A quantitative cytochemical study of UDP-D-glucose: NAD-oxidoreductase (E.C. 1.1.1.22) activity during stelar differentiation in Pisum sativum L. cv Meteor

A quantitative cytochemical assay for UDP-D-glucose dehydrogenase (UDPGD) activity employing scanning and integrating microdensitometry has been revised and applied to a study of this enzyme during the initiation of secondary cell wall biosynthesis during the formation of primary vascular tissues in roots of Pisum sativum L. cv Meteor. The reaction involves the use of NBT as final electron acceptor and is inhibited 10-fold by either 10 mM UDP-D-xylose or 25 mM UDP-D-glucuronic acid, two molecules involved in feed-back inhibition of UDPGD activity in vivo. UDPGD is a far-from equilibrium enzyme representing a flux-generating step in the biosynthesis of precursors for hemicelluloses involved in secondary cell wall construction, and can be demonstrated to increase sharply in activity in cells of the developing primary vascular elements. This changed activity occurs 18-20 cells back from the root cap junction and coincides with the first cells containing the activated programme for secondary cell wall synthesis.     

Effect of 2-deoxy-d-glucose on maintenance in culture of neonatal B cell of rat

Summary The effect of 2-deoxy-d-glucose on maintenance in culture of B cells of the neonatal rat was examined by supplementation of Medium 199 containing 5.5 mM glucose with 1 mM 2-deoxy-d-glucose. Islets maintained in medium with 5.5 mM glucose (basal medium) for 7 d underwent remarkable decreases in glucose sensitivity, and the levels of insulin in the medium dropped. By contrast, addition of 2-deoxy-d-glucose promoted a higher insulin content in medium and an increase in the glucose-induced insulin release and biosynthesis. Moreover, the addition of the deoxysugar caused a selective deletion of fibroblasts and prevented the deterioration of islet cells in basal medium, yielding clusters mostly consisting of islet cells at the end of culture.     

Alterations in the outer wall architecture caused by the inhibition of mycoside C biosynthesis inMycobacterium avium

Radiolabeled amino acids (l-U[C¹⁴]alanine,d-U[C¹⁴]alanine,l-U[C¹⁴]threonine, andl-U[C¹⁴]phenylalanine) were exponentially incorporated into the trichloroacetic acid (TCA)-insoluble material (whole cells) ofMycobacterium avium during the first 30–60 min of labeling. Bacteria labeled for 48 h were extracted with chloroform-methanol (21 vol/vol). The thin layer chromatography (TLC) analysis of native lipids showed that mycoside C was labeled by the amino acids used.d-cycloserine (d-CS) and other amino acid analogs were examined as potential inhibitors of mycoside C biosynthesis. It was found thatd-CS caused about 27% inhibition, whereaso-,p-, andm-fluoro-dl-phenylalanine (Fl-phe) caused 80%–90% inhibition of the mycoside C biosynthesis. Judging from the data on inhibition experiments, it was concluded that the mycoside C biosynthesis started from the fatty acyl end and proceeded by the stepwise addition ofd-phenylalanine,d-allo-threonine, andd-alanine. Thed-alanyl-d-alanine peptidoglycan intermediate did not seem to serve as a donor ofd-alanine for mycoside C biosynthesis. Ultrastructural observation of the bacteria treated withd-CS showed only partial alteration of the outer wall layer, whereasm-Fl-phe treatment caused profound alterations. Successive transfers of the bacteria in growth medium supplemented withm-Fl-phe resulted in extensive disorganization of the outer layer.     

Cloning and characterization of CalS7 from Micromonospora echinospora sp. calichensis as a glucose-1-phosphate nucleotidyltransferase

The deoxysugar biosynthetic gene cluster of calicheamicin contains the calS7, which encodes glucose-1-phosphate nucleotidyltransferase and converts glucose-1-phosphate and nucleotides (NTP) to NDP-glucose and pyrophosphate. calS7 was expressed in Escherichia coli BL21(DE3), and the purified protein had significant thymidylyltransferase and uridylyltransferase activities as well, with some guanidylyltransferase activity but negligible cytidyl and adenyltransferase activity. The functions of thymidylyltransferase and uridylyltransferase were also verified using one-pot enzymatic synthesis of TMK and ACK. The products were analyzed by HPLC and ESI/MS, which showed peaks at m/z = 563 and 565 for TDP-d-glucose and UDP-d-glucose, respectively, in negative mode.     

Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein

Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 –/– mice. In wild-type PCT cells in primary culture, a Cl^– conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I^– > Br^– > Cl^– >> gluconate. This conductance was sensitive to 1 mM 4,4-Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K⁺ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 µM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 µM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl^– and K⁺ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.     

Astrocytes as potential modulators of mercuric chloride neurotoxicity

Summary 1. MC has been shown to inhibit the uptake ofl-glutamate and increased-aspartate release from preloaded astrocytes in a dose-dependent fashion.2. Two sulfhydryl (SH-)-protecting agents; reduced glutathione (GSH), a cell membrane-nonpenetrating compound, and the membrane permeable dithiothreitol (DTT), have been shown consistently to reverse the above effects. MC-inducedd-aspartate release is completely inhibited by the addition of 1 mM DTT or GSH during the actual 5-min perfusion period with MC (5µM); when added after MC treatment, DTT fully inhibits the MC-inducedd-aspartate release, while GSH does not.3. Neither DTT nor GSH, in the absence of MC, have any effect on the rate of astrocyticd-aspartate release. Other studies demonstrate that although MC treatment (5µM) does not induce astrocytic swelling, its addition to astrocytes swollen by exposure to hypotonic medium leads to their failure to volume regulate.4. Omission of calcium from the medium greatly potentiates the effect of MC on astrocyticd-aspartate release, an effect which can be reversed by cotreatment of astrocytes with the dihydropyridine Ca²⁺-channel antagonist nimodipine (10µM), indicating that one possible route of MC entry into the cells is through voltage-gated L-type channels.     

Effects of fructose on human fibroblast metabolism: the application of DNA measurements as a basis for interpretation

Summary A fluorometric procedure for measuring DNA was used to study growth and metabolic responses of eight cell strains of human foreskin fibroblasts. In preliminary studies this procedure gave more precise specific activity changes inN-acetyl-β-d-glucosaminidase (NAG) than did a protein activity basis, when changes in this enzyme's specific activity were investigated as a function of experimental cell manipulation. When fibroblast growth in eight cell strains was compared in 134 mM d-fructose vs. 13.4 mM glucose-supplemented minimum essential media, a significant increase in cellular DNA (50%) and protein (45%) occurred over an 11-d period. No significant differences in media pH change, lactate production, or carbohydrate uptake occurred on a DNA basis when cell metabolism was compared over the last 24 h of culture in the two media. Cells grown in fructose-containing media tended to show a reduction in NAG specific activity when compared with those grown in glucose-containing media.     

Modification of cambial cell wall architecture during cambium periodicity in Populus tomentosa Carr.

Cambium periodicity is correlated with changes in the cambial cell wall, but the heterogeneity of cell wall structure and composition makes it difficult to give an accurate interpretation, especially for complex secondary vascular tissues. A combination of different methods is necessary to reveal the structure of this complex cell wall. In this study, the cell wall architecture and composition of active and dormant cambial cells in Populus tomentosa were investigated by a combination of light microscopy, rapid-freezing and deep-etching electron microscopy, Fourier-transform infrared microspectroscopy and immuno-histochemistry. The results showed that the architecture of dormant cambial cell walls displayed a multi-layered structure, denser fibril network, smaller pore size, and fewer crosslinks between microfibrils than active cambial cell walls. The FTIR spectra of cell walls from active and dormant cambium showed differences in the intensity of bands near 1,740, 1,629, 1,537, 1,240, and 830 cm⁻¹, which reflected differences in cell wall composition. Immuno-labeling indicated that high methyl-esterified homogalacturonan and (1 → 4)-β-d-galactan epitopes were abundant and distributed in active cambial cell walls, and relatively de-esterified homogalacturonan and (1 → 5)-α-l-arabinan epitopes had weak labeling in the active cambium, while almost no labeling or very weak labeling for high methyl-esterified homogalacturonan, (1 → 4)-β-d-galactan and (1 → 5)-α-l-arabinan epitopes occurred in dormant cambial cells, except for the de-esterified homogalacturonan epitope, which was abundant in dormant cambial cells. These results demonstrate that there are great differences, both in structure and composition, between active and dormant cambial cell walls, which reflect their dynamic changes during cambium activity.     

Biochemical Characterization of the Pneumococcal Glucose 1-Phosphate Uridylyltransferase (GalU) Essential for Capsule Biosynthesis

The glucose 1-phosphate uridylyltransferase (GalU) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of Streptococcus pneumoniae. The pneumococcal GalU protein was overexpressed in Escherichia coli, and purified. GalU showed a pI of 4.23, and catalyzed the reversible formation of UDP-glucose and pyrophosphate from UTP and glucose 1-phosphate with K_m values of 0.4 mM for UDP-glucose, 0.26 mM for pyrophosphate, 0.19 mM for glucose 1-phosphate, and 0.24 mM for UTP. GalU has an optimum pH of 8–8.5, and requires Mg²⁺ for activity. Neither ADP-glucose nor TDP-glucose is utilized as substrates in vitro. The purification of GalU represents a fundamental step to provide insights on drug design to control the biosynthesis of the main pneumococcal virulence factor.     

Purine nucleotide synthesis in lymphoblasts cultured from normal subjects and a patient with Lesch-Nyhan syndrome

Human lymphoblasts derived from normal and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient individuals have been maintained in permanent tissue culture, and comparative studies of their purine metabolism have been undertaken. In agreement with previous observations in fibroblasts, the HGPRT-deficient lymphoblasts (less than 2% normal HGPRT activity) demonstrate threefold increases in the production of purines by the de novo pathway and four- to eightfold increases in intracellular concentrations of 5-phosphoribosyl 1-pyrophosphate (PRPP). The activities of the enzymes of purine metabolism responsible for production and utilization of PRPP were measured under optimal conditions in each cell line. The activities of adenine phosphoribosyltransferase (APRT), PRPP synthetase, and PRPP amidotransferase were independent of cell density and were not significantly different in the two cell lines. The K _m values of the common substrate, PRPP, were determined in normal lymphoblast extracts for APRT (K _m of 0.033 mM), HGPRT (K _m of 0.074 mM), and PRPP amidotransferase (K _m of 0.3 m M). The relatively low affinity of PRPP amidotransferase for PRPP suggests that deficiency of the HGPRT enzyme with its attendant increase in PRPP concentration should be accompanied by increased in vivo activity of PRPP amidotransferase, the first and presumed rate-limiting enzyme of de novo purine biosynthesis.This work was supported in part by National Institutes of Health Grants AM-05646, AM-13622, and GM-17702.     

A cytochemical study on the effects of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells

Summary The effect of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells was studied using cytochemical techniques. Autophagocytosis was induced with vinblastine incubation (0.1 mM) and the cellular ATP-level was lowered with 2-deoxy-d-glucose (0.35 mM). Acid phosphatase was used as a marker for lysosomal enzymes and imidazole-buffered osmium tetroxide impregnation in order to study the effects of energy deprivation on the maturation of autophagic vacuole (AV) membranes.Control and vinblastine treated cells maintained their ATP-levels throughout the incubation period tested (120 min). 2-Deoxy-d-glucose alone and with vinblastine decreased the intracellular ATP-level significantly after only 3 min incubation. Most of the AV's in control and vinblastine treated cells contained degraded material and acid phosphatase activity. Their membranes were stained only slightly or not at all with imidazole-buffered osmium tetroxide. 2-Deoxy-d-glucose alone as well as with vinblastine induced in particular an accumulation of early stages of AV's. These vacuoles contained undegraded cytoplasmic material and no acid phosphatase activity and their membranes were stained usually partly with imidazole-buffered osmium tetroxide. The membranes of some early AV's resembled endoplasmic reticulum and still had attached ribosomes.It was concluded that the inhibition of cellular energy production used in the present study did not inhibit autophagic sequestration but retarded the maturation of AV membranes and impaired the functioning of lysosomal hydrolases.     

Recycle Use of Sphingomonas sp. CDH-7 Cells for Continuous Degradation of Carbazole in the Presence of MgCl2

Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.₆₆₀ 3.3) were shaken in 50 mM K₂HPO₄-KH₂PO₄ buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 mM MgCl₂. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl₂. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells. Received: 28 June 2001 / Accepted: 30 July 2001     

Effects of dibutyryl cAMP and bromodeoxyuridine on expression ofN-acetylglucosaminyltransferases III and V in GOTO neuroblastoma cells

The sugar chain structures of the cell surface change dramatically during cellular differentiation. A human neuroblastoma cell line, GOTO, is known to differentiate into neuronal cells and Schwannian cell-like cells on treatments with dibutyryl cAMP and bromodeoxyuridine, respectively. We have examined the expression of UDP-N-acetylglucosamine: -d-mannoside -1,4N-acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) and UDP-N-acetylglucosamine: -6-d-mannoside -1,6N-acetylglucosaminyltransferase V (GnT-V: EC 2.4.1.155), two major branch forming enzymes inN-glycan synthesis, in GOTO cells on two distinct directions of differentiation.In neuronal cell differentiation, GnT-III activity showed a slight increase during initial treatment with Bt₂cAMP for 4 days and decreased drastically after the fourth day, but the mRNA level of GnT-III did not show a decrease but in fact a slight increase. GnT-V activity increased to approximately two- to three-fold the initial level with increasing mRNA level after 8 days, and lectin blot analysis showed an increase in reactivity toDatsura stramonium (DSA) of the immunoprecipitated neural cell adhesion molecule (NCAM). In Schwannian cell differentiation, the activity and mRNA level of GnT-III showed no significant change on treatment with BrdU. GnT-V activity also showed no change in spite of the gradual increase in the mRNA level. These results suggest that the activation of GnT-V during neuronal cell differentiation of GOTO cells might be a specific change for branch formation in N-glycans, and this affects the sugar chain structures of some glycoproteins such as NCAM.Abbreviations and trivial names GnT N-acetylglucosaminyltransferase - Bt₂cAMP N ⁶,O ⁶-dibutyryl cAMP - BrdU bromodeoxyuridine - DSA Datsura stramonium - NCAM neural cell adhesion molecule - PAGE polyacrylamide gel electrophoresis     

Characterization of prolidase I and II purified from normal human erythrocytes: comparison with prolidase in erythrocytes from a patient with prolidase deficiency

The effect of various sulfur-containing amino acids on the activities of prolidase isoenzymes I and II isolated from erythrocytes of healthy individuals, and erythrocyte lysates from a patient with prolidase deficiency was investigated. The activity of prolidase I against glycylproline was strongly enhanced by d-methionine. l-Methionine and d,l-methionine slightly enhanced the activity at low concentration, but N-acetyl-l-methionine had no effect. d-Ethionine, l-ethionine, and d,l-ethionine also enhanced the activity of prolidase I. d,l-Homocysteine enhanced the activity at low concentration, but inhibited the activity at 50 mM. The activity of prolidase II against methionylproline was enhanced by d-methionine, d,l-methionine, and l-methionine, but N-acetyl-l-methionine had no effect. d-Ethionine and d,l-ethionine strongly enhanced the activity of prolidase II compared with l-ethionine; d,l-homocysteine weakly enhanced the activity. d,l-Homocysteine-thiolactone inhibited the activities of prolidase I and II in a concentration-dependent manner. The effect of various sulfur-containing amino acids on prolidase activity against methionylproline in erythrocyte lysates from a patient with prolidase deficiency was almost the same as that on prolidase II. The kinetics of the activities of prolidase I, II, and patient prolidase were also studied. Their K _m values were changed by adding sulfur-containing amino acids, but V _max values were unchanged.     

Microphotometric determination of enzymes in brain sections

Summary A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM d-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN₃, 10 mM MgCl₂, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 m. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1, 2-2)     

Identification of α-<Emphasis Type="SmallCaps">d</Emphasis>-glucose-1-phosphate cytidylyltransferase involved in Ebosin biosynthesis of <Emphasis Type="BoldItalic">Streptomyces</Emphasis> sp. 139

Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139 has antagonist activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo. Its biosynthesis gene cluster (ste) has been identified. In this study, gene ste17 was expressed in Escherichia coli BL21 and the recombinant protein was purified. With CTP and α-d-glucose-1-phosphate as substrates, the recombinant Ste17 protein was found capable of catalyzing the production of CDP-d-glucose and pyrophosphate, demonstrating its identity as an α-d-glucose-1-phosphate–cytidylyltransferase (CDP-d-glucose synthase). To investigate the function of ste17 in Ebosin biosynthesis, the gene was disrupted with a double crossover via homologous recombination. The monosaccharide composition of exopolysaccharide (EPS) produced by the mutant Streptomyces sp. 139 (ste17 ⁻) was found significantly altered from that of Ebosin, with glucose becoming undetectable. This gene knockout also negatively affected the antagonist activity for IL-1R of EPS. These results indicate that the CDP-d-glucose synthase encoded by ste17 gene is involved in the formation of nucleotide sugar (CDP-d-glucose) as glucose precursor in Ebosin biosynthesis. Xiao-Qiang Qi and Qing-Li Sun contributed equally to this work.     

Purification and characterization of an alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810

An alkaline cellulase from Bacillus sp. HSH-810 was purified 8.7-fold with a 30% yield and a specific activity of 71 U mg^–1 protein. It was optimally active at pH 10 and 50 °C and was stable from pH 6 to 10 with more than 60% activity remaining after heating at 60 °C for 60 min. The molecular mass of cellulase was 80 kDa. It was inhibited by 50% by Fe³⁺ (1 mM) and Mn²⁺ (0.1 mM) but was relatively insensitive to Hg²⁺ and Pb²⁺ at 1 mM.Revisions requested: 8 October 2004/1 December 2004; Revisions received 29 November 2004/5 January 2005     

Influence of monosaccharides on aerobactin production byAerobacter aerogenes 62-1

Supplementation of cultures ofAerobacter aerogenes 62-1, 43/4 h after initiation of growth withd-glucose (20 mM), resulted in a threefold increase in the production of aerobactin. Administration ofl-lysine under similar conditions led to a twofold incrasse in the yield of the siderophore. Studies with a cell-free system ofAerobacter aerogenes 62-1 revealed considerable stimulation of lysine-N⁶-hydroxylase activity by glucose and several of its derivatives. Inclusion of ferric chloride (0.1 mM) in the growth medium led to the repression of both lysine-N⁶-hydroxylase and aerobactin synthetase.     

<Emphasis Type="Italic">QUASIMODO1</Emphasis> is expressed in vascular tissue of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> inflorescence stems,and affects homogalacturonan and xylan biosynthesis

An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, homogalacturonan α-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of β-1-4-D-xylan synthase, β-1-4-D-galactan synthase and β-glucan synthase II activities were also measured in microsomal membranes. Of these, only β-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on α-1,4-D-galacturonosyltransferase and β-1,4-D-xylan synthase activities.