Metabolic signatures suggest o-phosphocholine to UDP-N-acetylglucosamine ratio as a potential biomarker for high-glucose and/or palmitate exposure in pancreatic ß-cells.

INTRODUCTION: Chronic exposure to high-glucose and free fatty acids (FFA) alone/or in combination; and the resulting gluco-, lipo- and glucolipo-toxic conditions, respectively, have been known to induce dysfunction and apoptosis of ß-cells in Diabetes. The molecular mechanisms and the development of biomarkers that can be used to predict similarities and differences behind these conditions would help in easier and earlier diagnosis of Diabetes. OBJECTIVES: This study aims to use metabolomics to gain insight into the mechanisms by which ß-cells respond to excess-nutrient stress and identify associated biomarkers. METHODS: INS-1E cells were cultured in high-glucose, palmitate alone/or in combination for 24 h to mimic gluco-, lipo- and glucolipo-toxic conditions, respectively. Biochemical and cellular experiments were performed to confirm the establishment of these conditions. To gain molecular insights, abundant metabolites were identified and quantified using 1H-NMR. RESULTS: No loss of cellular viability was observed in high-glucose while exposure to FFA alone/in combination with high-glucose was associated with increased ROS levels, membrane damage, lipid accumulation, and DNA double-strand breaks. Forty-nine abundant metabolites were identified and quantified using 1H-NMR. Chemometric pair-wise analysis in glucotoxic and lipotoxic conditions, when compared with glucolipotoxic conditions, revealed partial overlap in the dysregulated metabolites; however, the dysregulation was more significant under glucolipotoxic conditions. CONCLUSION: The current study compared gluco-, lipo- and glucolipotoxic conditions in parallel and elucidated differences in metabolic pathways that play major roles in Diabetes. o-phosphocholine and UDP-N-acetylglucosamine were identified as common dysregulated metabolites and their ratio was proposed as a potential biomarker for these conditions.

Borate-aided anion exchange high-performance liquid chromatography of uridine diphosphate-sugars in brain, heart, adipose and liver tissues.

In this paper we describe a method optimized for the purification of uridine diphosphate (UDP)-sugars from liver, adipose tissue, brain, and heart, with highly reproducible up to 85% recoveries. Rapid tissue homogenization in cold ethanol, lipid removal by butanol extraction, and purification with a graphitized carbon column resulted in isolation of picomolar quantities of the UDP-sugars from 10 to 30mg of tissue. The UDP-sugars were baseline separated from each other, and from all major nucleotides using a CarboPac PA1 anion exchange column eluted with a gradient of acetate and borate buffers. The extraction and purification protocol produced samples with few unidentified peaks. UDP-N-acetylglucosamine was a dominant UDP-sugar in all the rat tissues studied. However, brain and adipose tissue showed high UDP-glucose levels, equal to that of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine showed 2.3-2.7 times higher levels than UDP-N-acetylgalactosamine in all tissues, and about the same ratio was found between UDP-glucose and UDP-galactose in adipose tissue and brain (2.6 and 2.8, respectively). Interestingly, the UDP-glucose/UDP-galactose ratio was markedly lower in liver (1.1) and heart (1.7). The UDP-N-acetylglucosamine/UDP-glucuronic acid ratio was also constant, between 9.7 and 7.7, except in liver with the ratio as low as 1.8. The distinct UDP-glucose/galactose ratio, and the abundance of UDP-glucuronic acid may reflect the specific role of liver in glycogen synthesis, and metabolism of hormones and xenobiotics, respectively, using these UDP-sugars as substrates.

On-target labeling of intracellular metabolites combined with chemical mapping of individual hyphae revealing cytoplasmic relocation of isotopologues.

A microscale analytical platform integrating microbial cell culture, isotopic labeling, along with visual and mass spectrometric imaging with single-cell resolution has been developed and applied in the monitoring of cellular metabolism in fungal mycelium. The method implements open chips with a two-dimensional surface pattern composed of hydrophobic and hydrophilic zones. Two hydrophilic islands are used as medium reservoirs, while the hydrophobic area constitutes the support for the growing aerial hyphae, which do not have direct contact with the medium. The first island, containing (12)C(6)-glucose medium, was initially inoculated with the mycelium (Neurospora crassa), and following the initial incubation period, the hyphae progressed toward the second medium island, containing an isotopically labeled substrate ((13)C(6)-glucose). The (13)C atoms were gradually incorporated into cellular metabolites, which was revealed by MALDI-MS. The fate of the chitin-biosynthesis precursor, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), was monitored by recording mass spectra with characteristic isotopic patterns, which indicated the presence of various (12)C/(13)C isotopologues. The method enabled mapping the (13)C-labeled UDP-GlcNAc in fungal mycelium and recording its redistribution in hyphae, directly on the chip.

Enzymatic analysis of uridine diphosphate N-acetyl-D-glucosamine.

The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD(+)-dependent oxidation of the 3'' position of uridine diphosphate N-acetyl-D-glucosamine (UDP-GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP-GlcNAc oxidation, forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP-GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2 microM UDP-GlcNAc in a 1-ml sample. Using the method of standard addition, UDP-GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae, and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP-GlcNAc concentrations in time series experiments or inhibitor screens.

Novel acetylation-aided migrating rearrangement of uridine-diphosphate-N-acetylglucosamine in electrospray ionization multistage tandem mass spectrometry.

Uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) is the final product of hexosamine biosynthetic pathway (HSP) and the donor substrate for the modification of nucleocytoplasmic proteins at serine and threonine residues with N-acetylglucosamine (GlcNAc) catalyzed by O-GlcNAc transferase (OGT). Many analogs of UDP-GlcNAc were designed to interfere with the process of protein O-glycosylation by blocking OGT. A novel rearrangement reaction was observed in which phosphate-N-acetylglucosamine moiety migrated to 3' terminus of ribose in ESI-MS(n) of UDP-GlcNAc. Results from tandem mass spectrometry, control experiments and calculation showed that the phosphate-N-acetylglucosamine migration might undergo a pentacoordinate phosphoric intermediate. Furthermore, the acetylation of glucosamine in UDP-GlcNAc was essential in the migration process.

Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress.

In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division. We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three- to fourfold increase in cell wall chitin levels. We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone. Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool. This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips. In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls. Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane. We conclude that Chs3p-dependent chitin synthesis in S. cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane.

Palmitate-induced activation of the hexosamine pathway in human myotubes: increased expression of glutamine:fructose-6-phosphate aminotransferase.

The nutrient sensing capacity of the hexosamine biosynthetic pathway (HBP) has been implicated in the development of insulin resistance of skeletal muscle. To study the molecular mechanism of the free fatty acid (FFA)-induced activation of the HBP myotubes obtained from muscle biopsies of metabolically characterized, subjects were stimulated with different fatty acids for 20 h. Incubation with the saturated fatty acids palmitate and stearate (0.5 mmol/l) resulted in a three- to fourfold increase in mRNA expression of glutamine:fructose-6-phosphate aminotransferase (GFAT), the key and rate-limiting enzyme of the hexosamine pathway. Unsaturated fatty acids or 30 mmol/l glucose had little or no effect. Palmitate increased the amount of GFAT protein nearly two-fold, and subsequently, the concentration of UDP-N-acetylglucosamine, the end product of the HBP, was 1.3-fold enhanced in the palmitate-stimulated myotubes. The nonmetabolized fatty acid bromopalmitate had no effect. The DNA binding activity of the transcription factor Sp1, a target downstream of the HBP, was increased by palmitate and completely lost after enzymatic removal of O-GlcNAc. No correlation was found between the palmitate-induced increase in GFAT protein and the insulin resistance in the respective subjects. The findings reveal a new mechanism for how FFAs induce the activation of the HBP.

Simultaneous, quantitative analysis of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-glucose and UDP-galactose in human peripheral blood cells, muscle biopsies and cultured mesangial cells by capillary zone electrophoresis.

The aim of this study was to develop and evaluate a capillary zone electrophoretic (CE) procedure for the accurate quantification of the UDP-hexosamines as well as for the corresponding UDP-hexoses in samples from various biological origins. Testing different buffer conditions, voltages, capillary dimensions and temperatures, optimal results were achieved with a 90 mM borate buffer, pH 9.0, at 18 degrees C and 15.5 kV in an uncoated fused-silica capillary of 50 cm x 50 microm and a detection wavelength of lambda = 262 nm. The total procedure, i.e., including variations of the sample preparation, showed coefficients of variation for the peak areas between 4. 1% and 10.4% in mesangial cells (n = 7) and between 7.8 and 10.3% (n = 6) in leukocytes for the components of interest. To improve precision, an internal standard was used for calibration. The limit of detection for all compounds is an absolute amount of 180 fmol, sufficient for the precise analysis of UDP-sugars in a limited amount of biological samples, such as human leukocytes (obtained from a 10 mL blood sample), muscle biopsies (< or = 100 mg), and mesangial kidney cells (ca. 2.5 x 10(5) cells). This reproducible, quantitative analysis of all four UDP-sugars from various biomedically relevant origins by CZE is a definite improvement over the generally used high performance liquid chromatography (HPLC) procedures. The CZE method allows the study of the flux through the hexosamine pathway in diabetes mellitus and other diseases in a simple, quantitative and accurate way.

Discordant effects of glucosamine on insulin-stimulated glucose metabolism and phosphatidylinositol 3-kinase activity.

The impact of increased GlcN availability on insulin-stimulated p85/p110 phosphatidylinositol 3-kinase (PI3K) activity in skeletal muscle was examined in relation to GlcN-induced defects in peripheral insulin action. Primed continuous GlcN infusion (750 micromol/kg bolus; 30 micromol/kg.min) in conscious rats limited both maximal stimulation of muscle PI3K by acute insulin (I) (1 unit/kg) bolus (I + GlcN = 1.9-fold versus saline = 3.3-fold above fasting levels; p < 0.01) and chronic activation of PI3K following 3-h euglycemic, hyperinsulinemic (18 milliunits/kg.min) clamp studies (I + GlcN = 1.2-fold versus saline = 2.6-fold stimulation; p < 0.01). To determine the time course of GlcN-induced defects in insulin-stimulated PI3K activity and peripheral insulin action, GlcN was administered for 30, 60, 90, or 120 min during 2-h euglycemic, hyperinsulinemic clamp studies. Activation of muscle PI3K by insulin was attenuated following only 30 min of GlcN infusion (GlcN 30 min = 1.5-fold versus saline = 2.5-fold stimulation; p < 0.05). In contrast, the first impairment in insulin-mediated glucose uptake (Rd) developed following 110 min of GlcN infusion (110 min = 39.9 +/- 1.8 versus 30 min = 42.8 +/- 1.4 mg/kg.min, p < 0.05). However, the ability of insulin to stimulate phosphatidylinositol 3,4, 5-trisphosphate production and to activate glycogen synthase in skeletal muscle was preserved following up to 180 min of GlcN infusion. Thus, increased GlcN availability induced (a) profound and early inhibition of proximal insulin signaling at the level of PI3K and (b) delayed effects on insulin-mediated glucose uptake, yet (c) complete sparing of insulin-mediated glycogen synthase activation. The pattern and time sequence of GlcN-induced defects suggest that the etiology of peripheral insulin resistance may be distinct from the rapid and marked impairment in insulin signaling.

Cell lines with reduced UDP-N-acetylhexosamine pool in the presence of ammonium.

The glycosylation of pharmaglycoproteins from recombinant cell lines can be affected by an uncontrolled accumulation of ammonium in the medium. Glucosamine-6-phosphate isomerase (GPI) has been proposed as the key enzyme responsible for elevating the intracellular UDP-N-acetylhexosamine pool (UDPGNAc) by accepting ammonium from the medium of cultured mammalian cells. As previously reported, the increased UDPGNAc pool then affects the N-glycan complexity in glycoproteins. To understand the entry of extracellular ammonium into the cellular metabolism, GPI has been isolated to homogeneity from BHK-21 cells and characterized. Thus, the complete pathway by which ammonium enters the cellular metabolism was elucidated. To reduce the negative effects of ammonium, GPI was inhibited using two different strategies. First, the addition of mannose to the culture media and, second, antisense RNA expression. In both cases, the cellular UDPGNAc pool was suppressed in the presence of high ammonium concentrations in the medium. However, constant suppression of the UDPGNAc pool could not be achieved by antisense RNA expression because antisense clones were apparently unstable. Further studies showed that the main reason for instability was the inducibility of GPI by its substrate ammonium. GPI was induced to a factor of two under ammonium-containing medium conditions. We propose gene knockout technology for GPI repression to obtain cell lines consisting of an UDPGNAc pool unaffected by the presence of ammonium.

Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells.

Elevated ammonium concentrations in the medium of cultivated cells have been shown to increase the intracellular levels of uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc; Ryll et al., 1994). These sugar nucleotides are substrates for glycosyltransferases in the glycosylation pathway. In our experiments, recombinant Chinese hamster ovary cells producing an immunoadhesin glycoprotein (GP1-IgG) have been cultivated under controlled cell culture conditions in the presence of different ammonium concentrations.15N-Labeled ammonium chloride (15NH4Cl) was added exogenously to the cell culture media to determine if ammonium was incorporated into UDP-GlcNAc and cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NANA) pools, and subsequently incorporated into GP1-IgG as N-linked glycans. The intracellular pools of UDP-activated hexosamines (UDP-GNAc) were followed during the time course of the experiment. To assess the extent of15NH4+incorporation into the glycans of GP1-IgG, the glycoprotein was first purified to homogeneity by protein A chromatography. Enzymatically released N-glycans were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. N-Glycans synthesized in the presence of15NH4Cl revealed an N-glycan-dependent increase in mass-to-charge of 2.5-4.8 Da. These results indicate that 60-70% of the total nitrogen containing monosaccharides had incorporated15N. Presumably,15NH4+was incorporated into GlcNAc and N-acetylneuraminic acid as proposed earlier (Ryll et al., 1994). This might be a universal and previously not described reaction in mammalian cells when exposed to nonphysiological but in cell culture commonly found concentrations of ammonium. The data presented here are of significance for glycoprotein production in mammalian cell culture, since it has been shown previously that elevated levels of UDP-activated hexosamines affect N-glycan characteristics such as branching and degree of amino sugar incorporation. In addition, our results demonstrate that isotope labeling in combination with MALDI-TOF-MS can be used as an alternate tool to radioactive labeling of sugar substrates in metabolic studies.

Advantages in the analysis of UDP-sugars by capillary electrophoresis-comparison of the conventional HPLC method with two new capillary electrophoretic micro-procedures.

Two different capillary electrophoretic separation modes for the analysis of UDP-sugars have been tested. In comparison to the traditionally used HPLC method the micellar electrokinetic capillary chromatography (MECC) as well as the capillary zone electrophoresis (CZE) results in an improvement of the separation profile and the speed of the analysis.

Characterization of UDP amino sugars as major phosphocompounds in the hyperthermophilic archaeon Pyrococcus furiosus.

The archaeon Pyrococcus furiosus is a strictly anaerobic heterotroph that grows optimally at 100 degrees C by the fermentation of carbohydrates. It is known to contain high concentrations of novel intracellular solutes such as beta-mannosylglycerate and di-myo-inositol 1,1'-phosphate (DIP) (L. O. Martins and H. Santos, Appl. Environ. Microbiol. 61:3299-3303, 1995). Here, 31P nuclear magnetic resonance (NMR) spectroscopy was used to show that this organism also accumulates another type of phospho compound, as revealed by a major multiplet signal in the pyrophosphate region. The compounds were purified from cell extracts of P. furiosus by anion-exchange and gel filtration chromatographic procedures and were structurally analyzed by 1H, 13C, and 31P NMR spectroscopy. They were identified as two uridylated amino sugars, UDP N-acetylglucosamine and UDP N-acetylgalactosamine. Unambiguous characterizations and complete assignments of 1H and 13C resonances from such sugars have not been previously reported. In vitro 31P NMR spectroscopic analyses showed that, in contrast to DIP, which is maintained at a constant intracellular concentration (approximately 32 mM) throughout the growth phase of P. furiosus, the UDP amino sugars accumulated (to approximately 14 mM) only during the late log phase. The possible biochemical roles of these compounds in P. furiosus are discussed.

A new method for the determination of specific 13C enrichment in phosphorylated [1-13C]glucose metabolites. 13C-coupled, 1H-decoupled 31P-NMR spectroscopy of tissue perchloric acid extracts.

A 31P-NMR method for the determination of 13C enrichment in phosphorylated [1-(13)C]glucose metabolites was developed by taking advantage of the 13C satellites detectable for 31P-NMR signals of metabolites such as UDP-hexoses, UDP-N-acetylhexosamines and other phosphorylated compounds generated during glycolysis and subsequent anabolism. HT-29 cells were incubated in culture medium containing 4.5 g/l [1-(13)C]glucose for 24 h prior to cell extraction, and high-resolution 31P-NMR spectra were acquired from perchloric acid extracts. Since glucose and its phosphorylated products are key metabolites for many different metabolic processes, this method may be very helpful for studying specific metabolic pathways involving phosphorylated glucose metabolites.

Spontaneous mucolipidosis in a cat: an animal model of human I-cell disease.

A 7-month-old female cat was seen for abnormal facial features and abnormality of gait. Facial dysmorphism, large paws in relation to body size, dysostosis multiplex, and poor growth were noted, and mucopolysaccharidosis was suspected. A negative urine test for sulfated glycosaminoglycans and extreme stiffness of skin indicated a mucolipidosis hitherto unknown in animals. Deficiency of UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase, EC 2.7.8.17) activity was demonstrated in leukocytes and cultured fibroblasts, which had the appearance of inclusion cells (I-cells). Activities of a set of lysosomal hydrolases were abnormally low in fibroblasts and excessive in blood plasma. Postmortem morphology revealed lysosomal inclusions predominantly in fibroblasts but also in endothelial cells and chondrocytes, i.e., in cells of mesenchymal origin. Storage lysosomes contained oligosaccharides, mucopolysaccharides, and lipids. Tissues most affected were bones, cartilage, skin, and other connective tissues such as those in heart valves, aortic wall, and vocal cords. Parenchymal cells of liver and kidney were unaffected, as was skeletal muscle. Only a few of the cerebral cortical neurons had lipid inclusions; in sciatic nerve some axons were affected, but other peripheral nerves were normal. There were striking clinical, biochemical, and morphologic similarities between the disorder in this cat and the human I-cell disease.

A fluorescent assay for the determination of UDP-GlcNAc: Gal beta 1,3GalNAc-R (GlcNAc to GalNAc) beta 1,6-N-acetylglucosaminyltransferase activity.

A fluorescent assay for UDP-GlcNAc: Gal beta 1,3Gal-NAc-R beta 1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) activity has been developed involving dansylation of the enzyme reaction product. Core 2 GlcNAc-T detection was performed using unlabeled UDP-GlcNAc as the donor and Gal beta 1,3GalNAc alpha-pAp as the acceptor. The product, Gal beta 1,3(GlcNAc beta 1,6)-GalNAc alpha-pAp, was quantitatively derivatized with dansyl chloride at the NH2 moiety of the pAp group and the resultant fluorescent trisaccharide was separated on a Spherisorb ODS2 HPLC column. This method, rapid and economical, was found to be sensitive enough for the detection of 1 pmol of reaction product and therefore represents a reliable alternative to assays which use radiolabeled substrates. Additionally, the approach described here can be adapted for the assay of other glycosyltransferases, where the acceptor substrate has a pAp group as a hydrophobic aglycon linker.

UDP-N-acetylhexosamines and hypotaurine in human glioblastoma, normal brain tissue and cell cultures: 1H/NMR spectroscopy study.

NMR spectroscopy was used to analyse perchloric acid extracts of normal human brain, murine brain cell cultures, glioblastoma tissue and the glioblastoma cell line U-87. 1H NMR spectra revealed the presence of elevated levels of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine in glioblastoma extracts and the glioblastoma cell line U-87, in comparison with normal brain tissue and primary cell cultures of neurons and astrocytes. UDP-N-acetylhexosamines appear to accumulate in cells that are unable to differentiate. Furthermore, it was found that the culture medium had an effect on the concentration of UDP-N-acetygalactosamine in the glioblastoma cell line. Hypotaurine, previously only associated with oligodendrocytes, has been identified in astrocyte cultures and in cerebellar granule cells. In normal brain it was not observed by NMR spectroscopy, but was easily detectable in glioblastoma tissue extracts. UDP-N-acetylhexoseamines and hypotaurine might be useful markers for brain pathology and play a role in cell differentiation and cell division.

Analysis of murein and murein precursors during antibiotic-induced lysis of Escherichia coli.

Lysis of Escherichia coli induced by either D-cycloserine, moenomycin, or penicillin G was monitored by studying murein metabolism. The levels of the soluble murein precursor UDP-N-acetylmuramyl-L-alanyl-D-glutamyl-m-diaminopimelyl-D-alanyl- D-alanine (UDP-MurNAc-pentapeptide) and the carrier-linked MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol as well as N-acetylglucosamine-beta-1,4-MurNAc-(pentapeptide)-pyrophosphoryl- undecaprenol varied in a specific way. In the presence of penicillin, which is known to interfere with the cross-linking of murein, the concentration of the lipid-linked precursors unexpectedly decreased before the onset of lysis, although the level of UDP-MurNAc-pentapeptide remained normal. In the case of moenomycin, which specifically blocks the formation of the murein polysaccharide strands, the lipid-linked precursors as well as UDP-MurNAc-pentapeptide accumulated as was expected. D-Cycloserine, which inhibits the biosynthesis of UDP-MurNAc-pentapeptide, consequently caused a decrease in all three precursors. The muropeptide composition of the murein showed general changes such as an increase in the unusual DL-cross bridge between two neighboring meso-diaminopimelic acid residues and, as a result of uncontrolled DL- and DD-carboxypeptidase activity, an increase in tripeptidyl and a decrease in tetrapeptidyl and pentapeptidyl moieties. The average length of the glycan strands decreased. When the glycan strands were fractionated according to length, a dramatic increase in the amount of single disaccharide units was observed not only in the presence of penicillin but also in the presence of moenomycin. This result is explained by the action of an exo-muramidase, such as the lytic transglycosylases present in E. coli. It is proposed that antibiotic-induced bacteriolysis is the result of a zipperlike splitting of the murein net by exo-muramidases locally restricted to the equatorial zone of the cell.

Pyrimidine 5'-nucleotidase deficiency: improved detection of carriers.

The activities of pyrimidine 5'-nucleotidase (P5N) and the nucleotide pools in the erythrocytes from 19 members of a Dutch family with P5N deficiency were measured. In the erythrocytes of 5 (out of 6) apparent heterozygotes (based on P5N activities), an increased amount of UDP-N-acetylhexosamines was found. This increase was also found in the erythrocytes of 2 (out of 3) questionable heterozygotes (P5N activity below normal range, but not below normal mean--2 X SD) and not in the erythrocytes of family members with a normal P5N activity nor in erythrocytes from healthy donors. We conclude that analysis of the ribonucleotide patterns, in combination with determination of P5N activity, allows a more accurate diagnosis of heterozygosity for P5N.