Nucleotides and sugar nucleotides in early development of Bufo arenarum. Coelomic oocytes.

Nucleotides and sugar nucleotides from coelomic oocytes of Bufo arenarum were extracted with trichloroacetic acid and analyzed by ion-exchange chromatography. The hypoxanthine and guanine were sequencially eluted from the column with water. Nucleotides and sugar nucleotides were eluted with a linear gradient of ammonium chloride. The first peak of ultraviolet adsorption eluted from the resin was a complex mixture of at least three substances. The main component was identified as cytidine diphosphocholine by chemical, enzymatic, and chromatographic analyses. Preliminary experiments suggest a possible role for this compound during oogenesis, since immature oocytes incubated in vitro with [¹⁴C]choline showed an active metabolism of this substance with rapid incorporation in choline phosphate, cytidine diphosphocholine, and lecithin.     

Golgi-localized UDP-glucose transporter is required for cell wall integrity in rice

Cell wall-related nucleotide sugar transporters (NSTs) theoretically supply the cytosolic nucleotide sugars for glycosyltransferases (GTs) to carry out ploysaccharide synthesis and modification in the Golgi apparatus. However, the regulation of cell wall synthesis by NSTs remains undescribed. Recently, we have reported the functional characterization of Oryza sativa nucleotide sugar transport (Osnst1) mutant and its corresponding gene. OsNST1/BC14 is localized in the Golgi apparatus and transports UDP-glucose. This mutant provides us with a unique opportunity for evaluation of its broad impacts on cell wall structure and components. We previously examined cell wall composition of bc14 and wild type plants. Here, the spatial distribution of these cell wall alterations was analyzed by immunolabeling approach. Analysis of the sugar yield in different cell wall fractions indicated that this mutation improves the extractability of cell wall components. Field emission scanning electron microscopy further showed that the orientation of microfibrils in bc14 is irregular when compared to that in wild type. Therefore, this UDP-glucose transporter, making substrates available for polysaccharide biosynthesis, plays a critical role in maintaining cell wall integrity.Key words: UDP-glucose transporter, Golgi apparatus, cell wall polysaccharides, xylan, riceNucleotide sugars mainly generated in cytosol are the substrates for the synthesis of cell wall polysaccharides. Supply of nucleotide sugars is thus a key level for regulation of cell wall components and structure. Mutation in MUR1, an isoform of GDP-D-mannose-4,6-dehydratase, causes reduced amount of GDP-fucose and abnormal xyloglucan structure.^1,2 Disturbance of UDP-rhamnose synthesis via the mutation in RHM2/MUM4 decreases the rhamnogalacturonan I contents in Arabidopsis seeds. Cellulose synthase catalytic subunits (CESAs) generally use cytosolic UDP-glucoses to synthesize cellulose on the plasma membrane. UDP-glucose can be produced either via the catalysis of sucrose by sucrose synthase (SuSy) or through the phosphorylation of glucose-1-phosphate by UDP-glucose pyrophosphorylase (UGPase).³ Suppression of SuSy function in cotton inhibited fiber initiation and elongation.⁴ For the synthesis of noncellulosic polysaccharides occurring inside the Golgi lumen, the cytosolic nucleotide sugars should be translocated inwards by Golgi nucleotide sugar transporters (NSTs).⁵ However, this hypothesis remains to be confirmed, although transport activities have been identified in some plant NSTs.^6–10 Altering the precursor supply may also affect the overall carbon allocation in plants. It is reasonable that substrate regulation often causes pleiotropic effects on cell wall biosynthesis and plant growth. Without genetic resources or mutants on cell wall related NST, the exact evaluation of NSTs'' impacts on cell wall structure and composition is largely delayed. Until recently, we identified a Golgi-localized transporter OsNST1 mutant in rice. This transporter has been found to supply UDP-glucose for the formation of matrix polysaccharides, thereby modulating cellulose biosynthesis.¹¹ Here, we examine these alterations of cell wall polymers at the cellular level. The orientation of cellulose microfibrils and extractability of wall polysaccharides were also compared between the mutant and wild type. All those further our understandings of the functions of NSTs and the synergetic synthesis of different polymers.     

Sweet Sorghum Juice and Bagasse as Feedstocks for the Production of Optically Pure Lactic Acid by Native and Engineered <Emphasis Type="Italic">Bacillus coagulans</Emphasis> Strains

Sweet sorghum is a bioenergy crop that produces large amounts of soluble sugars in its stems (3–7 Mg ha^?1) and generates significant amounts of bagasse (15–20 Mg ha^?1) as a lignocellulosic feedstock. These sugars can be fermented not only to biofuels but also to bio-based chemicals. The market potential of the latter may be higher given the current prices of petroleum and natural gas. The yield and rate of production of optically pure d-(?)- and l-(+)-lactic acid as precursors for the biodegradable plastic polylactide was optimized for two thermotolerant Bacillus coagulans strains. Strain 36D1 fermented the sugars in unsterilized sweet sorghum juice at 50 °C to l-(+)-lactic acid (～150 g L^?1; productivity, 7.2 g L^?1 h^?1). B. coagulans strain QZ19-2 was used to ferment sorghum juice to d-(?)-lactic acid (～125 g L^?1; productivity, 5 g L^?1 h^?1). Carbohydrates in the sorghum bagasse were also fermented after pretreatment with 0.5 % phosphoric acid at 190 °C for 5 min. Simultaneous saccharification and co-fermentation of all the sugars (SScF) by B. coagulans resulted in a conversion of 80 % of available carbohydrates to optically pure lactic acid depending on the B. coagulans strain used as the microbial biocatalyst. Liquefaction of pretreated bagasse with cellulases before SScF (L + SScF) increased the productivity of lactic acid. These results show that B. coagulans is an effective biocatalyst for fermentation of all the sugars present in sweet sorghum juice and bagasse to optically pure lactic acid at high titer and productivity as feedstock for bio-based plastics.     

In-microbe formation of nucleotide sugars in engineered Escherichia coli

Numerous different nucleotide sugars are used as sugar donors for the biosynthesis of glycans by bacteria, humans, fungi, and plants. However, many of these nucleotide sugars are not available either in their native form or with the sugar portion labeled with a stable or radioactive isotope. Here we demonstrate the use of Escherichia coli metabolically engineered to contain genes that encode proteins that convert monosaccharides into their respective monosaccharide-1-phosphates and subsequently into the corresponding nucleotide sugars. In this system, which we designated “in-microbe”, reactions occur within 2 to 4 h and can be used to generate nucleotide sugars in amounts ranging from 5 to 12.5 μg/ml cell culture. We show that the E. coli can be engineered to produce the seldom observed nucleotide sugars UDP–2-acetamido-2-deoxy-glucuronic acid (UDP–GlcNAcA) and UDP–2-acetamido-2-deoxy-xylose (UDP–XylNAc). Using similar strategies, we also engineered E. coli to synthesize UDP–galacturonic acid (UDP–GalA) and UDP–galactose (UDP–Gal). ¹³C- and ¹⁵N-labeled NDP–sugars are formed using [¹³C] glucose as the carbon source and with [¹⁵N]NH₄Cl as the nitrogen source.     

Carbon assimilation in carrot cells in liquid culture

Assimilation of carbohydrates by carrot (Daucus carota L. cv Danvers) cells in liquid culture was studied to delineate the major metabolic pathways used in transformation of external carbohydrates to UDP-glucose. The cells grown on either sucrose or glucose for several years proved equally capable of utilizing each of these sugars. Sucrose was rapidly hydrolyzed extracellularly to glucose and fructose, and glucose was preferentially taken up. Uptake of fructose was slower and delayed until glucose was nearly depleted from the medium. Concentrations of cellular sugars, mainly glucose and sucrose, increased during late logarithmic phase of growth and decreased during the plateau phase. Continuous labeling of the cells with d-[¹⁴C]glucose resulted in rapid accumulation of radioactivity in glucose-6-phosphate and UDP-glucose. Because there was virtually no uptake of sucrose, UDP-glucose was likely derived from glucose-1-phosphate in a reaction catalyzed by UDP-glucose pyrophosphorylase and not directly from sucrose. Concentrations of major nucleotides and nucleotide sugars were maximal during the early logarithmic phase of growth and decreased several-fold in the stationary phase. A modified `energy charge' for adenylates calculated with the omission of AMP decreased steadily from 0.9 to 0.8 during the course of culture cycle. An analogous uracil nucleotide ratio was considerably lower (0.85) during early culture, decreased to about 0.7 for the entire logarithmic phase, and returned to initial values as cells entered stationary phase. The uracil nucleotide ratio may provide a useful index to assess the coupling between the energy available in phosphoanhydride bond in adenine nucleotides and the demand for sugar for polysaccharide synthesis through uridine diphosphate-sugar pools.     

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using ¹³C₆-glucose and ¹³C₂-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS.Metabolic labeling of cultured cells with ¹³C₆-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a ¹³C₆-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells.Using ¹³C₂-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.Protein glycosylation, which is the most abundant post-translational modification, has important roles in many biological processes by modulating conformation and stability, whereas its dysregulation is associated with various diseases such as diabetes and cancer (1, 2). Glycosylation is regulated by various factors including glucose metabolism, the availability and localization of nucleotide sugars, and the expression and localization of glycosyltransferases (3, 4). Thus, ideally all of these components should be considered when detecting changes in a dynamic fashion; namely, it is necessary not only to take a snapshot but also to make movies of the dynamic changes in glycan metabolism.Glucose is used by living cells as an energy source via the glycolytic pathway as well as a carbon source for various metabolites including nucleotide sugars (e.g. UDP-GlcNAc and CMP-NeuAc). These nucleotide sugars are transported into the Golgi apparatus, and added to various glycans on proteins. UDP-GlcNAc is the donor substrate for N-acetylglucosaminyl (GlcNAc)¹ transferases; alternatively, it is used in the cytosol for O-GlcNAc modification (i.e. O-GlcNAcylation) of intracellular proteins (5). The UDP-GlcNAc synthetic pathway is complex as it is a converging point of glucose, nucleotide, fatty acid and amino acid metabolic pathways. Thus, the metabolic flow of glucose modulates the branching patterns of N-glycans via UDP-GlcNAc concentrations because many of the key GlcNAc transferases that determine the branching patterns have widely different K_m values for UDP-GlcNAc ranging from 0.04 mm to 11 mm (6, 7). Indeed, it was demonstrated that the branching formation of N-glycans in T cells is stimulated by the supply from the hexosamine pathway, whereby it regulates autoimmune reactions promoted by T cells (8).UDP-GlcNAc is also used for the synthesis of CMP-NeuAc, the donor substrate for sialyltransferases (9). The CMP-NeuAc concentration is controlled by the feedback inhibition of UDP-GlcNAc epimerase/ManNAc kinase by the final product CMP-NeuAc, and hence a high CMP-NeuAc level reduces metabolic flow in CMP-NeuAc de novo synthesis (10). However, there is still only limited information about how the levels of nucleotide sugars dynamically change in response to the environmental cues, and how such changes are reflected in the glycosylation of proteins.Stable isotope labeling is a promising approach to quantify metabolic changes in response to external cues (11, 12). For example, the use of nuclear magnetic resonance to obtain isotopomer signals of metabolically labeled molecules has been applied to trace the flux in glycolysis and fatty acid metabolism (13). An approach based on the mass isotopomers of labeled metabolites with ¹³C₆-glucose has been developed to monitor the UDP-GlcNAc synthetic pathway (13–15). The method based on the labeling ratio of each metabolite related to UDP-GlcNAc synthesis has clarified the contribution of each metabolic pathway (14). Moseley reported a novel deconvolution method for modeling UDP-GlcNAc mass isotopomers (15).Previous studies into the use of nucleotide sugars in glycosylation have relied on the specific detection of metabolically radiolabeled glycans (16). It is possible not only to deduce the glycan structures but also to trace their relative contributions to glycan synthesis without MS. On the other hand, mass isotopomer analysis of glycans labeled with stable isotope provides the ratios of labeled versus unlabeled molecules from MS spectra and structural details of the glycans. However, there are only a limited number of publications reporting the application of stable isotope labeling of glycans for monitoring the dynamics of glycans (17). To date, there have been no reports describing a systematic method for tracing cellular GlcNAc biosynthesis and use based on mass isotopomer analysis.The aim of this study was to extend our knowledge of the synthesis and metabolism of UDP-GlcNAc as well as its use in the synthesis of CMP-NeuAc, N- and O-glycans. We recently developed a conventional HPLC method for simultaneous determination of nucleotide sugars including unstable CMP-NeuAc (18). We first established an LC-MS method for isotopomer analysis of ¹³C₆-glucose labeled nucleotide sugars for tracing UDP-GlcNAc metabolism from synthesis to use, because previous methods were not suitable for estimating UDP-GlcNAc use in CMP-NeuAc de novo synthesis (15). We also established a method for isotopomer analysis of labeled N- and O-glycan to monitor the metabolic flow of hexosamine into glycans. Using these two methods, we demonstrated the differences in the use of hexosamines between hepatoma and pancreatic insulinoma cell lines. Our approach may be useful for identifying a metabolic “bottleneck” that governs the turnover speed and patterns of cellular glycosylation, which may be relevant for various applications including glycoprotein engineering and discovery of disease biomarkers.     

Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes

The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca²⁺ channel, in store-operated Ca²⁺ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3 and 5 rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-1 polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca²⁺ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP₃F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC₅₀ = 0.5 M), indicating that InsP₃F and LPA principally activate store-operated Ca²⁺ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP₃F-, LPA- or thapsigargin-stimulated Ca²⁺ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP₃F-stimulated Ca²⁺ inflow and only a very small enhancement of LPA-stimulated Ca²⁺ inflow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca²⁺ inflow through the previously-characterised Ca²⁺-specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.     

Polymorphisms in the 5′-UTR of <Emphasis Type="Italic">PTEN</Emphasis> and other gene polymorphisms in normal Japanese individuals

Polymorphisms are distributed differently in populations, including those of regions, ethnic groups, and diseased patients. In order to investigate variation in nucleotide sequences in normal individuals, we isolated genomic DNA from the blood of healthy Japanese individuals and sequenced the 5′-untranslated region (5′-UTR) of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene and the gene promoter, intron, and exon nucleotides of p53, p14 ^ARF , murine double minute 2 (MDM2), and the β₂- and β₃-adrenoceptor (?AR). We found polymorphisms in these regions, including a deletion at positions ?465 to ?463 and a substitution at position ?404 in PTEN and a substitution at position ?4924 in p14 ^ARF , in normal individuals. Individuals with or without the PTEN polymorphism harbored a different distribution of polymorphisms, including simultaneous alterations in nucleotides of p53, MDM2, and β₃-AR, and also harbored some polymorphic nucleotides located in the same set of associatively altered nucleotides. Our results show that multiple nucleotides, including the PTEN nucleotides, are altered in normal Japanese individuals and provide useful information for genotyping studies in individuals and populations.     

The effect of growth temperatures on the in vivo ribose methylation of Bacillus stearothermophilus transfer RNA

When Bacillus stearothermophilus was cultured at 70 and at 50 °C, 1.4 times as many methyl groups were incorporated into tRNA produced at the higher temperature compared to that produced at the lower. This was due predominantly to a threefold increase in the 2′-O-methylribose moieties of the tRNA. The type and quantity of the base methylated nucleotides in the tRNAs produced in cultures grown at 70 and 50 °C were almost identical. The base methylated nucleotides found were: m²Ap, ms²Ap, ms²i⁶Ap, an unidentified i⁶Ap derivative, m⁶Ap, m₂⁶Ap, m¹Gp, m⁷Gp, m⁵Up-(Tp), and an unidentified methylated Up or Cp.³ The nucleotide m⁷Ap, never before reported to be a constituent of tRNA, has been tentatively identified as a component of B. stearothermophilus tRNA.     

Energy metabolism of human erythrocytes in psoriasis

• 1.1. Adenine nucleotide concentrations and metabolism in red blood cells (RBC)2 and RBC ghosts from psoriatic patients and healthy subjects were compared.
• 2.2. The ATP and total adenine nucleotide levels and the adenylate energy charge (EC) were elevated in the blood from psoriatic patients.
• 3.3. The rate of glycolytic production of ATP by intact RBC was unchanged, but the Na⁺, K⁺-ATPase activity of RBC ghosts was decreased significantly in psoriasis.
• 4.4. Results suggest that the defect in adenine nucleotide metabolism is a systemic manifestation of psoriasis, and that the quantification of adenine nucleotides in RBC and in whole blood samples may be of pathophysiological value in psoriatic lesion.

     

<Emphasis Type="Italic">Amycolatopsis vastitatis</Emphasis> sp. nov., an isolate from a high altitude subsurface soil on Cerro Chajnantor,northern Chile

The taxonomic position of a novel Amycolatopsis strain isolated from a high altitude Atacama Desert subsurface soil was established using a polyphasic approach. The strain, isolate H5^T, was shown to have chemical properties typical of members of the genus Amycolatopsis such as meso-diaminopimelic acid as the diamino acid in the cell wall peptidoglycan, arabinose and galactose as diagnostic sugars and MK-9(H₄) as the predominant isoprenologue. It also has cultural and morphological properties consistent with its classification in the genus, notably the formation of branching substrate hyphae which fragment into rod-like elements. 16S rRNA gene sequence analyses showed that the strain is closely related to the type strain of Amycolatopsis mediterranei but could be distinguished from this and other related Amycolatopsis strains using a broad range of phenotypic properties. It was separated readily from the type strain of Amycolatopsis balhymycina, its near phylogenetic neighbour, based on multi-locus sequence data, by low average nucleotide identity (92.9%) and in silico DNA/DNA relatedness values (51.3%) calculated from draft genome assemblies. Consequently, the strain is considered to represent a novel species of Amycolatopsis for which the name Amycolatopsis vastitatis sp. nov. is proposed. The type strain is H5^T (= NCIMB 14970^T = NRRL B-65279^T).     

Respiration-dependent calcium ion uptake by two preparations of cardiac mitochondria. Effects of palmitoyl-coenzyme A and palmitoylcarnitine on calcium ion cycling and nicotinamide nucleotide reduction state

Ca²⁺ uptake and the effect of the uptake inhibitors palmitoyl-CoA and palmitoylcarnitine were examined in two preparations of dog cardiac mitochondria. Mitochondria prepared by using the Nagarse technique was 2.5-fold more active in respiration-dependent Ca²⁺ uptake than were mitochondria isolated by using the Polytron procedure. Palmitoyl-CoA and palmitoylcarnitine inhibited Ca²⁺ uptake in both preparations uncompetitively, with K_i,app 0.4 and 20μm. Ca²⁺-uptake rates were related to, or influenced by, the concentration of mitochondrial reduced nicotinamide nucleotides, with uptake slowing as this concentration decreased. When most of the nicotinamide nucleotides was oxidized, Ca²⁺ release and respiratory stimulation were observed. In the presence of Ruthenium Red and palmitoyl-CoA, oxidation of nicotinamide nucleotides was abolished and the time to Ca²⁺ release was shortened corresponding to the time of onset of nicotinamide nucleotide oxidation in the absence of Ruthenium Red. The results suggest that NAD(P)H oxidation in the presence of rotenone was a consequence of Ca²⁺ re-uptake and that net Ca²⁺ release could be observed as reduced nicotinamide nucleotide concentrations declined. Although nicotinamide nucleotide oxidation occurred in the presence of rotenone, it was not linked in an apparent manner to acyl-group metabolism (palmitoylcarnitine was less effective than palmitoyl-CoA). Therefore either a by-pass of the rotenone block or a direct interaction of NAD(P)H with the Ca²⁺-uptake process was possible. Loss of NADH occurred before respiratory stimulation, and this loss may relate to decreased coupling efficiency at sites 2 and 3 of the respiratory chain, as suggested by others [Bhuvaneswaran & Wadkins (1978) Biochem. Biophys. Res. Commun. 82, 648–654].     

H V-ATPase-Energized Transporters in Brush Border Membrane Vesicles from Whole Larvae of Aedes aegypti

Brush border membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs) contain an H⁺ V-ATPase (V), a Na⁺/H⁺ antiporter, NHA1 (A) and a Na⁺-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles. AgNAT8 was cloned from Anopheles gambiae, localized in BBMs and characterized in Xenopus laevis oocytes. AgNHA1 was cloned and localized in BBMs but characterization in oocytes was compromised by an endogenous cation conductance. AeBBMVWs complement Xenopus oocytes for characterizing membrane proteins, can be energized by voltage from the V-ATPase and are in their natural lipid environment. BBMVs from caterpillars were used in radio-labeled solute uptake experiments but ∼10,000 mosquito larvae are needed to equal 10 caterpillars. By contrast, functional AeBBMVWs can be prepared from 10,000 whole larvae in 4 h. Na⁺-coupled ^3Hphenylalanine uptake mediated by AeNAT8 in AeBBMVs can be compared to the Phe-induced inward Na⁺ currents mediated by AgNAT8 in oocytes. Western blots and light micrographs of samples taken during AeBBMVW isolation are labeled with antibodies against all of the VAN components. The use of AeBBMVWs to study coupling between electrogenic V-ATPases and the electrophoretic transporters is discussed.     

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. II. Effects of adenine nucleotides

• 1.1. The native rat-kidney cortex Fructose-1,6-bisphosphatase is differentially regulated by adenine nucleotides in the presence of divalent cations.
• 2.2. Binding of AMP and ADP to the enzyme is co-operative. The inhibition by both nucleotides show an uncompetitive mechanism AMP being the most efficient inhibitor.
• 3.3. Mg²⁺ decreases the inhibition produced by AMP and ADP by enhancing their I_0.5 and completely annulates the inhibitory effect of ATP.
• 4.4. In the presence of Mn²⁺ ADP behaves as an inhibitor but no inhibition is evident with AMP, suggesting the existence of different allosteric sites for each nucleotide.

     

Inhibition of glucuronokinase by substrate analogs

Glucuronokinase from Lilium longiflorum pollen was purified 30- to 40- fold on a blue dextran-Sepharose column. Substrate analogs were tested for inhibitory effects, and nucleotide substrate specificity of the enzyme was determined. Nine nucleotides were tested, and all were inhibitory when the substrate was ATP. ADP was competitive with ATP and had a K_i value of 0.23 mm. None of the other nucleotide triphosphates could effectively substitute for ATP as a nucleotide substrate. Ten mm dATP and ITP reacted only 3% as rapidly as 10 mm ATP, while the rates for 10 mm GTP, CTP, UTP, and TTP were less than 1%. The glucuronic acid analogs, methyl α-glucuronoside, methyl β-glucuronoside, β-glucuronic acid-1-phosphate, and 4-O-methylglucuronic acid were tested as possible enzyme inhibitors. The three methyl derivatives showed little or no inhibition. The β-glucuronic acid-1-phosphate was inhibitory, with 50% inhibition obtained at 1 to 3 mm depending on the concentration of the glucuronic acid. It is concluded that the glucuronic acid-binding site on the enzyme is highly selective.     

Purine catabolism in isolated rat hepatocytes. Influence of coformycin

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [¹⁴C]adenine. The production of allantoin reached 32±5nmol/min per g of cells (mean±s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1μm-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50μm-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1μm-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50μm-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [¹⁴C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1μm-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50μm-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5′-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5′-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.     

<Emphasis Type="Italic">Nonomuraea insulae</Emphasis> sp. nov., isolated from forest soil

Strain H2R21^T, a novel actinobacterium, isolated from a forest soil sample collected from Heybeliada, Istanbul, Turkey, and a polyphasic approach was used for characterisation of the strain. Chemotaxonomic and morphological characterisation of strain H2R21^T indicated that it belongs to the genus Nonomuraea. 16S rRNA gene sequence similarity showed that the strain is closely related to Nonomuraea purpurea 1SM4-01^T (99.1%) and Nonomuraea solani CGMCC 4.7037^T (98.4%). DNA–DNA relatedness values were found to be lower than 70% between the isolate and its phylogenetic neighbours N. purpurea 1SM4-01^T, N. solani CGMCC 4.7037^T and Nonomuraea rhizophila YIM 67092^T. The whole cell hydrolysates of strain H2R21^T were found to contain meso-diaminopimelic acid as the diagnostic diamino acid and glucose, madurose, mannose and ribose as the cell sugars. The polar lipids were identified as phosphatidylglycerol, diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, dihydroxy-phosphatidylethanolamine, phosphatidylinositol, glycophosphatidylinositol, two glycophospholipids and two unidentified lipids. The predominant menaquinones were identified as MK-9(H₄) and MK-9(H₆). The major fatty acids were found to be iso-C_16:0, iso-C_16:0 2OH and C_17:0 10-methyl. On the basis of DNA–DNA relatedness data and some phenotypic characteristics, it is evident that strain H2R21^T can be distinguished from the closely related species in the genus Nonomuraea. Thus, it is concluded that strain H2R21^T represents a novel species of the genus Nonomuraea, for which the name Nonomuraea insulae sp. nov. is proposed. The type strain is H2R21^T (= DSM 102915^T = CGMCC 4.7338^T = KCTC 39769^T).     

Sequence diversity of MHC class-II <Emphasis Type="BoldItalic">DRB</Emphasis> gene in gazelles (<Emphasis Type="BoldItalic">Gazella subgutturosa</Emphasis>) raised in Sanliurfa of Turkey

In this study, we aimed to assess the sequence diversity of major histocompatibility complex (MHC) class-II DRB gene at exon 2 in gazelles raised in Sanliurfa Province of Turkey. Twenty DNA samples isolated from gazelles (Gazella subgutturosa) were used for sequencing exon 2 of MHC class-II DRB gene. Target region was amplified by polymerase chain reaction (PCR) and their products were directly sequenced. Nine of these 20 samples yielded unambiguously readable sequences. Three of the nine samples were homozygotes and each showed different sequences. A 262-bp sequence obtained from the three homozygote samples were submitted to GenBank (accession numbers: KC309405, KC309406 and KC309407). Using an allele specific PCR, we detected 10 additional haplotypes. Among 13 haplotypes, 45 nucleotide positions were polymorphic and most of the polymorphic nucleotide positions localized at peptide-binding region (PBR). Rates of nonsynonymous substitutions were significantly higher than synonymous substitutions at PBR. Phylogenetic analysis of the haplotypes showed that 10 haplotypes of the gazelles were clustered together while three were clustered with ovine and bovine haplotypes. The results indicated that at least 13 haplotypes at exon 2 of MHC class-II DRB gene were showing high degree of nucleotide and amino acid diversity, and certain haplotypes of G. subgutturosa were more similar to haplotypes from sheep or cattle than to each other. Rates of synonymous and nonsynonymous substitutions suggested that positive selection was a driving force for diversity at this locus in G. subgutturosa.     

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix

Introduction

Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed.

Objectives

To study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions.

Methods

We present a double-labelling LC–MS approach with a ¹³C-labeled yeast cellular extract as complex surrogate matrix, and ¹³C¹⁵N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates.

Results

While nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions.

Conclusions

Extraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study.

Graphical abstract

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