An accurate colorimetric method for measurement of sulfaminohexose in heparins and heparan sulfates

A modification of a method for hexosamine analysis is presented which adapts it to measurement of sulfaminohexose in heparins and heparan sulfates. Unlike methods of sulfaminohexose analysis based upon coupling with indole, the absorptivity of polymeric and monomeric hexosamines is identical. N-Sulfated hexosamines are specifically deaminated in 33% acetic acid to yield free 2,5-anhydromannose residues which are then coupled to the color reagent 3-methyl-2-benzothiazolinone hydrazone hydrochloride. The sulfaminohexose content of a variety of heparins and heparan sulfates was determined with this methodology and compared with the indole-coupling method. Interferences by amino acids, proteins, and neutral sugar were evaluated in the sulfaminohexose assay and in the originally reported procedure for total hexosamine analysis.     

A simple determination of hexosamines in column effluents

A simple method of determinations of three hexosamines is presented. The solutions of hexosamines heated in 1 n NaOH at 60°C for 60 min, absorb ultraviolet light with the maximum at 302 nm. A linear relationship exists between the concentration of hexosamine up to 100 μg and the absorbance at 302 nm. N-acetylated hexosamines, having been deacetylated, may be determined in this way. The characteristic absorption curve ranging from 225 to 360 nm with the maximum at 302 nm may be utilized as a qualitative criterion for identification of hexosamines and N-acetylated hexosamines after their deacetylation.     

High-throughput analysis of hexosamine using a colorimetric method

A 96-well plate method was developed for analysis of total hexosamine content in biological samples. Four hexosamine monomer derivatives—glucosamine hydrochloride, glucosamine sulfate, galactosamine hydrochloride, and mannosamine hydrochloride—were examined for the linearity of their spectra in the concentration range specified in the assay. The hexosamine concentration analysis range was linear from 0.1 to 1 mM. The quantification of hexosamines from chitin and chitosan upon acid hydrolysis was also tested. Accurate quantification of glucosamine content in chitin and chitosan with different molecular sizes and degrees of acetylation was demonstrated using the new method.     

Glycoproteines sulfates des membranes de l'oeuf de poule et de l'oviducte Isolement et caracterisation de glycopeptides sulfatesSulfated glycoproteins form egg shell membranes and hen oviduct. Isolation and characterization of sulfated glycopeptides

Sulfated glycopeptides were isolated from pronaisc and tryptic digests of egg shell membranes and hen oviduct. They were precipitated by cationic detergents and separated by preparative electrophoresis, after removal of small quantities of glucuronoglycosaminoglycans detected only in the oviduct (isthmus and magnum). The principal isolated sulfated glycopeptides were divided according to increasing electrophoretic mobilities into two groups A and B. The homogeneity of the purified glycopeptides was confirmed by gel filtration and polyacrylamide gel electrophoresis.Glycopeptides from pool preparation of tissue are analysed and carbohydrate and amino acids average values are estimated. Hexosamines (mainly N-acetylglucosamine), hexoses (galactose, glucose, mannose) and fucose were found in Glycopeptides A. The molar ratio of hexose/hexosamine was 0.4. N-Acetylneuraminic acid and sulfate were also present in Glycopeptides A. The molar ratio of sulfate/hexosamine ranged from 0.1 to 0.25. The Glycopeptides A composition indicated the presence of chains with many glycosyl groups and a few of amino acids residues. The carbohydrate components of Glycopeptides B from egg shell membranes and magnum were found to be hexosamines (N-acetylgalactosamine and N-acetylglucosamine in equimolar proportions), hexoses (galactose mainly and glucose), N-acetylneuraminic acid, and fucose. The molar ratio of hexose/hexosamine was 1. Sulfate was also present and the molar ratio of N-acetylneuraminic acid and sulfate to hexosamine was ranged from 0.8 to 1. The main amino acid residues in these glycopeptides were serine and threonine with destruction of these hydroxyamino acids during alkali treatment. Glycopeptides B probably consist of short carbohydrate chains, linked to the polypeptide through O-glycosidic bonds involving N-acetylgalactosamine and serine and threonine. Approximately 40% of the amino acid residues were linked to carbohydrate chains.Glycopeptides B from egg shell membranes magnum and egg white were very similar in their carbohydrate and amino acid composition and in their properties.Gylcopeptides A from egg shell membranes, isthmus and magnum showed similarities and divergences especially in the amino acid composition. These results suggest that magnum and isthmus in oviduct are both concerned with the synthesis of egg shell membrane glycoproteins.     

Acid hydrolysis and quantitative determination of total hexosamines of an exopolysaccharide produced by <Emphasis Type="Italic">Citrobacter</Emphasis> sp.

During the hydrolysis of an exopolysaccharide (EPS) produced by Citrobacter sp., the maximum liberation of hexosamine was obtained with 6 m HCl at 115 °C in an autoclave for 1 h. The glycosidic bond energy and degree of acetylation of the hexosamine in EPS were approximately 77 kJ mol^–1 and 61%, respectively. Thermal destruction of the hexosamines and the effect of salt on the hexosamine determination could be minimized under the optimized hydrolytic conditions. Using a modified Elson–Morgan method, maximum total hexosamine concentration was determined to be 3.2 g l^–1 (29% of crude EPS) after 96 h of fed-batch culture.Revisions requested 18 August 2004; Revisions received 2 November 2004     

HPLC analysis of hexosamine phosphates in biological samples.

Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation.     

Production of a New Type of Acid Carboxypeptidase of Molds of the Aspergillus niger Group

The ability of 88 fungi, which had been obtained as high-potency strains for acid proteinase production, to produce a new type of acid carboxypeptidase (having on optimal pH of about 3 for hydrolysis of benzyloxycarbonyl-glutamyltyrosine) in surface koji culture was determined. Among the aspergilli, substantial amounts of this new acid carboxypeptidase were produced by Aspergillus saitoi, A. usamii, A. awamori, A. inuii, and A. niger. Maximum yields of acid carboxypeptidase per gram of substrate were obtained by submerged culture in a medium containing 0.9% defatted soybean and 0.6% wheat bran. However, the maximum enzyme concentration per milliliter was obtained with a medium containing 3% defatted soybean and 2% wheat bran. The terminal pH could be controlled by varying the concentrations of soybean oil meal and wheat bran. The maximum enzyme production was reached after 4 days or more at 30 C.     

Production of heparin related glycosaminoglycans by an extablished mammalian cell line

A sulfated glycosaminoglycan has been isolated from the acid-soluble fraction of an established line of Chinese hamster fibroblasts grown in suspension culture. This material has a molecular weight between 5000 and 10,000, contains equimolar amounts of hexosamine and uronic acid (orcinol method), and about 0.6 sulfate groups per hexosamine residue. About 80% of the sulfate groups are N-sulfates on the basis of lability of the sulfate and the formation of equivalent numbers of free amino groups upon mild acid hydrolysis. The material is completely resistant to testicular hyaluronidase but is degraded to reducing monosaccharides and small oligosaccharides upon treatment with lyophilized cells of Flavobacterium heparinum that were grown on heparin. It is thought, therefore, to be related to the known N-sulfated glycosaminoglycans heparin and heparitin sulfate.     

Hexosamine Biosynthesis Is a Possible Mechanism Underlying Hypoxia’s Effects on Lipid Metabolism in Human Adipocytes

Introduction

Hypoxia regulates adipocyte metabolism. Hexosamine biosynthesis is implicated in murine 3T3L1 adipocyte differentiation and is a possible underlying mechanism for hypoxia’s effects on adipocyte metabolism.

Methods

Lipid metabolism was studied in human visceral and subcutaneous adipocytes in in vitro hypoxic culture with adipophilic staining, glycerol release, and palmitate oxidation assays. Gene expression and hexosamine biosynthesis activation was studied with QRTPCR, immunofluorescence microscopy, and Western blotting.

Results

Hypoxia inhibits lipogenesis and induces basal lipolysis in visceral and subcutaneous human adipocytes. Hypoxia induces fatty acid oxidation in visceral adipocytes but had no effect on fatty acid oxidation in subcutaneous adipocytes. Hypoxia inhibits hexosamine biosynthesis in adipocytes. Inhibition of hexosamine biosynthesis with azaserine attenuates lipogenesis and induces lipolysis in adipocytes in normoxic conditions, while promotion of hexosamine biosynthesis with glucosamine in hypoxic conditions slightly increases lipogenesis.

Conclusions

Hypoxia’s net effect on human adipocyte lipid metabolism would be expected to impair adipocyte buffering capacity and contribute to systemic lipotoxicity. Our data suggest that hypoxia may mediate its effects on lipogenesis and lipolysis through inhibition of hexosamine biosynthesis. Hexosamine biosynthesis represents a target for manipulation of adipocyte metabolism.     

Indole-3-acetic acid production,solubilization of insoluble metal minerals and metal tolerance of some sclerodermatoid fungi collected from northern Thailand

Sclerodermatoid fungi basidiomes were collected from northern Thailand and pure cultures were isolated. The morphology and molecular characteristics identified them as Astraeus odoratus, Phlebopus portentosus, Pisolithus albus and Scleroderma sinnamariense. This study investigated the in vitro ability of selected fungi to produce indole-3-acetic acid (IAA), to solubilize different toxic metal (Co, Cd, Cu, Pb, Zn)-containing minerals, and metal tolerance. The results indicated that all fungi are able to produce IAA in liquid medium. The optimum temperature for IAA production of all fungi was 30 °C, and the optimum concentration of L-tryptophan of Astraeus odoratus, Pisolithus albus and Scleroderma sinnamariense was 2 mg ml^?1. The highest IAA yield (65.29?±?1.17 μg ml^?1) was obtained from Phlebopus portentosus after 40 days of cultivation in culture medium supplemented with 4 mg ml^?1 of L-tryptophan. The biological activity tests of fungal IAA showed that it can simulate coleoptile elongation, and increase seed germination and root length of tested plants. In addition, the metal tolerance and solubilizing activities varied for different minerals and fungal species. The presence of metal minerals affected fungal growth, and cobalt carbonate showed the highest toxicity. The solubilization index decreased when the concentration of metal minerals increased. Astraeus odoratus showed the lowest tolerance to metals. This is the first report of in vitro IAA production, solubilization of insoluble metal minerals and metal tolerance abilities of the tested fungi.     

Biodegradation and bioconversion of endrin by white rot fungi,Phlebia acanthocystis and Phlebia brevispora

Endrin is persistent organic pollutants that contaminate soil in many parts of the world. In this study, endrin was used as the substrate for a degradation experiment with the white rot fungi of the genus Phlebia. The results of tolerance test showed that the tolerance level of Phlebia acanthocystis and Phlebia brevispora to endrin was higher than that of other fungi, and the tolerance coefficient of both strains to 1.0 mg/L endrin exceeded 0.9 in solid PDA medium. P. acanthocystis and P. brevispora could degrade endrin efficiently in pure culture, especially P. acanthocystis had the highest degradability of more than 80% after 20 d incubation. Compared with low-nitrogen medium, PDB medium is more suitable for the biodegradability of two fungi. Several hydroxylated products such as 8-hydroxyendrin and two monohydroxyendrin were detected, indicating that endrin was initially branched to different monohydroxylated products in fungal degradation. Moreover, a carboxylic acid product was obtained from P. acanthocystis culture, suggesting that the carboxylation reaction occurred in bioconversion of endrin. The fungal cytochrome P450 enzymes play significant role in the in the initial hydroxylation process on endrin degradation. This is the first report that endrin is converted to hydroxylated and carboxylated metabolites by microorganisms.     

Two-stage fungal biopulping for improved enzymatic hydrolysis of wood

A novel two-stage, whole organism fungal biopulping method was examined for increasing the yield of enzymatic hydrolysis of wood into soluble glucose. Liriodendron tulipifera wood chips (1 g) were exposed to liquid culture suspensions of white rot (Ceriporiopsis subvermispora) or brown rot (Postia placenta) fungi and incubated at 28 °C, either alone in single-stage 30 day (one fungal species applied) or two-stage 60 day (both fungal species applied in alternative succession) treatments. Fungi grew in all treatments, but did not significantly decrease the percent carbohydrate content of the wood. Two-stage treatments differed significantly in mass loss depending on order of exposure, suggesting additive or inhibitory fungal interactions occurred. Treatments consisting of C. subvermispora followed by P. placenta exhibited 6 ± 0.5% mass loss and increased the yield of enzymatic hydrolysis by 67-119%. This significant hydrolysis improvement suggests that fungal biopulping technologies could support commercial lignocellulosic ethanol production efforts if further developed.     

Degradation of lindane and endosulfan by fungi, fungal and bacterial laccases

The ability of two white-rot fungi (Trametes versicolor and Pleurotus ostreatus) and one brown-rot fungus (Gloeophyllum trabeum) to degrade two organochlorine insecticides, lindane and endosulfan, in liquid cultures was studied and dead fungal biomass was examined for adsorption of both insecticides from liquid medium. Lindane and endosulfan were also treated with fungal laccase and bacterial protein CotA, which has laccase activities. The amount of degraded lindane and endosulfan increased with their exposure period in the liquid cultures of both examined white-rot fungi. Endosulfan was transformed to endosulfan sulphate by T. versicolor and P. ostreatus. A small amount of endosulfan ether was also detected and its origin was examined. Degradation of lindane and endosulfan by a brown rot G. trabeum did not occur. Mycelial biomasses of all examined fungi have been found to adsorb lindane and endosulfan and adsorption onto fungal biomass should therefore be considered as a possible mechanism of pollutant removal when fungal degradation potentials are studied. Bacterial protein CotA performed more efficient degradation of lindane and endosulfan than fungal laccase and has shown potential for bioremediation of organic pollutants.     

Development of a Defined Medium for Growth of Cercospora rosicola Passerini

A simple synthetic liquid medium containing a single amino acid, glucose, salts, trace metals, and thiamine was developed for cultivation of Cercospora rosicola Passerini. Thiamine was shown to be important to growth. Culture of C. rosicola Passerini in a chemically defined medium makes possible studies of (+)-abscisic acid biosynthesis and regulation.     

Production of phosphatates by fungi isolated from desert soils

Twelve fungal cultures isolated from Indian desert soils belonging toAspergillus, Penicillium, Acrophialophora andAlternaria were found to produce both acid and alkaline phosphatases in liquid medium, their amounts varying from culture to culture. Maximum production of these enzymes was observed withA. niger. In general, acid phosphatase activity was much higher as compared to alkaline phosphatase. The optimum incubation period for the production of these enzymes was found to be 14 d and thereafter it started declining. There was a significant and positive correlation between biomass production and acid phosphatase activity but not with alkaline phosphatase.     

Succinic acid production from wheat using a biorefining strategy

The biosynthesis of succinic acid from wheat flour was investigated in a two-stage bio-process. In the first stage, wheat flour was converted into a generic microbial feedstock either by fungal fermentation alone or by combining fungal fermentation for enzyme and fungal bio-mass production with subsequent flour hydrolysis and fungal autolysis. In the second stage, the generic feedstock was converted into succinic acid by bacterial fermentation by Actinobacillus succinogenes. Direct fermentation of the generic feedstock produced by fungal fermentation alone resulted in a lower succinic acid production, probably due to the low glucose and nitrogen concentrations in the fungal broth filtrate. In the second feedstock production strategy, flour hydrolysis conducted by mixing fungal broth filtrate with wheat flour generated a glucose-rich stream, while the fungal bio-mass was subjected to autolysis for the production of a nutrient-rich stream. The possibility of replacing a commercial semi-defined medium by these two streams was investigated sequentially. A. succinogenes fermentation using only the wheat-derived feedstock resulted in a succinic acid concentration of almost 16 g l^–1 with an overall yield of 0.19 g succinic acid per g wheat flour. These results show that a wheat-based bio-refinery employing coupled fungal fermentation and subsequent flour hydrolysis and fungal autolysis can lead to a bacterial feedstock for the efficient production of succinic acid.     

Degradation of Coal by the Fungi Polyporus versicolor and Poria monticola

We report that two species of basidiomycete fungi (Polyporus versicolor and Poria monticola) grow in minimal liquid or solid medium when supplemented with crushed lignite coal. The fungi also grow directly on crushed lignite coal. The growth of both fungi was observed qualitatively as the production and extension of hyphae. No fungal growth occurred in minimal agar medium without coal. The fungi degraded solid lignite coal to a black liquid product which never appeared in cultures unless fungi and coal were present together. Apparently, lignite coal can serve as the principal substrate for the growth of the fungi. Infrared analyses of the liquid products of lignite degradation showed both similarities to and differences from the original lignite.     

Growth and sporulation of Beauveria bassiana and Metarrhizium anisopliae on media containing various amino acids

Natural isolates of two entomogenous fungi, Beauveria bassiana and Metarrhizium anisopliae, were cultured in liquid culture media containing 24 amino acids and KNO₃ to determine their effect on growth and sporulation. In addition, the growth and medium pH changes for each isolate grown on an asparagine-containing medium were compared. Tryptophan and alanine were most effective for growth and sporulation of B. bassiana, although glutamine and KNO₃ also produced large numbers of regularly shaped spores. Tryptophan, glutamic acid, and histidine were all well utilized for both growth and sporulation of M. anisopliae. Nitrogen sources containing sulfur were poorly utilized for sporulation by M. anisopliae. In general, B. bassiana produces greater mycelial mass and much larger numbers of spores than M. anisopliae. Both fungi attained nearly the same growth maximum on asparagine medium though B. bassiana exhibited an initially more rapid growth rate. In both fungi this rapid growth phase was accompanied by a decline in medium pH followed by a rise in pH during the decline phase of growth.     

Arginine metabolism in developing soybean cotyledons : I. Relationship to nitrogen nutrition

The free and protein amino acid composition of Glycine max (L.) Merrill cotyledons was determined for the entire developmental period using high performance liquid chromatography. Arginine constituted 18% of the total protein nitrogen throughout development, and there was a linear arginine nitrogen accumulation rate of 1212 nanomoles per cotyledon per day between 16 and 58 days after anthesis. Arginine and asparagine were major constituents of the free amino acid pool, constituting 14 to 62% and 2 to 41% of the total free amino acid nitrogen, respectively. The urea cycle intermediates, citrulline, ornithine, and argininosuccinate were also detected in the free pool. A comparison of the amino acid composition of cotyledonary protein and of seedcoat exudate suggested that 72% of the cotyledon's arginine requirement is satisfied by in situ biosynthesis, and that 20% of the transformed nitrogen is incorporated into arginine. Also, [1-¹⁴C]glutamate and [U-¹⁴C]glutamine were fed to excised cotyledons. After 4 hours, ¹⁴C was incorporated into protein and released as ¹⁴CO₂, but none was incorporated into the C-1 and C-6 positions of free and protein arginine, determined using arginine-specific enzyme-linked assays. It is not currently known whether arginine biosynthesis in the cotyledon involves glutamate delivered from the mother plant or glutamate derived in situ.     

Cellobiose dehydrogenase from the ligninolytic basidiomycete Phlebia lindtneri

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in the culture supernatant of the selective delignifier Phlebia lindtneri maintained on a cellulose-based liquid medium. Cellobiose dehydrogenase was purified to homogeneity by a rapid procedure, using ammonium sulfate precipitation, ion-exchange chromatography, and chromatofocusing. The enzyme was recovered with a 61.2 fold increased specific activity and a yield of 47.5%. As determined by SDS-PAGE, the molecular mass of the purified enzyme was found to be 104.5 kDa and its isoelectric point was 4.0. The carbohydrate content of the purified enzymes was 22%. In this work, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from fungi Phlebia lidnteri were isolated, cloned, and characterized. The 2319 bp full-length cDNA of cdh1 encoded a mature CDH protein containing 755 amino acids, which was preceded by a signal peptide of 17 amino acids. The deduced protein sequence of cdh1 shared significant similarity with other known fungal cellobiose dehydrogenase.