Galactosamine inhibition of protein synthesis in Bacteroides thetaiotaomicron

Galactosamine does not support growth of Bacteroides thetaiotaomicron. Despite this, galactosamine was more effective than utilizable carbohydrates such as glucose in preventing synthesis of the inducible enzymes alpha-glucosidase and chondroitin lyase. Galactosamine also stopped overall protein synthesis. By contrast glucose and other utilizable carbohydrates increased the rate of protein synthesis. Addition of glucose to bacteria which had been treated with galactosamine restored the ability of the bacteria to synthesize protein and to produce inducible enzymes. Moreover, when B. thetaiotaomicron was incubated with [1-14C]galactosamine for 30 min at 37 degrees C, about one-third of the label which was taken up by the cells comigrated with glucosamine-6-phosphate on a thin-layer chromatogram. Thus galactosamine appears to be phosphorylated by the bacteria. After 2 h incubation of the bacteria with [1-14C]galactosamine, there was a significant increase in the amount of label which could be extracted from acidified extracellular fluid with diethyl ether. This indicates that galactosamine can be metabolized to the level of volatile fatty acids. The rate of uptake of galactosamine and the amount of labeled fatty acids produced from galactosamine were both much lower than the values obtained when glucosamine was the substrate. Thus, although some metabolism of galactosamine occurs, the rate is apparently too slow to enable galactosamine to support growth of B. thetaiotaomicron.     

A modification of determination for glucosamine and galactosamine in glycoprotein with the amino acid analyzer: Application to total acid hydrolyzate of rat renal glomerular basement membrane

A procedure was described for the rapid determination of glucosamine and galactosamine in total acid hydrolyzate of rat renal glomerular basement membrane (GBM) by the use of amino acid analyzer. Glucosamine and galactosamine, as well as other amino acids in glycoprotein hydrolyzate, were well identified and estimated simultaneously by using a short column of HITACHI I-PF-B spherical resin, eluted with a pH 6.09 buffer containing 8% methanol at 38°C.Total time consumed for elution of galactosamine was 60 min. This method is ideal for the separation of small amount of galactosamine from hydroxylysine-rich materials.     

Analysis of hog thyroglobulin. Identification of galactosamine and absence of lysinoalanine, a potential product of tyrosine coupling

A small peak in the amino acid analysis of hog thyroglobulin was observed in the region reported for lysinoalanine and galactosamine. Since galactosamine had been previously reported absent in hog thyroglobulin, the possibility that this peak was lysinoalanine, a potential product of the coupling of two iodotyrosines, was investigated. Tests, however, showed that the material was galactosamine and that hog thyroglobulin contains no significant amount of lysinoalanine. Approximately 5 moles of galactosamine were found per mole of thyroglobulin.     

Targeted antagonism of galactosamine toxicity in normal rat hepatocytes in vitro

We present evidence that normal hepatocytes can be specifically protected from galactosamine toxicity in vitro by targeting an antagonist to these cells via receptor-mediated endocytosis. The strategy is based upon the following principles: 1) galactosamine is a highly selective hepatotoxin that causes a dose-dependent depletion of uridine intermediates; 2) galactosamine toxicity can be antagonized by supplemental administration of uridine; 3) normal hepatocytes possess unique cell-surface receptors that can internalize galactose terminal (asialo-)glycoproteins with subsequent degradation of the glycoprotein ligand. Based on these facts, we hypothesized that chemical coupling of a galactosamine antagonist to an asialoglycoprotein could result in cell-specific delivery and protection of normal hepatocytes by targeting the antagonist via asialoglycoprotein receptors. Using a model system consisting of freshly isolated rat hepatocytes (receptor (+)) and Morris 7777 rat hepatoma (receptor (-)) cells, sensitivity to galactosamine in vitro was determined and found to be similar for both types of cells. A targetable antagonist was synthesized by coupling uridine monophosphate to asialoorosomucoid in a molar ratio of 5 to 1. Exposure of Morris 7777 cells to the targetable antagonist in the presence of a toxic concentration of galactosamine did not protect these cells as evidenced by a steady decline in the number of viable cells in a fashion identical to cells treated with galactosamine alone. However, normal hepatocytes that received the conjugate in the presence of galactosamine were protected as their viable cell number remained the same as control (untreated) cells. Competition by an excess of asialoglycoprotein inhibited the protective effect of the conjugate, supporting the concept that the asialoglycoprotein component of the conjugate was responsible for the specific delivery of the antagonist to the target cells.     

Studies on the inhibition of glycogenolysis by D-galactosamine in rat liver hepatocytes.

Effect of galactosamine on glycogenolysis was studied in isolated hepatocytes. It was found that addition of galactosamine strongly inhibited glycogenolysis in normal hepatocytes. Galactosamine-inhibited glycogenolysis was not stimulated by epinephrine or glucagon. This inhibition was specific as no such inhibition was observed with galactose, 2-deoxy-glucose or glucosamine. The glucagon-stimulated cyclic AMP formation in galactosamine-treated hepatocytes was the same as in normal cells; Glc-1-P and Glc-6-P did not accumulate nor was lactate formation enhanced. The glucose production by hepatocytes from regenerating liver was only slightly inhibited by galactosamine and glucagon addition stimulated glycogenolysis in the presence of the amino sugar.     

Gas chromatographic analysis of hexosamines in glycoproteins

A gas chromatographic method for the analysis of hexosamines in glycoproteins was described which uses the alditol acetate derivatives of the sugars. A polyamide (Poly A 103) liquid phase was used which effectively separates glucosamine, galactosamine, and mannosamine from each other. Mannosamine served as internal standard to facilitate accurate quantitation of glucosamine and galactosamine.     

Structure of the polysaccharide isolated from the sheath of Sphaerotilus natans

A polysaccharide was isolated from the sheath of a sheathed bacterium, Sphaerotilus natans. The sheath polysaccharide (SPS) was composed of D-glucose and D-(N-acetyl)galactosamine in molar ratios of 1:4. Methylation linkage analysis revealed the presence of the residues of 4-linked glucose, 4-linked (N-acetyl)galactosamine, and 3-linked (N-acetyl)galactosamine in molar ratios of 1:3:1. The oligomer of SPS was prepared with an SPS-specific degrading enzyme from a sheath-degrading bacterium, Paenibacillus koleovorans. The oligomer was derivatized and subjected to fast atom bombardment-mass spectrometry to investigate the monosaccharide sequence of SPS. The structure of SPS was confirmed by nuclear magnetic resonance. The resulting data showed that SPS is a straight-chained basic polysaccharide constructed of a pentasaccharide repeating unit.     

Biochemical and genetic studies on galactosamine metabolism in Neurospora crassa.

In Neurospora, galactosamine can be released from the cell wall and from an alcohol-soluble compound by acid hydrolysis. All of the detectable alcohol-soluble galactosamine was present as uridine diphospho-2-acetamido-2-deoxy-D-galactose (UDPGalNAc). The results of pulse-labeling studies and enzymatic assays indicated that UDPGalNAc was synthesized via the epimerization of uridine diphospho-2-acetamido-2-de+xy-D-glucose (UDPGlcNAc). A single-gene morphological mutant, doily (do), which grew at less than 4% the rate of the wild-type strain, had 3% of the wild-type UDPGalNAc content and 0.5% of the wild-type level of cell wall galactosamine but normal levels of UDPGlcNAc and cell wall glucosamine. Cell extracts of the doily cultures containing only 20% of the specific activity of UDPGlcNAc-4-epimerase found in the extracts of wild-type cultures. Two types of faster-growing partial revertants of the doily strain were isolated. One type had an intermediate level of both alcohol-soluble and cell wall galactosamine. A second type had an intermediate level of alcohol-soluble galactosamine but low levels of cell wass galactosamine. Genetic analyses indicated that the reverse mutations had occurred at the do locus in both types. This finding that cell wall glucosamine synthesis and growth rate can be separated genetically indicates that mutations at the do lucus lead to pleiotropic effects.     

Biochemical study of cardiac valvular tissue. Biosynthesis in vitro of hexosamine-containing substances in bovine heart valve

1. Two distinct hexosamine-containing substances have been obtained from bovine cardiac valvular tissue which was incubated with labelled glucosamine. These were identified as mucopolysaccharides and glycopeptides respectively, both by the elution pattern from a Sephadex G-50 column and by chemical analysis. 2. In the mucopolysaccharide fraction about 80% of both the total hexosamine and total radioactivity were present; the galactosamine/glucosamine ratio of the amounts was about 1.36. 3. The glycopeptide fractions had about 20% of both total hexosamine and total radioactivity; the galactosamine/glucosamine ratio of amounts was about 0.44. In this fraction, over 15% of radioactivity was present in sialic acid. 4. In contrast with the concentrations of the several chemical components, there were remarkable differences in the biosynthetic activities among the four valves; the tricuspid valve had highest specific radioactivity in all components of both substances, followed then in turn by mitral, aortic and pulmonary valves. 5. Glucosamine in mucopolysaccharides had a rapid rate of turnover, followed then in turn by turnover rates of both glucosamine and galactosamine in glycopeptides, and of galactosamine in mucopolysaccharides. The possible significance of these findings is discussed.     

Beneficial effect of somatostatin on galactosamine induced liver injury

The purpose of our investigation was to study the effect of somatostatin on acute experimental liver injury induced in rats by galactosamine (1.2 g/100 g body wt.). Somatostatin (125 micrograms/100 g body wt.) was administered subcutaneously in a protamine sulphate/ZnCl2 suspension either 2 h prior to the injection of galactosamine or 2 h and again 12 h following the injection. Serum transaminases (GOT, GPT) and serum concentrations of triiodothyronine and thyroxine were determined 28 h after the injection of galactosamine. Histology of the liver was performed by light microscopy. Our results showed that the administration of somatostatin significantly (P less than 0.02) reduced the elevation of GOT and GPT activity and diminished the degree of necrosis, and that although the administration of dibutyryl-cAMP (5 mg/100 g body wt.) intensified galactosamine induced liver injury, this effect of dibutyryl-cAMP could be completely prevented by somatostatin treatment. There was no difference in the serum concentrations of triiodothyronine and thyroxine in controls as compared to galactosamine and galactosamine plus somatostatin treated rats. At present the mechanism of this cytoprotection by somatostatin is unknown.     

The oligosaccharide units of rabbit immunoglobulin G. Asymmetric attachment of C2-oligosaccharide

Two populations of immunoglobulin G (IgG) molecules were isolated from pooled rabbit serum. The first was almost devoid of galactosamine and was obtained in a yield of 7% of the total IgG; the second contained on average a single residue of galactosamine per molecule and was obtained in a yield of 30% of the total IgG. The galactosamine, which is present solely in the C2-oligosaccharide, appeared to be present on one H-chain and not the other in the four-chain structure. Evidence was obtained by the isolation of a glycopeptide linked through a disulphide bridge to a second peptide of the same sequence; the oligosaccharide attached to the first peptide was absent from the complementary peptide. Further evidence was obtained by degradation and analysis of the 5S IgG fragment, which comprises an intact half-molecule coupled through a disulphide bridge to the Fc fragment derived from the opposing half molecule (Goodman, 1965); only the intact H-chain carried the C2-oligosaccharide.     

The carbohydrate components of hydrolysates of gastric secretion and extracts from mucous glands of the gastric body mucosa and antrum

1. The sugars and amino sugars of hydrolysates of gastric secretion were determined by gas-liquid chromatography. 2. All the gastric aspirations examined showed on hydrolysis the presence of fucose, galactose, mannose, glucose, galactosamine, glucosamine, N-acetylneuraminic acid and sulphate. 3. Galactose and glucosamine were always found in equimolar amounts, but the galactose/galactosamine ratio in different aspirations was 2:1, 3:1, 4:1 or 5:1. Repeated gastric aspirations of each subject examined showed constant ratios of these carbohydrate components. 4. Fucose and sialic acid appear to be related to glucosamine and galactosamine respectively. 5. The carbohydrate components of extracts from the mucous glands of the body mucosa and antrum did not differ from those of gastric secretion.     

The effect of scurvy on glycosaminoglycans of granulation tissue and costal cartilage

1. The effect of ascorbic acid deficiency on glycosaminoglycans of granulation tissue and cartilage of guinea pigs was investigated by determination of the changes in the glucosamine and galactosamine contents 12 days after tendonectomy. 2. In normal granulation tissue, the glucosamine and galactosamine contents rose to a peak at 5 and 10 days respectively, whereas the hydroxyproline and proline contents continued to rise throughout the 20 days after tendonectomy. 3. The galactosamine in scorbutic granulation tissue, but not in that of pair-fed controls, decreased significantly in absolute amount and relatively to glucosamine, which remained practically unchanged; the cartilage galactosamine did not decrease during the 22 days of deficiency owing to the presence of excess of preformed galactosaminoglycans, which masked the small amount of newly formed glycosaminoglycans. 4. The chemical results were confirmed by radioactivity studies in vivo of incorporation of [U-(14)C]glucose into galactosamine and glucosamine of scorbutic granulation tissue and cartilage. The incorporation of (14)C into galactosamine decreased significantly in scurvy in both tissues. 5. The results indicated in both tissues a decreased formation of galactosamine during scurvy, although an increased degradation of polymerized glycosaminoglycans could not be entirely ruled out. It is concluded that, if lack of ascorbic acid causes an impaired galactosamine formation, the most likely position for the block may be in the UDP-N-acetylglucosamine 4-epimerase reaction.     

Purification and Chemical Properties of a Flocculant Produced by Paecilomyces

A new flocculant for microbial cells was purified by ethanol precipitation and gel chromatography from the culture fluid of Paecilomyces sp. I-1. Isoelectric focusing of the flocculant (PF-101) showed a single band at pH 8.5, and its molecular weight was estimated to be over 300,000 daltons by the ultra-filtration method. The results of elemental analysis, the IR spectrum and investigation of the acid hydrolysate by gas and liquid chromatography and colorimetrie analysis suggested that PF-101 was a polysaccharide composed of galactosamine. About 80% of the galactosamine residues were N-unsubstituted and 8% were N-acetylated. Studies on deaminative cleavage, periodate oxidation and Smith degradation suggested that the galactosamine residues were mainly linked by α (→4)-linkaees.     

Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: mechanism of suppression

We have shown that nitric oxide production is dramatically decreased in rat primary hepatocyte cultures exposed to galactosamine. Cotreatment of the cells with uridine, which is known to prevent cytotoxicity, was found to also attenuate NO loss. In the present study, two possible mechanisms for the decreased nitric oxide production were examined. First, we examined the possibility that galactosamine could interfere with the uptake of extracellular arginine by the cultured hepatocytes. Cellular uptake of arginine was determined after addition of 14C-arginine at the time of hepatocyte attachment. Uptake of arginine was rapid in control cultures, and both the rate and level of uptake were unchanged by the addition of a cytotoxic concentration of galactosamine (4 mM). In addition, increased concentrations of arginine in the cell culture medium did not ameliorate the galactosamine-induced decrease in production of nitric oxide. Second, we determined whether the synthesis of inducible nitric oxide synthase in the hepatocyte cultures was inhibited by addition of galactosamine. Hepatocyte levels of inducible nitric oxide synthase were determined immunochemically at various times after the addition of galactosamine (4 mM). In control cultures, inducible nitric oxide synthase was detectable at 7 and 24 hours after attachment. In contrast, no nitric oxide synthase protein was detectable at any time in the galactosamine-treated cultures. Furthermore, addition of galactosamine after inducible nitric oxide synthase had already been synthesized (6.5 h after attachment) did not result in suppression of nitric oxide production in the hepatocyte cultures. The present studies suggest that galactosamine suppresses nitric oxide production in hepatocyte cultures by inhibiting synthesis of inducible nitric oxide synthase, rather than by interference in cellular uptake of arginine.     

Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: lack of involvement in cytotoxicity

Galactosamine hepatotoxicity in vivo has long been associated with rapid and extensive depletion of hepatic uridine nucleotides. Depletion of uridine nucleotides is considered to be causal in the toxicity, as evidenced by the protective effect of uridine administration. However, the exact mechanism of galactosamine-induced hepatic necrosis is still unclear. We have previously shown that the addition of galactosamine to rat primary hepatocyte cultures dramatically decreases production of nitric oxide, as measured in the 24 hour culture medium. The present study investigates whether decreased nitric oxide production contributes to the toxicity of galactosamine in primary hepatocyte cultures. Similar concentration-response curves were observed for the decrease in nitric oxide production and galactosamine cytotoxicity, raising the possibility that there is a similar mechanism for these effects. Suppression of NO synthesis was a direct effect of galactosamine, rather than an indirect effect due to loss of cells from the cultures. Both cytotoxicity and the decrease in nitric oxide production were attenuated by coaddition of 3 mM uridine. However, galactosamine cytotoxicity was not enhanced by prior inhibition of hepatocellular NO synthesis nor was it attenuated by maintenance of culture NO levels with molsidomine or diethylamine NONOate. These data do not support a role for decreased hepatocyte nitric oxide production in galactosamine hepatocyte toxicity.     

Influence of D-galactosamine upon the NAD-metabolism in rat liver

After application of D-galactosamine a hepatitis develops in the rat liver. This can be prevented by different agents, including tryptophan. Yet it has not been possible to give definitive conclusions about the mechanism of galactosamine hepatitis. In this paper we report about the influence of galactosamine on the NAD metabolism. D-galactosamine inhibits the NAD synthesis initiated by nicotinamide in normal and adrenalectomized animals. The NAD synthesis from tryptophan is prevented in normal animals, in adrenalectomized ones however there is an increase of NAD in the presence of D-galactosamine reduces the activity of the ADPR transferase. Inhibitors of the ADPR transferase prevent the galactosamine hepatitis. From the results presented we conclude that the ADPR transferase plays an important role in the development of the galactosamine hepatitis.     

The effect of scurvy on hexosamine-containing substances in healing wounds in guinea pigs

1. Granulation tissue from healing tendonectomy wounds in guinea pigs was analysed and the effects of inanition and ascorbic acid deficiency on this tissue were investigated. 2. Inanition produced no significant effect on either the glucosamine or the galactosamine content of the tissue. Ascorbic acid deficiency decreased the galactosamine content without affecting the glucosamine content. 3. Fractionation of papain-digested granulation tissue gave three major fractions, which behaved respectively as glycopeptide, hyaluronic acid and a sulphated glycosaminoglycan mixture. At least half of the sulphated glycosaminoglycan mixture behaved as dermatan sulphate. 4. Inanition produced no consistent effect on the fractions examined. In ascorbic acid deficiency, a decrease in the sulphated glycosaminoglycan fraction was observed, which accounted for the decreased galactosamine content of the tissue. This was accompanied by a decrease in hyaluronic acid and a slight increase in the glycopeptide fraction.     

Comparative studies of the effects of galactosamine and actinomycin D on nuclear ribonucleoprotein particles from rat liver

Nuclear ribonucleoprotein particles of 75S were obtained from rat liver nuclei after mild sonication and isotonic salt extraction only when the preparation was carried out in the presence of a cytosolic ribonuclease inhibitor. Particles of 38S were isolated in the absence of inhibitor. The 38S nuclear ribonucleoprotein (nRNP) particles showed a protein/RNA ratio of 8, and a buoyant density of 1.39 g/ml in cesium chloride solution. They were further characterized by the pattern of their proteins on sodium dodecylsulfate (SDS)-acrylamide gel electrophoresis. Incorporation of [³H]cytidine into nuclear RNA was reduced to approx. 20% of controls 3 and 6 h after administration of galactosamine or actinomycin D. However, when [³H]cytidine was administered 30 min prior to the drugs a decrease of radioactivity in 38S nRNP particles to 43 and 81% of controls was found after 3 h. The yield of 38S particles 3 h after galactosamine or actinomycin D dropped to 41% and 78% of controls, and after 6 h to 43 and 70%, respectively. Six hours after galactosamine or actinomycin D treatment, the protein to RNA ratio increased to 13.3 and 9.1. No significant changes in protein patterns 3 h after treatment with galactosamine or actinomycin D were observed. Possible mechanisms, such as impaired transport of 38S nRNP particles after actinomycin D treatment or increased loss of particles due to a defective nuclear membrane after galactosamine administration are discussed.     

In vivo and in vitro studies on the protective effects of 9 beta-methylcarbacyclin, a stable prostacyclin analogue, in galactosamine-induced hepatocellular damage

Early treatment with prostacyclin (PGI2) was previously shown to reduce mortality in the galactosamine model of acute hepatic failure in the rat, with a decreased release of hepatic cytosolic and lysosomal enzymes. In this study, 9 beta-methylcarbacyclin, a chemically stable analogue of PGI2, had similar protective effects to PGI2 in vivo but required approximately 100-fold higher concentrations (2 mg kg-1). These effects were only obtained when 9 beta-methylcarbacyclin was given early (0 to 6 h post-galactosamine) but not later (24 to 30 h). In isolated rat hepatocytes in vitro galactosamine up to a concentration of 100 mM caused a dose-dependent inhibition of L-[U-14C] leucine incorporation into protein and increase in the release of the cytoplasmic enzyme lactate dehydrogenase. Studies on the short-term effects of 9 beta-methylcarbacyclin using isolated hepatocytes treated with galactosamine (5 mM) showed that this agent, at an optimum concentration of 30 ng ml-1, was capable of significantly reducing the inhibition of protein synthesis caused by galactosamine but did not alter the rate of release of lactate dehydrogenase. The results demonstrate that the protective effects of 9 beta-methylcarbacyclin occur early in the time course of galactosamine action, and include direct effects on the hepatocytes.