Immunological properties of N-acetyl-β-d-glucosaminidase of normal human liver and of GM2-gangliosidosis liver

Antisera were raised to a partially purified preparation of human liver hexosaminidase and to highly purified preparations of hexosaminidase isoenzymes A and B. All the antisera precipitated the enzyme in an enzymically active form, which could be located on immunodiffusion and immunoelectrophoretic gels by using a histochemical substrate. The antisera to the purified isoenzymes were shown to react with hexosaminidase from human liver, kidney, brain and spleen, but did not cross-react with human liver beta-glucosidase, beta-galactosidase, alpha-mannosidase, beta-xylosidase, arylsulphatase or acid phosphatase. Hexosaminidases A and B were immunologically identical. The immunological properties of the hexosaminidases from livers of patients with three types of GM(2)-gangliosidoses were closely similar. No evidence could be found for cross-reacting material in enzyme-deficient states.     

Full-size form of human liver AMP-deaminase?

AMP-deaminase from human liver was purified by two-step phosphocellulose chromatography, and SDS-PAG electrophoresis of the most active enzyme fraction eluted has been performed. The largest of the protein fragments revealed had a size (92 kDa) of an apparent full-size enzyme subunit, and reacted positively with antibodies produced against specific human ampd2 gene product. Three-day storage at cold room temperature modified significantly the electrophoretical pattern of the enzyme, evidencing continuous and progressive degradation of its structure. This is a first report evidencing the presence of apparent full-size form of human liver AMP-deaminase in preparation obtained from endogenous source.     

Are some progestins genotoxic liver carcinogens?

Progestins (progestogens) are classified by the International Agency for Research on Cancer (IARC) as possibly carcinogenic to humans. In the last decade evidence has shown that a synthetic drug of this family, cyproterone acetate, is activated to a reactive species by the liver, and forms DNA adducts and elicits DNA repair in hepatocytes from both rats and humans. The response is similar in humans of both genders but markedly higher in female than in male rats. The promutagenic character of DNA lesions is indicated by the increase in liver of female rats of the frequency of micronucleated cells, of mutations, and of enzyme-altered preneoplastic foci. Two other synthetic progestins, chlormadinone acetate and megestrol acetate, and an aldosterone antagonist, potassium canrenoate, share with cyproterone acetate the 17-hydroxy-3-oxopregna-4,6-diene structure. While less extensively studied, results so far obtained indicate that they are capable of inducing genotoxic effects qualitatively similar to those of cyproterone acetate. The majority of progestins have not been systematically tested for genotoxicity and the generally negative responses obtained with the standard battery of genotoxicity tests might be the consequence of the use of inappropriate target cells and/or metabolic activation systems. Cyproterone acetate, is activated by the hepatocytes to reactive species of such short half-life that they react only with the DNA of the cell in which are formed. Therefore, it cannot be excluded a priori that other progestins will not display genotoxic effects when tested adequately. This hypothesis is supported by the knowledge that estrogen-progestin combinations used as oral contraceptives are classified by the IARC as carcinogenic to humans due to the increased risk of hepatocellular carcinoma. This risk should probably be ascribed to the progestin component, since estrogens are carcinogenic to humans due to the increased risk of endometrial and possibly of breast cancer but not liver cancer.     

TGF-β in progression of liver disease

Transforming growth factor-β (TGF-β) is a central regulator in chronic liver disease contributing to all stages of disease progression from initial liver injury through inflammation and fibrosis to cirrhosis and hepatocellular carcinoma. Liver-damage-induced levels of active TGF-β enhance hepatocyte destruction and mediate hepatic stellate cell and fibroblast activation resulting in a wound-healing response, including myofibroblast generation and extracellular matrix deposition. Being recognised as a major profibrogenic cytokine, the targeting of the TGF-β signalling pathway has been explored with respect to the inhibition of liver disease progression. Whereas interference with TGF-β signalling in various short-term animal models has provided promising results, liver disease progression in humans is a process of decades with different phases in which TGF-β or its targeting might have both beneficial and adverse outcomes. Based on recent literature, we summarise the cell-type-directed double-edged role of TGF-β in various liver disease stages. We emphasise that, in order to achieve therapeutic effects, we need to target TGF-β signalling in the right cell type at the right time.     

χ alcohol dehydrogenase isozymes of horse liver

Horse liver contains previously unrecognized isozymes of alcohol dehydrogenase. In contrast to the molecular forms identified up to now, under the conditions employed these variants migrate toward the anode on starch gel electrophoresis and were separated from the cathodic isozymes by DEAE-cellulose chromatography. They were then purified on agarose-hexane-AMP. Their physicochemical and compositional characteristics are similar to those x alcohol dehydrogenases from human liver. Like these and similar ones from simian liver, they retain most of their activity in the presence of10 mm 4-methylpyrazole, oxidize short-chain primary alcohols very poorly, and appear to prefer longer chain primary alcohols and ω-hydroxy fatty acids as substrates.     

NF-κB induced the donor liver cold preservation related acute lung injury in rat liver transplantation model

Background

We have observed at our clinical work that acute lung injury (ALI) often occurs in patients transplanted with donor livers persevered for long time. So, we conducted this study to investigate the influence of cold preservation time (CPT) of donor liver on ALI induced by liver transplantation (LT), and further study the role of nuclear factor-κB (NF-κB) in the process.

Methods

Wistar rats were used as donors and recipients to establish orthotopic rat liver transplantation models. Donor livers were preserved at 4°C for different lengths of time. The effect of NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC), on ALI was detected. All samples were harvested after 3 h reperfusion. The severity of liver injury was evaluated first. The expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in liver tissue and liver outflow serum were measured respectively. The severity indexes of ALI, the activity of NF-κB and inhibitor-κBα (I-κBα) in lung/liver were measured accordingly.

Results

With the prolonged liver CPT, the liver damage associated indexes and ALI-related indexes all increased significantly. TNF-α and IL-1β in liver outflow serum increased accordingly, and the activity of NF-κB in liver/lung increased correspondingly. All these ALI-associated indexes could be partially reversed by the use of PDTC.

Conclusions

Extended CPT aggravates the damage of donor liver and induces the expressions of TNF-α and IL-1β in liver. These inflammatory factors migrate to lung via liver outflow blood and activate NF-κB in lung, inducing ALI finally. NF-κB may play a critical role in LT-related ALI. Patients with or at risk of ALI may benefit from acute anti-inflammatory treatment with PDTC.     

Are oral contraceptive-associated liver cell adenomas premalignant?

From January 1977 to June 1990, 1,670 patients with a liver mass or masses underwent transabdominal fine needle aspiration biopsy of the liver. Of those cases, 99 were diagnosed cytologically as "hepatocellular carcinoma" and 9 as "consistent with liver cell adenoma." Among the 99 patients with hepatocellular carcinoma, 3 were users of oral contraceptives. The nine patients with liver cell adenoma were all users of oral contraceptives. Of them, two developed foci or areas of liver cell dysplasia within the adenomas. The mean periods of oral contraceptive use among these three groups of patients were different. It appeared that patients started to develop liver cell adenoma after five years of oral contraceptive use (mean, 6.3 years). Foci or areas of liver cell dysplasia began to arise within liver cell adenomas after 8 years of oral contraceptive use (mean, 9 years), and the patients started to develop hepatocellular carcinoma after 10 years of contraceptive use (mean, 11 years). The cytologic features of liver cell dysplasia strikingly mimicked those of hepatocellular carcinoma. From this study, the foci or areas of liver cell dysplasia appear to be the missing link responsible for the transformation of liver cell adenoma to carcinoma. It is believed that liver cell adenomas are not premalignant and may undergo reversible change after withdrawal of causative agents, whereas foci or areas of liver cell dysplasia within the adenomas are irreversible, premalignant changes and may transform into hepatocellular carcinoma.     

Why does liver fibrosis occur in schistosomiasis?

Schistosomiasis is one of the most prevalent of several chronic inflammatory diseases in which morbidity results primarily from tissue scarring. New concepts regarding the molecular pathogenesis of scar formation are being applied in research efforts to define the basis of liver fibrosis in schistosomiasis. Such investigations have led to the identification of an apparently novel lymphokine, fibroblast stimulating factor-I (FsF-I), produced in the egg granulomas. FsF-1 and other granulomo-derived fibrogenic cytokines may represent the molecular links between periovular granulomotous inflammation and hepatic fibrosis. Here, David Wyler postulates that the unmodified production of these f brogenic signals may be responsible for the development of severe hepatic fibrosis in the subpopulotion of infected individuals who develop this complication.     

Wilson's disease: a common liver disorder?

In two sibships 7 of 24 siblings were homozygous for Wilson''s disease. In family A, the largest kindred of this recessively inherited disease thus far reported, the proband presented with chronic active hepatitis, one sibling died of cirrhosis, a second had clinical evidence of chronic liver disease and two others had biochemical and histologic changes in liver biopsy specimens. In family B the proband had cirrhosis and portal hypertension and one sibling had biochemical and histologic evidence of liver disease. All six living patients had low serum concentrations of ceruloplasmin and copper and a high 24-hour urinary excretion of copper, which was greatly increased by administration of D-penicillamine. None showed neurologic abnormalities and only one had Kayser-Fleischer rings (detectable only by slit-lamp examination). Each patient had an erythrocyte sedimentation rate (ESR) of 8 mm/h or less. After 3 and 2 years, respectively, of D-penicillamine therapy the conditions of the two probands had improved. Liver function became normal in three siblings, and no abnormalities developed in the remaining one. Thus, since Wilson''s disease may present with chronic active hepatitis or cirrhosis with a normal ESR and without ocular or neurologic signs, it may be a more common cause of liver disease in young people than has been appreciated.     

Augmenter of liver regeneration (ALR) gene therapy attenuates CCl₄-induced liver injury and fibrosis in rats

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Augmenter of liver regeneration (ALR) has been shown to protect hepatocytes from various toxins. The aim of this study was to investigate the effects of ALR gene therapy on liver injury and fibrosis induced by CCl(4) in rats and further explore the underlying mechanisms. Human ALR expression plasmid was delivered via the tail vein. ALR gene therapy might protect the liver from CCl(4)-induced injury and fibrogenesis by attenuating the mitochondrial dysfunction, suppressing oxidative stress, and inhibiting activation of HSCs. This report demonstrated that ALR gene therapy protected against the ATP loss, increased the activity of ATPase, decreased intrahepatic reactive oxygen species level, and down-regulated transforming growth factor-β1, platelet-derived growth factor-BB, and α-smooth muscle actin expression. Following gene transfer liver function tests were significantly improved. In brief, ALR gene therapy might be an effective therapeutic reagent for liver fibrosis with potential clinical applications.     

Carbohydrate composition of purified human liver α-L-Fucosidase

Summary Human liver -L-fucosidase was purified to apparent homogeneity and analyzed for carbohydrate content primarily by gas-liquid chromatography (glc). The enzyme is about 7% carbohydrate by weight and contains the following sugars (residues per 50,000 molecular weight subunit): mannose (8.3), glucosamine (4.3) (presumably N-acetylated), sialic acid (1.6) and glucose (1.6). Galactose (0.8) and L-fucose (1.8) were also found but their presence may be due to artifacts of the purification procedure.     

α-tocopherol β-oxidation localized to rat liver mitochondria

Approximately 40% of Americans take dietary supplements, including vitamin E (α-tocopherol). Unlike other fat-soluble vitamins, α-tocopherol is not accumulated to toxic levels. Rather tissue levels are tightly regulated, in part via increased hepatic metabolism and excretion that could, theoretically, alter metabolism of drugs, environmental toxins, and other nutrients. To date, in vivo subcellular location(s) of α-tocopherol metabolism have not been identified. The proposed pathway of α-tocopherol metabolism proceeds via ω-hydroxylation to 13′-OH-α-tocopherol, followed by successive rounds of β-oxidation to form α-CEHC. To test the hypothesis that α-tocopherol ω-hydroxylation occurs in microsomes while β-oxidation occurs in peroxisomes, rats received daily injections of vehicle, 10 mg α-tocopherol, or 10 mg trolox/100 g body wt for 3 days, and then microsomes, mitochondria, and peroxisomes were isolated from liver homogenates. Homogenate α-tocopherol levels increased 16-fold in α-tocopherol-injected rats, while remaining unchanged in trolox- or vehicle-injected rats. Total α-tocopherol recovered in the three subcellular fractions represented 93 ± 4% of homogenate α-tocopherol levels. In α-tocopherol-injected rats, microsome α-tocopherol levels increased 28-fold, while mitochondria and peroxisome levels increased 8- and 3-fold, respectively, indicating greater partitioning of α-tocopherol to the microsomes with increasing liver α-tocopherol. In α-tocopherol-injected rats, microsome 13′-OH-α-tocopherol levels increased 24-fold compared to controls, and were 7-fold greater than 13′-OH-α-tocopherol levels in peroxisome and mitochondrial fractions of α-tocopherol-injected rats. An unexpected finding was that α-CEHC, the end product of α-tocopherol metabolism, was found almost exclusively in mitochondria. These data are the first to indicate a mitochondrial role in α-tocopherol metabolism.     

How does hexachlorobenzene treatment affect liver uroporphyrinogen decarboxylase?

The aims of the present work were: (1) to investigate whether the strong decrease of liver uroporphyrinogen decarboxylase (UroD) activity observed in experimental porphyria cutanea tarda is due to alteration of the enzymatic protein and (2) to improve the knowledge about the normal liver enzyme. With these purposes, several physicochemical studies for enzymatic characterization were carried out comparatively on the 12-fold purified liver enzyme of both normal and hexachlorobenzene porphyric rat. The study shows that the enzyme from porphyric rats has a higher activation energy, lower reactivity index and lower optimum pH than the normal one. In addition, it did not reach the Vmax at any of the substrate concentrations assayed (up to 28 microM uroporphyrinogen III), while the normal enzyme reached the plateau around 14 microM. The porphyric enzyme appears to be more protected than the normal against the inhibitory action of several metals, particularly Cu2+ and Pb2+, and against thermal inactivation. Zn2+ did not affect enzymatic activity, whereas Cu2+, Hg2+, Fe2+, Pb2+, and Cd2+ lowered the activities of both normal and porphyric enzyme in a dose-related way. It was also observed that the larger the atomic radius in its hydrated state, the lower the effect of the metal. Neither glutathione nor dithiothreitol significantly altered enzymatic activity in the range of concentrations assayed. beta-Mercaptoethanol had diverse effects, as regards both the concentration assayed and the enzymatic sample used. Assays with cystine showed a dual behaviour of both normal and porphyric enzymatic activity. Western blots for both preparations revealed a single band (65 kDa) with a similar intensity.This study show that hexachlorobenzene treatment modifies the physicochemical properties of liver UroD leading to a sharp decrease of its activity, without affecting its antigenic reactivity probably as a consequence of changes at the conformational level promoted by the binding of its reported inhibitor.     

β-Lapachone alleviates alcoholic fatty liver disease in rats

Alcohol-induced liver injury is the most common liver disease in which fatty acid metabolism is altered. It is thought that altered NAD⁺/NADH redox potential by alcohol in the liver causes fatty liver by inhibiting fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. β-Lapachone (βL), a naturally occurring quinone, has been shown to stimulate fatty acid oxidation in an obese mouse model by activating adenosine monophosphate-activated protein kinase (AMPK). In this report, we clearly show that βL reduced alcohol-induced hepatic steatosis and induced fatty acid oxidizing capacity in ethanol-fed rats. βL treatment markedly decreased hepatic lipids while serum levels of lipids and lipoproteins were increased in rats fed ethanol-containing liquid diets with βL administration. Furthermore, inhibition of lipolysis, enhancement of lipid mobilization to mitochondria and upregulation of mitochondrial β-oxidation activity in the soleus muscle were observed in ethanol/βL-treated animals compared to the ethanol-fed rats. In addition, the activity of alcohol dehydrogenase, but not aldehyde dehydrogenase, was significantly increased in rats fed βL diets. βL-mediated modulation of NAD⁺/NADH ratio led to the activation of AMPK signaling in these animals. Conclusion: Our results suggest that improvement of fatty liver by βL administration is mediated by the upregulation of apoB100 synthesis and lipid mobilization from the liver as well as the direct involvement of βL on NAD⁺/NADH ratio changes, resulting in the activation of AMPK signaling and PPARα-mediated β-oxidation. Therefore, βL-mediated alteration of NAD⁺/NADH redox potential may be of potential therapeutic benefit in the clinical setting.