Influence of D-galactosamine on the synthesis of sugar nucleotides and glycoconjugates in rat hepatocytes.

Rat hepatocytes were incubated in the presence of a high concentration of the hepatopathogenic agent D-galactosamine (GalN), and the effect on the cellular concentrations of pyrimidine nucleotides and nucleotide sugars was determined. The UTP pool became depleted. The pools of UMP and CMP in RNA decreased to 72%, indicative for an inhibition of RNA synthesis. UDP-HexNAc (where HexNAc is GlcNAc+GalNAc) and UDP-HexN (where HexN is GlcN+GalN) levels increased, and those of UDP-hexose and UDP-GlcA (where GlcA is glucuronic acid) decreased. The cellular concentration of CTP did not change, whereas that of CMP-NeuAc (where NeuAc is N-acetylneuraminic acid) showed a 2-fold increase. Labelling with [14C]orotic acid and [3H]cytidine showed that the metabolic flow via the de novo pathway was not changed. The depletion of the so-called overflow pool of UTP [Pels Rijcken et al., Biochem. J., 293, 207-213, 1993] caused a release of the feedback inhibition by UTP and thus an increased flow through the salvage pathway. Finally, it appeared that GalN, when added to hepatocytes, gives rise to a pool of UDP-GlcNAc (where GlcNAc is N-acetylglucosamine) that is separate from the pool of UDP-GlcNAc that is derived from GlcN.

The effect of increasing nucleotide-sugar concentrations on the incorporation of sugars into glycoconjugates in rat hepatocytes.

Treatment of rat hepatocytes with 0.5 mM concentrations of uridine and cytidine results in increased cellular concentrations of UTP, UDP-sugars and CTP, whereas that of CMP-N-acetylneuraminate remained unchanged [Pels Rijcken, Overdijk, Van den Eijnden and Ferwerda (1993) Biochem. J. 293, 207-213]. The incorporation of radioactivity from 3H-labelled sugars into the cell-associated and secreted glycoconjugate fraction was influenced by these altered cellular concentrations of the nucleotides. For [3H]glucosamine, pretreatment with uridine resulted in a reduction of the glycosylation in both fractions. Increases in the secreted fractions were observed for fucose with both uridine and cytidine and for N-acetylglucosamine with uridine only. With [3H]N-acetylglucosamine, similar specific radioactivities for UDP-N-acetylhexosamine and CMP-N-acetylneuraminate were found, regardless of the pretreatment conditions. With [3H]N-acetylmannosamine, the specific radioactivity of CMP-N-acetylneuraminate showed an almost 2-fold increase on pretreatment. The latter increase did not result in an increased incorporation of radioactivity into the glycoconjugates. It was estimated that, in untreated cells, the ratio of radioactivity incorporated from [3H]glucosamine into glycoconjugate-bound N-acetylhexosamine and N-acetylneuraminate amounted to 2:3. In pretreated cells this ratio changed to approx. 2:1. Overall, the data show that pretreatment resulted in an increased incorporation of N-acetylhexosamine into cell-associated and secreted glycoconjugates, accompanied by a reduction in sialylation. It was concluded that an increased availability of UDP-N-acetylhexosamine caused the increased incorporation of N-acetylhexosamine. The elevated cytosolic level of UDP-N-acetylhexosamine (and of compounds like CMP) is suggested to impair the transport of CMP-acetylneuraminate to the Golgi, resulting in reduced sialylation. This study demonstrates that protein glycosylation can be regulated at the level of the availability of the various nucleotide-sugars in the Golgi lumen.

Pyrimidine nucleotide metabolism in rat hepatocytes: evidence for compartmentation of nucleotide pools.

Pyrimidine nucleotide metabolism in rat hepatocytes was studied by measurement of the labelling kinetics of the various intermediates after double labelling with [14C]orotic acid and [3H]cytidine, the precursors for the de novo and the salvage pathways respectively. For the uridine nucleotides, differences were found for the 14C/3H ratios in the UDP-sugars, in UMP (of RNA) and in their precursor UTP, suggesting the existence of separated flows of the radioactive precursors through the de novo and the salvage pathways. Higher ratios in the UDP-sugars, which are synthesized in the cytoplasm, and a lower ratio in UMP (of RNA) relative to the 14C/3H ratio in UTP indicated that UTP derived from orotic acid is preferentially used for the cytoplasmic biosynthesis of the UDP-sugars. Uridine, derived from cytidine, is preferentially used for the nuclear-localized synthesis of RNA. In contrast to these findings, the 14C/3H ratios in the cytidine derivatives CMP-NeuAc and CMP (of RNA), and in the liponucleotides CDP-choline and CDP-ethanolamine, were all lower than that in the precursor CTP. This indicates a preferential utilization of the salvage-derived CTP for the synthesis of the liponucleotides as well as for RNA and CMP-NeuAc. Similar conclusions could be drawn from experiments in which the intracellular amounts of several uridine- and cytidine-nucleotide-containing derivatives were increased by preincubating the hepatocytes with unlabelled pyrimidine nucleotides or ethanolamine. Based on these data, we propose a refined model for the intracellular compartmentation of pyrimidine nucleotide biosynthesis in which three pools of UTP are distinguished: a pool of de novo-derived molecules and a pool of salvage-derived molecules, both of which are channelled to the site of utilization; in addition an 'overflow' pool exists, consisting of molecules having escaped from channelling. An overflow pool could also be distinguished for CTP, but no discrimination between de novo and salvage-derived molecules could be made.

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver.

With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 14C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.

[Reconstruction of the urinary bladder following cystectomy]. / Urineblaasreconstructie uit ileum na cystectomie.

In 8 male patients a bladder substitute was made of 40 cm detubularized ileum after cystectomy done because of invasive bladder cancer (pT2-3N0M0). The results of this operation as regards bladder functions and morbidity were reasonably good, in accordance with data from the recent literature. Follow-up data over periods longer than 3 years are not yet available.

Glycosylation of three molecular forms of human alpha 1-acid glycoprotein having different interactions with concanavalin A. Variations in the occurrence of di-, tri-, and tetraantennary glycans and the degree of sialylation.

Human alpha 1-acid glycoprotein (AGP) was separated into a non-bound (AGP-A; 46%), a retarded (AGP-B; 39%) and a bound fraction (AGP-C; 15%) using concanavalin A (ConA)-Sepharose chromatography. The apparent molecular masses, as determined by SDS-PAGE, of the three fractions were 43.5, 42.3 and 41.2 kDa, respectively. The occurrence of N-linked di-, tri- and tetraantennary glycans on these three molecular forms (AGP-A, -B, and -C) was studied by sequential lectin-affinity chromatography of the 14C-labelled glycopeptides. These were obtained by extensive pronase treatment followed by N-[14C]acetylation of the peptide moieties. The glycopeptides of AGP-A did not bind to ConA-Sepharose whereas for AGP-B and AGP-C 18% and 44%, respectively, of the glycopeptides were bound as diantennary structures. Glycopeptide fractions of all three forms of AGP which were not bound to ConA-Sepharose were shown to contain equal amounts of both tri- and tetraantennary glycans by chromatography with Phaseolus vulgaris leukoagglutinating lectin (L-PHA). With the assumption that each molecule contains five glycosylation sites, it could be shown that AGP-A contains no diantennary structures whereas AGP-B and AGP-C contain one and two diantennary structures, respectively. In addition each of the molecular forms contains equal amounts of tri- and tetraantennary structures on the remaining glycosylation sites. The results of this study, therefore, exclude a uniformity of glycan chains in the three molecular forms of AGP. The degree of sialylation of each of the molecular forms was investigated by chromatography on L-PHA-agarose and Ricinus communis agglutinin-I--agarose both before and after desialylation of the glycopeptides. It was shown that about 90% of the biantennary glycans of both AGP-B and AGP-C were disialylated while the remainder were monosialylated. The degree of sialylation of the tri- and tetraantennary glycans was identical for the three molecular forms. In each case, one or more terminal galactose residues occurred on at least 20% of the tri- and 65% of the tetraantennary chains. It is suggested that the decrease in the exposure of galactose residues from AGP-A to AGP-C is related to the concomittant decrease in branching of the glycans of the three molecular forms. The relevance of these findings to studies on the function of AGP during inflammatory and liver diseases is discussed.

Treatment of chronic bacterial prostatitis with ciprofloxacin.

Thirty two patients with proven chronic bacterial prostatitis were treated with ciprofloxacin 500 mg twice daily orally for four weeks. The causative organisms, cultured from prostatic fluid were Enterobacteriaceae (19 patients), enterococci (9), staphylococci (4), streptococci (3), non-fermentative Gram-negative rods (2) and anaerobic bacteria (9). Nineteen patients had pure cultures, 13 mixed cultures. The susceptibility of all organisms to ciprofloxacin, sulfamethoxazole, trimethoprim and doxycyclin was determined by agar dilution. The effect of therapy was measured by clinical parameters and by repeated cultures of prostatic fluid during and after therapy up to six months. Clinical cure (at one month after therapy) was obtained in 22 patients, improvement in seven; two patients did not respond, one patient had to stop during therapy because of severe nausea. No other side effects were noted. Ciprofloxacin may be a useful alternative drug in the treatment of prostatitis.

Subcellular localization and tissue distribution of sialic acid-forming enzymes. N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase.

The activities of N-acetylneuraminate 9-phosphate synthase and N-acetylneuraminate 9-phosphatase, the two enzymes involved in the final steps of the biosynthetic pathway of N-acetylneuraminic acid, were measured with the substrates N-acetyl[14C]mannosamine 6-phosphate and N-acetyl[14C]neuraminic acid 9-phosphate respectively. Subcellular localization studies in rat liver indicated that both enzymes are localized in the cytosolic fraction after homogenization in sucrose medium. To test the possibility of misinterpretation due to the hydrolysis of N-acetylneuraminic acid 9-phosphate by non-specific phosphatases, the hydrolysis of various phosphate esters by the cytosolic fraction was tested. Only p-nitrophenyl phosphate was hydrolysed; however, competition studies with N-acetylneuraminic acid 9-phosphate and p-nitrophenyl phosphate indicated that two different enzymes were involved and that no competition existed between the two substrates. In various other rat tissues N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase activities were detected, suggesting that N-acetylmannosamine 6-phosphate is a general precursor for N-acetylneuraminic acid biosynthesis in all the tissues studied.

Synthesis of N-acetylneuraminic acid and of CMP-N-acetylneuraminic acid in the rat liver cell.

Adult male rats, under starving and normal conditions, were injected intravenously with N-acetyl[3H]mannosamine and after various time intervals the specific radioactivities of free N-acetylneuraminic acid (NeuAc) and CMP-N-acetylneuraminic acid were determined in the liver. The specific radioactivity of free NeuAc was high even within 20s after injection; the maximum was reached between 7 and 10 min. The specific radioactivity of CMP-NeuAc showed a lag phase of approx. 1 min. Thereafter it increased quickly and rose above the specific radioactivity of free NeuAc, reaching a maximum about 20 min after injection. These results point to a channelling of the newly synthesized NeuAc molecules into a special compartment, from which they are preferentially used by the enzyme CMP-sialic acid synthetase. It is suggested that the cytosolic enzyme N-acetylneuraminic acid 9-phosphate phosphatase is working in concert with the nuclear localized enzyme CMP-N-acetylneuraminic acid synthetase. Incorporation of radioactive sialic acid into sialoglycoproteins in liver occurred 2 min after injection, and after 10 min bound radioactivity began to appear in the circulation, indicating a transport time of 8 min of sialoglycoproteins from the point of attachment of sialic acid to the point of excretion.

Subcellular localization and tissue distribution of sialic acid precursor-forming enzymes.

The enzymes UDP-N-acetylglucosamine pyrophosphorylase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine kinase, N-acetylglucosamine kinase and N-acetylglucosamine 2-epimerase, which are involved in the metabolism of N-acetylneuraminic acid, were studied in rat with regard to their subcellular localization and tissue distribution. The subcellular distribution studies in liver indicated that the enzymes are localized in the soluble cell fraction. In other tissues the comparison of enzyme activities in homogenates with that in high-speed supernatants led to a similar conclusion. UDP-N-acetylglucosamine pyrophosphorylase, N-acetylglucosamine kinase and N-acetylglucosamine 2-epimerase were detected in almost all tissues studied. UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase, two enzymes considered to be key enzymes in the N-acetylneuraminic acid biosynthesis, were detected only in sialoglycoprotein-secreting tissues, i.e. liver, salivary gland and intestinal mucosa. The low activity of the key enzymes in other tissues suggests that the biosynthetic pathway of N-acetylneuraminic acid is not the same in various tissues.

Turnover of free sialic acid, CMP-sialic acid, and bound sialic acid in rat brain.

Adult male rats were injected intraventricularly with N-[3H]acetylmannosamine. After different time intervals the rats were killed and free sialic acid, CMP-sialic acid, lipid- and protein-bound sialic acid were isolated from brain and the specific radioactivities determined. Maximal specific radioactivity was reached after approximately 4 h for CMP-sialic acid, after 10-12 h for free sialic acid and after approximately 42 h for lipid- and protein-bound sialic acid. After some days the specific radioactivities of all four pools were the same and decreased equally, with a calculated turnover rate of approximately 3.5 weeks. The conclusion was that this phenomenon was the result of reutilisation of sialic acid and/or precursors. Therefore, the calculated turnover is not the turnover of bound sialic acid, but merely the rate of leakage of sialic acid and/or precursors out of the brain, so that no real turnover can be measured by this method. The first few hours after injection the specific radioactivity of CMP-sialic acid rose above that of free sialic acid. It is supposed that a compartmentalization exists of free sialic acid. The newly synthesised sialic acid molecules are not secreted into the cytoplasmic pool but are preferentially used for the synthesis of CMP-sialic acid. The results and conclusions are discussed in view of the general problems concerning turnover measurements of glycoconjugates.

Immunological properties of glomerular basement membrane antigens solubilized by elastase digestion.

Glomerular basement membrane (GBM) was solubilized by hog pancreas elastase and fractionated on Bio-Gel P-2. The composition and antigenic properties of elastase-solubilized GBM were compared with those of whole GBM. Elastase-solubilized bovine GBM (ES-BoGBM) and whole bovine GBM (BoGBM) showed similar amino acid and carbohydrate compositions, suggesting that neither the glycoprotein nor the collagen component of the GBM was selectively solubilized. Administration of rabbit antibodies against elastase-solubilized rat GBM (ES-RtGBM) and whole rat GBM (RtGBM) to rats showed that these preparations had similar nephritogenic properties although the kidney saturation dose was higher with antibodies to ES-RtGBM, suggesting that rabbits may recognize antigenic sites in ES-RtGBM which are not exposed in RtGBM. Radiolabeled ES-BoGBM or ES-RtGBM could be used in a radioimmunoassay to measure the heterologous immune response and the autoimmune response in rats immunized with BoGBM.

Preparation and chemical characterisation of calf Tamm-Horsfall glycoprotein.

Tamm-Horsfall glycoprotein preparations were obtained from calf urine by 1.0 M NaCl precipitation followed by 4 M urea/Sepharose 4B chromatography. By using 0.1% sodium dodecyl sulfate polyacrylamide gel electrophoresis a molecular weight of 86 500 +/- 4500 (n = 12) was calculated for the glycoprotein. Amino acid and carbohydrate analyses were performed, the carbohydrate composition being (in residues per 100 amino acid residues in the glycoprotein): fucose, 0.90; galactose, 4.82; mannose, 4.63;N-acetylglucosamine, 7.36; N-acetylgalactosamine, 1.38; sialic acid, 2.93. Under conditions of mild acid hydrolysis (0.05 M H2SO4, 80 degrees C, 1 h) the calf Tamm-Horsfall glycoprotein preparations were degraded partially into two lower molecular weight fragments (approximate Mr 66 000 and 51 000), as shown by polyacrylamide gel electrophoresis, both fragments being periodic acid-Schiff reagent positive.

Experimental study on the mechanism of hyperphosphatasaemia in bile flow obstruction.

Extrahepatic bile flow obstruction is followed by a several-fold increase in alkaline phosphatase activity in the liver. This activity passes into the blood. The increase in the activity of the enzyme in the liver can be prevented by inhibitors of RNA and protein synthesis. Inducible alkaline phosphatase was purified from rat liver and 1,000-fold purification was achieved. The incorporation of labbelled precursors (14C amino acids and 14C glucosamine) into the liver alkaline phosphatase was evaluated. No significant difference were found between specific alkaline phosphatase activities and specific radioactivities in the control rats and rats with 7.30 h after bile duct obstruction. The incorporation of 14C-amino acids into the total liver proteins was 20% lower in rats with cholestasis than in the control group. The results are evidence that the liver is the source of elevated alkaline phosphatase activity in serum in cholestasis, and that the production of alkaline phosphatase in the liver is prevented by inhibitors of RNA a protein synthesis. However, the enzyme might be synthesized before the 14C-labelled precursors were applied.

Epithelial basement membrane of bovine renal tubuli. Isolation and analysis of the carbohydrate chains.

The carbohydrate chains present in the tubular basement membrane of bovine kidney were studied. Digestion with collagenase followed with pronase resulted in a complete solubilization of the basement membrane. The different glycopeptides were purified by gel filtration and ion-exchange chromatography. Two kinds of carbohydrate chains could be characterized: oligosaccharides composed of glucosamine, mannose, galactose, fucose and sialic acid, and glucosylgalactose disaccharides. A very small portion of the oligosaccharide chains (ca. 4%) appeared to be free of sialic acid. The bulk of these chains contained sialic acid and fucose, although in small amounts. Only traces of galactosamine were found.