Oxygenomics in environmental stress.

Environmental stressors such as chemicals and physical agents induce various oxidative stresses and affect human health. To elucidate their underlying mechanisms, etiology and risk, analyses of gene expression signatures in environmental stress-induced human diseases, including neuronal disorders, cancer and diabetes, are crucially important. Recent studies have clarified oxidative stress-induced signaling pathways in human and experimental animals. These pathways are classifiable into several categories: reactive oxygen species (ROS) metabolism and antioxidant defenses, p53 pathway signaling, nitric oxide (NO) signaling pathway, hypoxia signaling, transforming growth factor (TGF)-beta bone morphogenetic protein (BMP) signaling, tumor necrosis factor (TNF) ligand-receptor signaling, and mitochondrial function. This review describes the gene expression signatures through which environmental stressors induce oxidative stress and regulate signal transduction pathways in rodent and human tissues.

Heterogeneous Sp1 mRNAs in human HepG2 cells include a product of homotypic trans-splicing.

Sp1 is one of the well documented transcription factors, but the whole structure of human Sp1 has not been determined yet. In the present study, we isolated several cDNAs representing two forms of human Sp1 mRNA with different 5'-terminal structures in HepG2 cells. Isolation of a genomic clone established that one of the cDNAs represents the mRNA having consecutive alignment of exons, which allowed deducing the complete amino acid sequence for human Sp1. Another cDNA clone had a surprising structure that possessed an alignment of exons 3-2-3. Both reverse transcriptase-polymerase chain reaction and RNase protection assays confirmed accumulation of the two forms of Sp1 mRNA in HepG2 cells. Because Southern blot analysis suggested that exon 3 is of a single copy in the genome, the cDNA clone having the duplicated sequences for exon 3 appeared to reflect the trans-splicing between pre-mRNAs of human Sp1.

N-linked oligosaccharide processing enzyme glucosidase II produces 1,5-anhydrofructose as a side product.

alpha-1,4-Glucan lyase cleaves alpha-1,4-linkages of nonreducing termini of alpha-1,4-glucans to produce 1,5-anhydrofructose (1,5-AnFru). The enzymes isolated from fungi and algae show high homology with glycoside hydrolase family 31. Purification of alpha-1,4-glucan lyase from rat liver using DEAE Cellulose chromatography resulted in separation of two enzymatic active fractions, one was bound to the column and the other was in the flow-through. Partial amino acid sequence determined from the lyase, retained on the anion exchange column, were identical with that of the N:-linked oligosaccharide processing enzyme glucosidase II. The lyase showed similar enzymatic properties as the microsomal glucosidase such as inhibition by 1-deoxynojirimycin and castanospermine. On the other hand, glucosidase II purified from rat liver microsomes produced not only glucose but also a small amount of 1,5-AnFru using maltose as substrate. Furthermore, CHO cells overexpressing pig liver glucosidase II showed a 1.5- to 2-fold higher lyase activity compared to the nontransfected CHO cells. Conversely, no lyase activity was detectable either in PHAR2.7, the glucosidase II-deficient mutant from a mouse lymphoma cell line, or in Saccharomyces cerevisiae strain YG427 having the glucosidase II gene disrupted. These data demonstrate that glucosidase II possesses an additional enzymatic activity of releasing 1,5-AnFru from maltose.

1,5-Anhydroglucitol promotes glycogenolysis in Escherichia coli.

Glycogen is a storage compound that provides both carbon and energy, but the mechanism for the regulation of its metabolism has not been fully clarified. Recently, we found a new glycogenolytic pathway in rat liver in which glycogen is first metabolized to 1, 5-anhydrofructose (AnFru) and then to 1,5-anhydroglucitol (AnGlc-ol). Because the amounts of glycogen and AnFru are closely related in various rat organs and the second reaction, AnFru to AnGlc-ol, is strongly inhibited in the presence of glucose, we expected that this pathway might play a regulatory role in glycogen metabolism. Here we evaluate the expected involvement of AnGlc-ol and AnFru in the regulatory mechanism in Escherichia coli C600. Having established the presence of this new glycogenolytic pathway in E. coli C600, we further show that the conversion of AnFru to AnGlc-ol is activated only after the exhaustion of glucose in the medium, and that as little as 5 microM AnGlc-ol in the medium acutely accelerates the degradation of glycogen by 40%. We consider the role of AnGlc-ol in the mechanism that controls glycogen metabolism in E. coli to be as follows. When glucose is abundant, E. coli accumulate glycogen, a fraction of which is steadily degraded so that the amount of AnFru is about 1/1,000 of glycogen on a weight basis. When glucose is depleted and the demand for glycogen utilization is elevated, AnFru, which has accumulated mostly in the medium, is promptly taken up and converted to AnGlc-ol, which accelerates glycogen degradation. We also suggest the possibility that AnGlc-ol is one of the extracellular signaling molecules for bacteria.

[A case of severe status epilepticus of frontal lobe origin successfully treated with corticosteroids].

A 17-year-old girl was admitted to our hospital due to low-grade fever, confusion, numbness in her right hand and automatism. On admission, she was slightly disoriented but there were no meningeal signs. Weakness and sensory disturbance were observed in her right hand. Automatism and clonic seizures frequently appeared. Electroencephalography revealed frequent delta bursts in her left frontal lobe. 123I-IMP-SPECT study showed abnormally increased isotope uptake in the left cerebral hemisphere. She was diagnosed as status epilepticus of left frontal lobe origin and treated with anti-convulsants including carbamazepine, phenytoin, diazepam, phenobarbital, and thiopental, which were not effective. Then we started corticosteroid therapy. Three cycles of intravenous injections of methylprednisolone, followed by oral prednisolone led to marked improvement in her symptoms. It is known that corticosteroid decreases the threshold of seizure, so we do not use it for idiopathic epilepsy. On the other hand, in some secondary epilepsy due to vasculitis in the brain, corticosteroid is very effective for seizures. It is still unclear whether our patient actually had vasculitis or not. However, it is important to recognize that steroid therapy might be effective in a certain portion of epilepsies resistant to anti-convulsants, especially in young patients with non-infectious fever.

Binding of human cytomegalovirus to sulfated glucuronyl glycosphingolipids and their inhibitory effects on the infection.

Interactions between human cytomegalovirus (HCMV) and various carbohydrate structures were analysed using sulfated glucuronyl glycosphingolipids (SGGLs) and the structurally related glycosphingolipids (GLs). A thin-layer chromatography-overlay assay and a solid-phase binding assay revealed that HCMV strongly bound to sulfated glucuronyl lactosaminylparagloboside, one of the SGGLs having the repeating lactosamine structure (3Gal beta1-4GlcNAc1-)2 in addition to the 3-O-sulfated glucuronyl moiety. The virus bound less strongly to other 3-O-sulfated GLs, which included sulfated glucuronyl paragloboside and cerebroside sulfate ester, and also to (3Gal beta1-4GlcNAc1-)2-containing GLs that included nLc6Cer. Thus, a (3Gal beta1-4GlcNAc1-)2 and a 3-O-sulfated saccharide seem to be important structures for the binding by HCMV. When virus particles were preincubated with these GLs, inhibitory effects were observed both on expression of the viral immediate-early gene and on plaque formation by HCMV. These effects were very well correlated with the abilities of the GLs to bind to the virus. Pretreatment of host cells with HNK-1 monoclonal antibody, which specifically recognizes SGGLs, resulted in partial inhibition of plaque formation by HCMV. These results clearly show that HCMV recognizes and binds to the sulfated carbohydrate structure in SGGL and also suggest that binding of HCMV to the specific sugar structure may play an important role in HCMV infection.

Comparison of calpains from rabbit, monkey, human and rat.

Two isozymes of calpain, mu-calpain and m-calpain, were purified from rabbit, monkey, human and rat tissues to homogeneity and the apparent molecular masses of the large and small subunits of each calpain species were compared directly. While the molecular masses of the small subunits were the same (28 kDa), those of the large subunits were different depending on the calpain type and animal species: Rabbit mu (79 kDa), rabbit m (75 kDa), monkey mu (79 kDa), monkey m (74 kDa), human mu (78 kDa), human m (73 kDa), rat mu (75 kDa), and rat m (74 kDa). Ca2+-sensitivity of monkey mu-calpain was lower than that of rabbit mu-calpain, but m-calpains from rabbit and monkey shared a similar Ca2+-dependency. Immunoreactivities of rabbit, monkey and rat m-calpains towards anti-rabbit m-calpain monoclonal antibodies were different depending on the antibody species, showing the existence of common and different antigenic sites in these three m-calpains. While monkey mu-calpain still showed weak cross-reactivity with anti-rabbit mu-calpain monoclonal antibodies, rat mu-calpain failed to react with any antibody examined, except for the monoclonal 1D10A7 which reacted with mu- and m-calpains from any animal species. Peptides generated by V8 protease digestion were similar between mu-calpains or m-calpains from rabbit and monkey but, again, the reactivity of monkey calpain peptidesto anti-rabbit calpain antibodies was weak, especially to those of mu-calpain.

Purification and some properties of a hepatic NADPH-dependent reductase that specifically acts on 1,5-anhydro-D-fructose.

Glycogen gives rise to 1,5-anhydro-D-fructose (AF), which is then reduced to 1,5-anhydro-D-glucitol (AG) in animal livers. An enzyme that catalyzes NADPH-dependent reduction of AF to AG was isolated and purified to homogeneity from porcine liver. Its apparent molecular mass was about 38 kDa on the basis of SDS-PAGE, and its monomeric dispersion in aqueous solution was indicated by gel filtration on a Superose 12 column. Amino acid sequences were determined for four peptides obtained from the purified enzyme. The resulting sequences covered about 50% of the whole sequence and indicated a remarkable similarity between the enzyme and aldose reductase. The purified enzyme showed molecular activity of 8.7 s(-1) on the basis of a molecular mass of 38 kDa, and a Km value of 0.44 mM for AF at the optimum pH of 7.0. It reduced pyridine-3-aldehyde and 2,3-butanedione effectively, acetaldehyde, glucosone, and glucuronic acid poorly and showed no detectable action on glucose, mannose and fructose. It was inactivated by p-chloromercuribenzoic acid to a considerable extent, and the inactivation was partially reversed by 2-mercaptoethanol treatment. It was also sparingly inhibited by relatively high concentrations of glucose, glucose-1(6)-phosphate and 1,5-anhydroglucitol. The reverse reaction, i.e., NADP+-dependent AG oxidation, was not observed. The observed catalytic properties and partial amino acid sequences rule out the possibility that the isolated protein is identical with any known reductase.

Identification and characterization of positive regulatory elements in the human glyceraldehyde 3-phosphate dehydrogenase gene promoter.

The gene for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is expressed at high levels in almost all tissues. However, the molecular mechanism which sustains high-level expression of this house-keeping enzyme is still unknown. Here we show that transcriptional activity is reduced by deletion of the nucleotides from -181 to -144 (relative to the transcriptional start site) in the promoter of human GAPDH gene, both in CHO (derived from Chinese hamster ovary) and HepG2 (derived from human hepatoma) cells. Gel retardation assays revealed that at least two nuclear factors, termed GAPBF1 and GAPBF2, bind to this region. Mutations in the GAPBF1 binding site (-178 to -169) or the GAPBF2 binding site (-168 to -163) reduced this promoter activity in vivo, showing that these two sites contribute to the activity of the GAPDH gene promoter. Since mutations in the region from -162 to -146 also reduced the promoter activity, this region seemed to function as an added cis-element, although we failed to find a factor that interacted specifically with this region in vitro. Accordingly, we propose that there are multiple cis-elements in the region from -181 to -144, each of which contributes to the promoter activity of GAPDH gene; the GAPBF1 binding site has the unique feature of having a stretch of repeated A nucleotides.

Hepatic production of 1,5-anhydrofructose and 1,5-anhydroglucitol in rat by the third glycogenolytic pathway.

A unique anhydrohexulose, 1,5-anhydrofructose (1,5AnFru) has been detected in rat livers. Here we describe a microanalytical method for 1,5AnFru using GC/MS and report results on the distribution and production of 1,5 AnFru in rats. The highest levels of 1,5AnFru were found in the liver (0.43 microgram/g wet tissue) and appreciable amounts were detected in adrenal gland and spleen (0.12 microgram/g and 0.09 microgram/g, respectively). Other organs contained lower amounts while plasma contained virtually no detectable 1,5AnFru. We also demonstrated that 1,5AnFru is produced in the cytosol fraction of rat liver homogenate when an alpha-1,4-glucan or glycogen was added; 1,5AnFru was readily reduced to 1,5-anhydroglucitol with NADPH or at a reduced efficiency with NADH in the presence of a Mono Q chromatographic fraction obtained from the same cytosol preparation. Based on these results, we propose the existence of a third degradation pathway, in addition to the phosphorolytic and hydrolytic reaction sequences, from glycogen to 1,5-anhydroglucitol via 1,5AnFru in mammals. However, the physiological significance of 1,5AnFru and this putative minor glycogenolytic pathway in mammals remains obscure.

Production of 1,5-anhydroglucitol from 1,5-anhydrofructose in erythroleukemia cells.

The pyranoid polyol 1,5-anhydroglucitol (1,5AnGlc-ol) occurs in a wide variety of organisms. In humans, it is present as one of the major monosaccharide components in body fluids and serves as an indicator for glycemic control in diabetic care. However, its metabolic origin and fate have been poorly understood. Here we demonstrate that 1,5AnGlc-ol is produced from glucose in erythroleukemia cells, K-562. We show the occurrence of 1,5-anhydrofructose (1,5AnFru), a derivative of 1,5AnGlc-ol oxidized at the C2 position, in K-562 cells. In addition, several pieces of evidence indicated that 1,5AnFru, rather than glucose, was the immediate precursor in 1,5AnGlc-ol production in erythroleukemia cells: exogenous 1,5AnFru was readily taken up into the cells and reduced to 1,5AnGlc-ol, but the reverse reaction, oxidation of 1,5AnGlc-ol to 1,5AnFru, was scarcely observed. The apparent K(m) of the overall cellular reduction for 1,5AnFru was estimated as 70 mg/l. This reduction was markedly inhibited by glucose in the culture medium but not by 1,5AnGlc-ol or glucitol. Since 1,5AnFru arises from alpha-1,4-glucans through lyase reactions in fungi and algae, we suggest the possibilities that glycogen in the precursor of 1,5AnFru and, therefore, 1,5AnGlc-ol originates from glycogen in mammals.

Escherichia coli phosphorylates 1,5-Anhydroglucitol and releases 1,5-Anhydroglucitol 6-phosphate when glucose is absent in the medium.

The cyclic polyol 1,5-anhydro-D-glucitol (AG) is detected in most organisms, but little is known about its metabolism and physiological roles. Our previous study demonstrated that Escherichia coli C600 synthesizes AG when glucose is exhausted in the medium and that it temporarily releases AG into and then takes it back from the medium, thus forming a sharp peak in AG concentration in the medium a few hours after reaching stationary growth phase. The present study demonstrates that when glucose is absent in the culture medium, E. coli C600 takes up and phosphorylates AG and releases a large portion of it back into the medium in the form of a phosphate ester. [U-13C]AG was added to the medium after the exhaustion of glucose and the resulting [U-13C]AG phosphate was partially purified by several steps of anion exchange chromatography and identified as AG 6-phosphate by 13C-NMR. The identity of the phosphate ester was also confirmed by GC-MS analysis after further purification.

NMR of all-carbon-13 sugars: an application in development of an analytical method for a novel natural sugar, 1,5-anhydrofructose.

Of the all-carbon-13 compounds, glucose is one of the most easily accessible, and therefore we applied 13C-NMR technique to the metabolic study of glucose-related compounds, 1,5-anhydro-D-glucitol and 1,5-anhydro-D-fructose (AF). Applying an INADEQUATE method to the substitutes of these novel sugars fully labeled with carbon-13, we could trace out the entire carbon skeleton with high sensitivity and confirm the chemical structures of these sugars. The method also provided a much easier way to optimize the enzymatic oxidation for AF preparation: we selectively and continuously monitored the quantities, as well as their structures in aqueous solution, of the substrate and products in a noninvasive manner. Similarly relying upon information from the 13C-NMR, we developed a valuable derivatization method of AF for its GC-MS application, which was so sensitive that we were able to demonstrate the natural occurrence of AF in rat liver.

Phosphorylation of 1,5-anhydro-D-glucitol in mammalian cells.

A cyclic polyol, 1,5-anhydro-D-glucitol (AG), is generally present in animals, although little is known about the metabolic and physiological roles of AG in any type of animal cells. The present metabolic study demonstrated phosphorylation of AG in human chronic myelogenous leukemia cells, K-562. Phosphorylated AG (AGP) was also proved to be present in various rat organs; its level in most organs ranged between 2 and 5 nmol/g wet tissue, which amounted to 5 to 10% of the AG levels in the respective organs. In the spleen and brain, however, the AGP levels were especially high, 13.4 and 8.3 nmol/g, respectively, or 24.4 and 20.6% of the respective AG levels. These data suggest that AGP is an intermediary metabolite related to AG in animal cells.

Transport and accumulation of 1,5-anhydro-D-glucitol in the human erythroleukemia cell line K-562.

The transport and intracellular accumulation of 1,5-anhydro-D-glucitol (AG) was studied in the human erythroleukemia cell line K-562 by gas chromatography-mass spectrometry in conjunction with liquid scintillation spectrometry. K-562 cells contained 106 +/- 6 nM/10(6) cells of free AG, primarily in the cytosol. Addition of physiologic amounts of AG to the extracellular medium resulted in rapid intracellular incorporation of AG, with a half-saturation time of 5 s. Intracellular accumulation was linear for 2 h and subsequently reached saturation. AG uptake was temperature and concentration dependent with an apparent Km of 127 mM. AG uptake and accumulation was not inhibited by fructose, fucose, galactose, mannose, glucose, or 3-O-methyl-D-glucose and was less affected by cytochalasin B or phloretin than that of 2-deoxyglucose. Phloridzin did not affect AG uptake but did inhibit 2-deoxyglucose uptake. Efflux of AG from K-562 cells depended on external AG concentration alone and was not affected by extracellular glucose concentration. Intracellular AG concentration decreased rapidly and reached zero within 10 min following removal of AG from the external medium. We therefore propose that both transport and countertransport of AG in K-562 cells are mediated by a specific carrier system.

Synthesis of 1,5-anhydro-D-glucitol from glucose in rat hepatoma cells.

A pyranoid polyol, 1,5-anhydroglucitol (AG), generally occurs in the human body as a humoral component. The plasma AG concentration in healthy individuals is maintained at a constant level, but it is markedly decreased in diabetes mellitus. This is due to hyperglycemia-dependent abolishment of renal AG retention. Hence, the plasma AG concentration has been established as a clinical marker for duration of hyperglycemia and since 1991 it has been practically applied to diabetic care in Japan. However, the details of the metabolism of AG and its physiological significance generally remain to be studied. In this study, we confirmed AG synthesis in cultured cells of a rat hepatoma line, Reuber H-35, in which AG was found to be derived from glucose, with retention of all six carbon atoms in the pyranoid structure. The fraction of the total glucose consumed by the cells, which was converted to AG (conversion efficiency) was at most 5 x 10(-6). The conversion efficiency increased at higher glucose concentrations (mM orders) where the glucose consumption rate was saturated. Since the rate of the hexokinase reaction, one of the rate-limiting steps in glucose consumption, has been estimated to be saturated at microM orders of glucose concentration, this observation was interpreted as indicating that AG is synthesized through a pathway which does not share the hexokinase reaction with glucose utilization. The presence of precursors other than glucose was also indicated in the time-course study of AG synthesis. Further, the amount of AG synthesized daily in humans is significant in comparison with the amount obtained from the diet.

Conditional synthesis and utilization of 1,5-anhydroglucitol in Escherichia coli.

A cyclic polyol, 1,5-anhydro-D-glucitol (AG), is widely detected in most organisms, although little is known about its metabolism and physiological roles. The present study demonstrates the synthesis of AG in Escherichia coli C600. The major portion of the synthesized AG was indicated to be derived from glucose retaining all the six carbon atoms, and only 5% was attributed to AG synthesized from C3 compounds. AG synthesis is apparent in an early stage of the stationary phase, and accumulation is transient both in cells and in medium. Evidence is also presented for AG uptake and metabolism and for effects of cyclic AMP.

High concentration of glucitol in fetal serum and glucitol permeable cells.

We found that the glucitol concentration was extraordinarily high in bovine fetal serum, which is routinely used for cell culture in laboratories: it was as much as two orders of magnitude higher than that reported for human adult serum. We also confirmed that the serum glucitol concentration in new born babies was on average 5.5-fold higher than in the maternal serum. These observations raise the possibility that some tissue(s) demand extracellular glucitol during embryogenesis and this indicates that the cells in such tissues could be permeable to glucitol. Since the hepatic metabolism of glucitol had been reported, we investigated glucitol permeability and its metabolism in rat hepatoma cells, Reuber H-35. The cells rapidly incorporated glucitol but the mode of incorporation was unusual: the incorporation rate was still proportional to the ambient glucitol concentration at 100 mM. The major part of the incorporated glucitol underwent metabolic conversion to probably negatively charged metabolites. Active synthesis of glucitol was also observed in the same cells. The cells proliferated normally in medium containing glucitol instead of glucose. This observation may indicate that glucitol can substitute for glucose in the culture of H-35 cells.

Determination of 1,5-anhydroglucitol in urine by high performance liquid chromatography and an enzyme sensor.

A simple high performance liquid chromatographic method combined with an enzyme sensor has been developed to measure 1,5-anhydroglucitol in urine. The enzyme sensor consists of a hydrogen peroxide electrode and a chitosan membrane of an immobilized pyranose oxidase. As the system does not resist interfering substances, urine samples are first purified by passing them through a two-layer column packed with (1) strongly basic anion (OH- form, the upper layer) and (2) strongly acidic cation (H+ form, the lower layer) exchange resins. 1,5-Anhydroglucitol is efficiently recovered in the flow-through fraction of the column. In this system, the minimum detectable concentration of 1,5-anhydroglucitol is 0.1 mg/L, and the measurable range extends from 0.1 to 60 mg/L. The coefficient of variation values of the within-day and day-to-day precisions are 3.0-6.5% and and 4.4-6.7% respectively, and there is good agreement between the results measured by our method and those obtained by the gas-liquid chromatographic/mass spectrometric method (r = 0.994). The method we have described here has been successfully used to elucidate a mechanism for the reducing 1,5-anhydroglucitol level in the serum and plasma of patients.