful liver recipients, while it changed more drastically or precipitously in unsuccessful ones, accompanying or unaccompanying elevation of AST and ALT levels. A higher elevation pattern of the arginase level (above 100 ng ml-1) was observed in each of the unsuccessful recipients with critical condition, except for one patient. Other hepatic markers (LDH, ALP, and T-BIL) remained relatively unchanged until the terminal stage of deceasing patients. The finding that the liver-type arginase emerged in large quantity in the blood stream immediately after reperfusion of the liver graft indicates that the enzyme leaks out of hepatocytes damaged, presumably, by storage in the absence of circulation. A half-life of the liver-type arginase in the human blood was estimated to be 1 h, that is clearly shorter than that of AST. The short half-life of the arginase appears to be ascribable, at least partly, to formation of an immune complex with circulating autoantibody which appears in many liver recipients. These results suggest that liver-type arginase behaves uniquely in the serum among many hepatic enzymes, and could serve as a distinct marker of hepatic lesions, particularly during and after liver transplantation.

The beneficial effect of phosphocreatine accumulation in the creatine kinase transgenic mouse liver in endotoxin-induced hepatic cell death.

Purpose. The purpose of this study was to investigate the relationship between hepatic energy status and liver injury during sepsis, using transgenic mice which express creatine kinase in the liver catalyzing the phosphocreatine/creatine system. Methods. Creatine kinase transgenic mice were fed with normal rodent chow or chow containing 10% creatine for 5 days. Lipopolysaccharide (0.2 mg/kg) combined with d-galactosamine (600 mg/kg) was administered intraperitoneally. Results. Eighty percent of the creatine-fed transgenic mice had survived at 48 h post-d-galactosamine and lipopolysaccharide administration, compared with none of the normally fed transgenic mice. Hepatic phosphocreatine and ATP levels in the normally fed transgenic mice were significantly lower than those in the creatine-fed transgenic mice before and after lipopolysaccharide combined with d-galactosamine was administered. Massive hepatic hemorrhagic necrosis with apoptosis was seen in response to d-galactosamine and lipopolysaccharide in normally fed transgenic mice. These results are consistent with a significant increase in serum aminotransferase at 8 h. In contrast, there were faint necrotic changes in the liver with minimal cellular infiltration in creatine-fed transgenic mice. Conclusions. Maintenance of hepatic ATP levels protects from sepsis-induced liver injury and mortality.

Signal transduction pathway of human fibroblast growth factor receptor 3. Identification of a novel 66-kDa phosphoprotein.

Stimulation of fibroblast growth factor receptor 3 (FGFR3) results in a variety of functional effects, including regulation of epithelial cell growth and differentiation. In order to characterize the signaling pathway through which FGFR3 regulates cell growth, L6 cells lacking any endogenous FGFR were stably transfected with the two different human isoforms, FGFR3 IIIb and FGFR3 IIIc, that result from alternative splicing of exon III of the FGFR3 gene encoding the ligand binding domain. Expression of FGFR3 IIIc in stably transfected L6 cells conferred growth responses to several members of the FGF family including FGF-1, -2, -4, and -6, while FGFR3 IIIb-expressing cells responded only to FGF-1. Activation of FGFR3 upon ligand binding resulted in activation of mitogen-activated protein kinase pathway. FGFR3 utilizes two different pools of adapter protein GRB2 to link to Ras. Activated FGFR3 predominantly interacts with GRB2.Sos in complex with a previously identified 90-kDa protein and designated protein 80K-H. In addition, 80K-H.GRB2. Sos complex was found to contain a novel 66-kDa protein. Tyrosine phophorylation of the 66-kDa protein was dependent on ligand activation of FGFR3, suggesting that the 66-kDa protein may play an important role in FGFR3-specific signaling. In addition to this unique pathway, FGFR3 also links to GRB2.Sos complex via the adapter protein Shc. Furthermore, activated FGFR3 was not able to induce dissociation of GRB2.Sos complex following Sos phosphorylation. In summary, FGFR3 signaling pathway utilizes two GRB2-containing complexes; Shc.GRB2.Sos and 80K-H.pp66.GRB2.Sos; these two complexes may alternatively link FGFG3 to mitogen-activated protein kinase. Finally, activated FGFR3 was also found to result in phosphorylation of phospholipase C-gamma but reduced phosphorylation of c-Src.

Postoperative monitoring of the oxygenation state of the graft liver in cases with hepatopulmonary syndrome.

Postoperative changes in the oxygen saturation of hemoglobin in the graft liver (graft SO2) were monitored by near-infrared spectroscopy in four cases complicated by hepatopulmonary syndrome. A plastic cylinder was placed in the abdominal wall, and optical measurements of the graft liver were obtained through this window. Our findings were as follows; (1) graft SO2 decreased after abdominal wall closure, and decreased further 1 day after surgery. (2) Graft SO2 was maintained despite severe hypoxemia, with partial pressure of oxygen in arterial blood as low as 50 mmHg. High hematocrit was beneficial for oxygenating the graft. (3) Graft livers could tolerate hypoxia with a graft SO2 as low as 20%. (4) It may be useful to monitor graft SO2 during the critical period after transplantation for the assessment of graft function.

Induction of endotoxin tolerance in transgenic mouse liver expressing creatine kinase.

The liver plays an important role in maintaining homeostasis during endotoxin-triggered systemic inflammatory response. To study the effects of phosphocreatine on hepatic energy metabolism after endotoxin administration, we used transgenic mice whose livers express creatine kinase (CK). CK catalyzes a phosphocreatine/creatine reaction, that is, an adenosine triphosphate (ATP) reservoir system. Because dietary supplementation with creatine leads to an accumulation of creatine and phosphocreatine in transgenic livers, we compared the CK transgenic mice fed with creatine with the normally fed CK transgenic mice. In the creatine-fed mice, hepatic ATP, energy charge ([ATP + 0.5 adenosine diphosphate (ADP)]/[ATP + ADP + adenosine monophosphate (AMP)]), and mitochondrial oxidative phosphorylation activities remained at high levels after injection of 10 mg/kg of lipopolysaccharide (LPS) as compared with those in normally fed mice. Furthermore, there were beneficial effects on the functional reserve for ATP synthesis and work-cost performance, as calculated by free cytoplasmic ADP and the Michaelis constant (Km). Interestingly, a reduction of tissue necrosis factor alpha and interleukin-lalpha (IL-lalpha), and suppression of the decrease in glucose levels after LPS injection were observed in the creatine fed mice. Survival rates at 72 hours after injection of 10 mg/kg of LPS significantly increased in the creatine fed mice compared with the normally fed mice (80% vs. 24%, P < .001). Therefore, we concluded that the presence of phosphocreatine in the liver maintains energy metabolism and attenuates cytokine response, resulting in endotoxin tolerance.

Extension of surgical indication for advanced hepatocellular carcinoma: is it possible to prolong life span or improve quality of life?

BACKGROUND/AIMS: We have tried to break through the limitations of treatment for advanced hepatocellular carcinoma (HCC), which has been regarded as a contraindication for surgical treatment. MATERIALS AND METHODS: In 640 cases of hepatic resection for Liver cancer, we analyzed 55 cases of HCC with tumor thrombi in the trunk or first branch of the portal vein (PV) which required additional PV thrombectomy, 5 cases with direct invasion or compression to the inferior vena cava (IVC) which required replacement of IVC with a polytetrafluoroethylene (PTFE) tube, 9 cases with involvement of the extrahepatic bile duct (BD) which required additional extirpation of tumor fragments in the BD, 6 cases with tumor thrombi in the IVC which required IVC thrombectomy, and 4 cases of huge main tumor with intrahepatic metastasis in the entire liver which required intraoperative ethanol injection. RESULTS: Mean survival times in these groups were 796, 717, 650, 220, and 147 days, respectively. All patients with IVC thrombi and large tumor with intrahepatic metastasis in the entire liver died of early recurrence in spite of surgical treatment. By contrast, half of the patients with PV thrombi, BD involvement and IVC invasion or compression survived approximately 500 days because of a combination of hepatic resection, additional intraoperative treatment and postoperative treatment, and some patients could enjoy a longer life. CONCLUSIONS: Multimodality treatment including hepatic resection should be encouraged for advanced HCC patients with PV thrombi, BD involvement or compression or invasion of the IVC, as long as the remnant liver can overcome postoperative liver failure.

Energy metabolism and regeneration in transgenic mouse liver expressing creatine kinase after major hepatectomy.

BACKGROUND & AIMS: The catalysis of a creatinine/phosphocreatine system by creatine kinase is not expressed in the liver. The aim of this study was to examine the energy energy metabolism and regeneration after hepatectomy using transgenic mouse liver expressing creatine kinase to clarify the effects of phosphocreatine on liver regeneration. METHODS: Transgenic mice were divided into two groups: group 1 was fed normal chow, and group 2 was fed 10% creatine chow for 5 days. Hepatic energy metabolism was evaluated before and after hepatectomies. Changes in remnant liver weight gain and bromodeoxyuridine labeling index were measured after 70% and 80% hepatectomies. RESULTS: Hepatic adenosine triphosphate level 24 hours after 70% hepatectomy was significantly higher in group 2 than group 1 (P<0.05). In group 2, mitochondrial adenosine triphosphate synthesis was enhanced because of elevated intramitochondrial adenine nucleotide content before hepatectomy, leading to sufficient adenosine triphosphate synthesis after a 70% hepatectomy. Bromodeoxyuridine DNA labeling index 24 hours after a 70% hepatectomy was significantly higher in group 2 than group 1. Rapid liver weight gain was observed in group 2 after a 70% hepatectomy. CONCLUSIONS: Abundant phosphocreatine promotes liver regeneration by reinforced hepatic energy metabolism. Gene transfer of creatine kinase to the liver may be a potential method in liver surgery.

Identification of a novel variant form of fibroblast growth factor receptor 3 (FGFR3 IIIb) in human colonic epithelium.

Although several tyrosine kinase-type growth factor receptors have been demonstrated in human colonic epithelial cells, the full spectrum of growth factor receptors has not been identified. Low stringency screening of a complementary DNA library prepared from the human colon cancer-derived cell line HT-29 with a probe containing the tyrosine kinase domain of human c-src kinase led to the identification and isolation of a clone containing a receptor class tyrosine kinase. This putative receptor was found to be identical to the human fibroblast growth factor receptor 3 (FGFR3) except for a region of 150 nucleotides (50 amino acids) encoding the presumptive ligand-binding domain, where it exhibited only 32% homology with the previously described FGFR3. The variant domain corresponded precisely to the splicing junctions of the exon encoding the carboxyl terminal half of the third immunoglobulin-like domain, suggesting that two forms of FGFR3 result from splicing of alternate exons in a manner similar to that previously found for FGFR1 and FGFR2. By prior convention, the previously reported from of FGFR3 was designated IIIc due to its high degree of homology with the IIIc domain of FGFR1 (83% homology) and the IIIc domain of FGFR2 (81% homology). However, the ligand-binding domain of FGFR3 found in the HT-29 cell line was more highly divergent from all previously reported FGFR immunoglobulin-like domain IIIs than any other two members of this receptor family. Therefore, we propose to designate the newly reported form as the FGFR3 IIIb variant. Genomic polymerase chain reaction confirmed that the IIIb-containing exon occupies a position 5' relative to the IIIc-containing exon within the FGFR3 gene. Northern blot analysis using a probe encompassing sequences unique to the FGFR3 IIIb mRNA confirmed the expression of a 4.4-kilobase transcript in two colon cancer-derived cell lines as well as normal human colonic mucosa. Using a technique combining reverse transcriptase polymerase chain reaction with restriction endonuclease digestion, cell lines, primary cells, and tissues were assessed for IIIb and IIIc transcripts; expression of the IIIb variant was associated with an epithelial lineage, while the IIIc variant was expressed predominantly in nonepithelial cells and tissues.

Fibroblast growth factors modulate intestinal epithelial cell growth and migration.

BACKGROUND/AIMS: Various peptide growth factors have been found to exert functional effects among epithelial cell populations. This study assessed the role of certain fibroblast growth factors (FGFs) (acidic FGF, basic FGF, and keratinocyte growth factor) in the regulation of intestinal epithelial cell proliferation and restitution. METHODS: Recombinant growth factors were added to subconfluent cultures of IEC-6, Caco-2, and HT-29 cell lines with subsequent assessment of [3H]-thymidine incorporation. The effects on an in vitro model of restitution were assessed by quantitation of cells migrating into standard wounds established in confluent monolayers of IEC-6 cells. Transforming growth factor beta (TGF-beta) content of growth factor-treated wounded monolayers was assessed by Northern blot and bioassay. RESULTS: Acidic FGF, basic FGF, and keratinocyte growth factor caused a modest increase in proliferation of IEC-6, Caco-2, and HT-29 cell lines. Acidic FGF and basic FGF promoted intestinal epithelial cell restitution in vitro up to 10-fold, in conjunction with the enhanced expression of TGF-beta messenger RNA and protein. Promotion of IEC-6 restitution by acidic and basic FGF could be blocked by addition of immunoneutralizing anti-TGF-beta antisera. CONCLUSIONS: FGFs that exert effects on fibroblast cells also exert effects on intestinal epithelial cell populations and may help promote epithelial cell restitution, the initial step of intestinal wound healing through a TGF-beta-dependent pathway.

Enzyme immunoassay of liver-type arginase and its potential clinical application.

We developed an efficient enzyme-linked immunosorbent assay (ELISA) system for measurement of human liver-type arginase in serum. A conjugate of the Fab' fragment of anti-human liver (recombinant) arginase IgG and horseradish peroxidase was used as the second antibody. This assay is highly specific, sensitive, and reproducible, enabling us to detect arginase at concentrations as low as several micrograms per liter without any prior processing of serum. The reaction is linear up to 200 micrograms/L. The arginase concentration in serum, as determined by this method, increased markedly and temporarily at the time of surgical operation or later injury to the liver. The increase was accompanied or followed by increases in serum concentrations of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, suggesting that the arginase emerged from damaged hepatocytes. In view of a limited tissue distribution of liver-type arginase, our ELISA system may be useful in diagnosis of various hepatic disorders as well as follow-up of postoperative conditions of patients.

Molecular damage to rat liver mitochondrial H(+)-ATPase during cold preservation with UW solution.

Deterioration of rat liver mitochondrial function during cold preservation with UW solution was studied. Mitochondria were isolated from control liver (0-hr preservation), 24-hr preserved liver, and 48-hr preserved liver with UW solution at 4 degrees C. Respiration assay revealed that phosphorylation was damaged more rapidly than oxidation. Inside-out submitochondrial particles prepared from each sample by sonication in the presence of EDTA were subjected to ATPase assay. ATP hydrolyzing activity of H(+)-ATPase (ATP synthase), which plays a key role in phosphorylation in mitochondria, decreased markedly to 58% after 24-hr preservation. After 48-hr preservation, reduction to 40% of control was noted. When an intrinsic H(+)-ATPase inhibitor protein was removed from ESMP by Sephadex gel filtration, decrease of the ATPase activity was enhanced down to 49% and 29% of the control for 24-hr and 48-hr preserved liver, respectively. Molecular damage of the enzyme was confirmed by SDS-PAGE. Alpha subunit of the enzyme decreased time-dependently, and H(+)-ATPase molecules that lost alpha subunit seemed to lose their catalytic activity. Although the cause of the molecular damage of H(+)-ATPase is not clear yet, some mitochondrial protease(s) may be involved.

Molecular diversity of erythrocyte calpastatin.

According to the difference of molecular masses estimated by SDS-polyacrylamide gel electrophoresis, calpastatins are classified into two types, i.e. muscle type (110 kDa) and erythrocyte type (70 kDa). Muscle type calpastatin contains four internally repetitive sequences (Domains 1-4) and one nonhomologous sequence on the amino-terminal side (Domain L), whereas erythrocyte type lacks Domains L and 1. By immuno-blot analysis, chicken erythrocytes, nucleated cells, were found to contain muscle type calpastatin. In avian erythrocytes, diminution of the calpastatin molecule as in mammalian erythrocytes was not observed.