Changes in Liver Ganglioside Metabolism in Obstructive Cholestasis - the Role of Oxidative Stress.

Bile acids have been implicated in cholestatic liver damage, primarily due to their detergent effect on membranes and induction of oxidative stress. Gangliosides can counteract these harmful effects by increasing the rigidity of the cytoplasmic membrane. Induction of haem oxygenase (HMOX) has been shown to protect the liver from increased oxidative stress. The aim of this study was to determine the changes in the synthesis and distribution of liver gangliosides following bile duct ligation (BDL), and to assess the effects of HMOX both on cholestatic liver injury and ganglioside metabolism. Compared to controls, BDL resulted in a significant increase in total as well as complex gangliosides and mRNA expression of corresponding glycosyltransferases ST3GalV, ST8SiaI and B3GalTIV. A marked shift of GM1 ganglioside from the intracellular compartment to the cytoplasmic membrane was observed following BDL. Induction of oxidative stress by HMOX inhibition resulted in a further increase of these changes, while HMOX induction prevented this effect. Compared to BDL alone, HMOX inhibition in combination with BDL significantly increased the amount of bile infarcts, while HMOX activation decreased ductular proliferation. We have demonstrated that cholestasis is accompanied by significant changes in the distribution and synthesis of liver gangliosides. HMOX induction results in attenuation of the cholestatic pattern of liver gangliosides, while HMOX inhibition leads to the opposite effect.

Chromatography and spectrofluorometry of brain fluorophores in neuronal ceroid lipofuscinosis (NCL).

The aim of the present work was to develop a chromatographic system for the separation of individual fluorophores extracted from neuronal ceroid lipofuscinosis (NCL) brain and isolated storage bodies. Extracts from gray matter were best resolved on silica-gel HPTLC plates using a mixture of chloroform/methanol/water (55:45:10 by vol.). Two other chromatographic systems were tested which gave poorer separation. Corrected fluorescence spectra were obtained on the original extract and fluorescence intensity, especially at longer wavelengths was increased in both samples. Yellow and blue fluorophores were detected on HPTLC plates using a primary violet and secondary yellow filter with cut-off levels of 400 and 520 nm, respectively. Plates were photographed at 20 min, 2 h and 1 week after chromatography. With this filter system, up to 12 yellow bands of differing intensity were observed at 20 min but with time, some of these changed to blue as a result of autoxidation. NCL tissues emit yellow fluorescence when viewed under light microscopy, however extracted material did not demonstrate a distinct peak in this region of the spectrum which should be around 575 nm. HPTLC confirmed this observation and time studies revealed that autoxidation changes occur and must be carefully controlled to reduce artifacts. The discrepancy between extracted and non-extracted observations may be the result of superposition of multiple fluorophores with differing maxima and/or a self-absorption phenomenon. The combination of chromatographic separation and spectral analysis as described in this study, may be a valuable technique to further clarify the characteristics of compound fluorescent lipopigments. It is suggested that NCL fluorophores of human brain differ in their properties from other models.

Mutagenicity of glycerol chlorohydrines and of their esters with higher fatty acids present in protein hydrolysates.

3-chloro-1,2-propanediol and 1,3-dichloro-2-propanol caused base substitutions in Salmonella typhimurium TA1535 both with and without metabolic activation. Metabolic activation seemed to act mainly by decreasing the toxicity of these compounds. A difference in the growth of the wild-type and repair-deficient strains of Escherichia coli was observed only for 1,3-dichloro-2-propanol with S9 mix. Esters of both chlorohydrines with fatty acids has smaller mutagenic effects than unesterified compounds.

Ganglioside composition in experimental tumors with different growth properties.

The composition of gangliosides was studied in four fibrosarcomas (FL, FLA, FLB, FLC) induced in the Lewis rat by Ferridextran, and in two clones of a spontaneous Lewis rat mammary sarcoma (C-1-SAM LEW and C-2-SAM LEW) and two supertransformed clones (S-174 and S-271) derived from these clones, using the B77 virus. The malignancy of the Lewis tumors was tested in terms of their ability to outgrow the RT-5 barrier in LEW.1x rats and expressed as the loss-rate of LEW.1x rats. As for the Ferridextran-induced tumors, the FL was the only one to have been rejected in nearly 100% of the LEW.1x recipients. Following presensitization with FL the other tumors were also rejected, though not at a rate of 100%. The rat loss-rate was: FL--0%, FLC--23.5%, FLB--36.5%, and FLA--82.6%. As malignancy increased, the composition of gangliosides showed signs of progressive simplification indicating a step-by-step repression of ganglioside biosynthesis involving first the disialoganglioside and subsequently also the monosialoganglioside pathways. Some less distinct decrease (but significant at the 5% level of probability) of gangliosides of the disialoganglioside pathway and an increase of the simplest ganglioside (GM3) were observed between the C-1 clone of SAM LEW and its S-174 supertransformant. However, the changes, especially in the C-2 clone and its supertransformant, were not such as would suggest a marked defect in the biosynthesis of gangliosides.

Adrenal changes in Niemann-Pick disease: differences between sphingomyelinase deficiency and type C.

Structural, chemical, and histochemical analyses of adrenal tissue performed in 8 cases of Niemann-Pick disease (NPD) revealed stark differences of storage between spingomyelinase (SMase) deficiency (6 cases) and type C (2 cases). In all the full-blown cases of the SMase deficiency group, pronounced sphingomyelin (SM) storage was found in all the zones of the cortical epithelium with slightly increasing centripetal gradient. The storage resulted in the reduction or even disappearance of lipofuscinogenesis in the reticular zone, in the reduction of the physiological fat content, in the generalized foamy transformation of the epithelium, and in moderate organomegaly. The storage was expressed in both A and B types and was roughly proportional to the storage in other viscera. The stromal storage was confined to the vascular endothelium, and in particular, to the macrophages. One of the cases showed the presence of typical spirolactone bodies unmodified in fine structure by the lysosomal storage. Their most conspicuous enzymatic activity was that of non-specific esterase and NADH tetrazolium reductase. The adrenals in type C were macroscopically and histologically normal except for a variable population of stromal foam cells. Chemically, there was slight increase in all phospholipids with borderline or moderate percentual increase of SM. There was also slight increase in some of the lower neutral glycosphingolipids. Electron microscopy dislosed rudimentar storage in lower cortical layer epithelium which by its fine structure and according to results of lipid histochemistry was qualitatively different from that in SMase deficiency. The stromal storage was expressed mainly in macrophages in which there was histochemically detectable amount of SM. There was no storage detectable in medullary cells in neither group of NPD complex. The results point not only to striking quantitative differences in storage intensity between the 2 basic groups of NPD showing the cortical epithelium in type C as being remarkably resistant to the metabolic disorder, but also to difference in quality of the storage very much like that found in other tissues, too.

Fine specificity of anti-Le(X) monoclonal antibody TEC-01.

TEC-01 monoclonal antibody recognizes the oligosaccharide structure Galbeta1-->4[Fucalpha1-->3] GlcNAc (Le(X) hapten). To determine its fine specificity, reactivity of TEC-01 antibody with Le(X) glycosphingolipids, isolated from human liver metastasis of adenocarcinoma, and Le(X) neoglycolipid were analysed. Immunostaining of Le(X) glycosphingolipids, fractionated by thin-layer chromatography, showed that TEC-01 reacted with difucosyl and trifucosyl Le(X) glycosphingolipids but not with Le(X) pentasaccharide ceramide. Interestingly, TEC-01 also reacted with Le(X) neoglycolipid prepared by reductive amination from Le(X) pentasaccharide, lacto-N-fucopentaose III, and L-1,2-dipalmitoyl-sn-glycerophosphoethanolamine. The combined data suggest that TEC-01 recognizes Le(X) hapten in glycolipids with longer oligosaccharide chain moiety.

Factors influencing the activity of cellular alkaline phosphatase during growth and sporulation of Bacillus cereus.

Alkaline phosphatase (EC 3.1.3.1) is synthesized in media with a low phosphate concentration (0.37 mM of total and 19 microM of inorganic phosphate, respectively) already during the exponential phase of growth of Bacillus cereus. The enzyme is repressed by higher phosphate concentrations (3.7 mM) during the whole growth period; during sporogenesis the enzyme activity in cells slightly increases even under these conditions. During growth the enzyme is not secreted into the medium, a minor amount being released after cessation of growth. The enzyme activity can be increased by adding Zn2+ ions (10 microM). When during growth without phosphate the pH of the medium decreases below 5.0, the enzyme activity temporarily decreases and growth is slowed down, followed by a subsequent increase of the enzyme activity. In this case the onset of sporulation is also delayed.

[Glycosphingolipids as tumor markers]. / Glykosfingolipidy jako nádorové markery.

Interest in research in the sphere of tumours has concentrated for many years on finding tumour specific antigenic structures which can be used in immunodiagnosis and immunotherapy. During the last 30 years a number of monoclonal antibodies against tumours was prepared by means of which evidence was provided that antigens associated with tumourous processes have very often the nature of carbohydrates, in particular those linked to lipids, i.e. glycolipids. Carbohydrate structures with which anti-tumour antibodies react are usually found (at least in small concentrations) also in normal tissues. In the strict sense, from the chemical aspect specific tumour antigens do not exist but rather antigens associated with the tumourous process-tumour markers. Factors which influence the presence of a glycolipid of a tumour marker may be a) a simplification of the glycolipid spectrum by incomplete biosynthesis with concurrent deficiency of higher complex glycolipids and cumulation of the glycolipid precursor, b) activation of new pathways of biosynthesis, c) changes in the ceramide portion, d) lactonisation in the carbohydrate portion of the molecule, e) changes in the accessibility of the glycolipid molecule, their conformation and density on the cell surface. The author reviews the main glycolipid tumour markers which can be used in immunodiagnostics.

[Vesicular and pronuclear glycoproteins in the pathogenesis of cholesterol lithiasis]. / Vezikulární a pronukleacní glykoproteiny v patogenezi cholesterolové litiázy.

BACKGROUND: Several biliary proteins have been known to accelerate fusion of cholesterol rich phospholipid vesicles. Some of them are present in vesicular membrane, localisation of other proteins is unknown. Biliary glycoprotein has not been studied in consequence with pathogenesis of cholesterol lithiasis. METHODS AND RESULTS: Low molecular extravesicular proteins were separated from vesicles by gel filtration on a 1200mm column of Sephacryl S-300 HR. Immunoglobulins IgM, IgA, haptoglobin, biliary glycoprotein I (BGP I) and nonspecific crossreactive antigen were eluted along with vesicles. Albumin and alpha 1-acid glycoprotein were eluted later and must be extravesicular. CONCLUSIONS: Fact that BGP I (85 kDa membrane glycoprotein) eluted along with vesicles and not in albumin fraction suggests that it might be bound in vesicular membrane. As a known adhesion molecule it could thus play an important role in pathogenesis of cholesterol cholelithiasis.

[Pronuclear and antinuclear factors in the pathogenesis of cholesterol cholelithiasis]. / Pronukleacní a antinukleacní faktory v patogenezi cholesterolové cholelitiázy.

Contrary to hitherto published results, the authors provided evidence of significant pronucleation activity in the protein fraction which is not linked to concanavaline A. Delipidation or proteolysis markedly reduce the pronucleation activity of this fraction. Albumin was identified as the main protein in this fraction. The lipid-protein complex formed by albumin and lipids had a high pronucleation and crystallization activity in relation to cholesterol. Calcium ions increased the crystalization activity. Complexes formed by proteins and lipids can be vectors of the main pronucleation activity in bile. In investigations of the main cholesterol fraction the authors provided evidence that only part of so-called pronucleation proteins is linked to vesicles--i.e. IgM, IgA and biliary glycoprotein BGP I and II. The authors assume that only proteins firmly linked to vesicles can participate in the process of cholesterol crystallization. Biliary glycoprotein BGP I and II was present in vesicles and when added into a model bile it presented a high pronucleation activity. Biliary glycoprotein is a new hitherto not identified pronucleation protein in bile.

[Changes in the composition of glycolipids in N-nitrosomorpholine-induced hepatoma. The role of such substances in the plasma membrane of normal and neoplastic cells]. / Zmeny ve slození glykolipidu v hepatomu indukovaném n-nitrózomorfolínem. Uloha techto látek v plazmatické membráne normálních a nádorových bunek.

Malignant hepatomas induced in the rat by N-Nitrosomorpholine showed an absence of trisialogangliosides GT1 and, in some cases, also a decrease in disialoganglioside GD1a and monoganglioside GM1. Simultaneously there occurred an increase in a monoganglioside with a short oligosacharide chain GM3. Such deviations in the composition of glycolipids are in accordance with the literary data mentioning, as a general change, the presence of only some glycolipids as a result of a deficient synthesis of the oligosacharide part of their molecule. The differences in the glycolipid composition of the cell membrane as well as their different arrangement have been discussed with respect to some present views concerning the different behaviour of the neoplastic cell surfaces.

[Phosphoglyceridosis]. / Fosfoglyceridóza

On the basis of a bioptic examination of the appendix, skin and a liver specimen, the diagnosis of phospholipidosis was made in a girl aged 27 months. In contrast to the Niemann-Pick's complex of sphingomyelinoses, phosphoglycerides were stored in larger amounts than spingomyelin. The disease should be undoubtedly included under one heading with the so-called "kephalinosis" [11] and with the cases described by Wiedemann et al. [10]. The terms "Type II phospholipidosis [Baar-Wiedemann's disease]" or, briefly, phosphoglyceridosis, appear to be most adequeate for designating the diseases in question. The disorder can be diagnosed on the basis of iron hematoxylin staining, visualizing all phospholipids. In Niemann-Pick's sfingomyelinosis, alkaline hydrolysis does not alter the colour, whereas in the phosphoglyceridosis under discussion the colour is substantially reduced or even desappears after alkaline hydrolysis.

[Composition of gangliosides in experimental rat tumors]. / Slození gangliosidu v nekolika experimentálních krysích nádorech.

Spectrum of gangliosides was studied in some tumors. It was the same in a hepatocellular carcinoma induced by N-nitrosomorpholine in Wistar rats as in control liver In addition, several tumors contained an unidentified fraction between GD1b and GT1b. There were total ganglioside differences both in tumours and controls. Lymphatic leukemia samples had lower contents of GD1a and higher contents of GM1 ganglioside. Spontaneous breast sarcoma of Lewis rats (SAM) failed to differ from a sarcoma of low grade malignancy induced by ferridextran (FL) with the exception of slight increase in GD1a and GD2. All the tumours contained higher gangliosides in concentration at least the same as controls.

The effect of heme oxygenase on ganglioside redistribution within hepatocytes in experimental estrogen-induced cholestasis.

Cholestasis is characterized by the elevation of serum total bile acids (TBA), which leads to the production of both free radicals and oxidative stress. Although they do not share the same mechanisms, membrane glycosphingolipids (GSL) and the antioxidant enzyme heme oxygenase-1 (HMOX1) both act against the pro-oxidative effect of TBA. The aim of the study was to assess the role of HMOX on GSL redistribution and composition within hepatocytes in the rat model of estrogen-induced cholestasis. Compared to the controls, an increase of total gangliosides in the liver homogenates of the cholestatic group (P=0.001) was detected; further, it paralleled along with the activation of their biosynthetic b-branch pathway (P<0.01). These effects were partially prevented by HMOX activation. Cholestasis was accompanied by a redistribution of GM1 ganglioside from the cytoplasm to the sinusoids; while HMOX activation led to the retention of GM1 in the cytoplasm (P=0.014). Our study shows that estrogen-induced cholestasis is followed by changes in the synthesis and/or distribution of GSL. These changes are not only triggered by the detergent power of accumulated TBA, but also by their pro-oxidant action. Increases in the antioxidant defenses might represent an important supportive therapeutic measure for patients with cholestatic liver disease.

Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction.

A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde), which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit. Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at -20 degrees C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0 +/- 0.3% only. The loss from dried brain homogenate was 9.5 +/- 1.1%. Thus, anhydrous conditions (dry tissue samples and anhydrous acetone) are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion), and removal of cholesterol, which can act as a hydrophobic blocking barrier.

Lysosomal non-lipid component of Gaucher's cells.

An ultrastructural, histochemical and chemical analysis of storage elements in the infantile form of Gaucher's disease showed that in addition to cerebroside the lysosomes also included a non-lipid component of protein, or possibly glycoprotein nature. This component, easily removable with trypsin, was present in such quantities that it conditioned the typical solid and fibrillar appearance of storage elements even after they had been substantially delipidized. Another noteworthy finding was that the ultrastructural appearance of tubular structures generally regarded as stored cerebrosides persisted in all the extracted specimens without any noticeable change. The findings are compared with available data from the literature and their significance briefly discussed.