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The cell wall (CW) of fungi exhibits a complex structure and a characteristic chemical composition consisting almost entirely of interacting crystalline and amorphous polysaccharides. These are synthesized by a number of sugar polymerases and depolymerases encoded by a high proportion of the fungal genome (for instance, 20% in Saccharomyces cerevisiae ). These enzymes act in an exquisitely coordinated process to assemble the tridimensional and the functional structure of the wall. Apart from playing a critical role in morphogenesis, cell protection, viability and pathogenesis, the CW represents a potential target for antifungals as most of its constituents do not exist in humans. Chitin, β-glucans and cellulose are the most frequent crystalline polymers found in the fungal CW. The hexosamine biosynthesis pathway (HBP) is critical for CW elaboration. Also known as the Leloir pathway, this pathway ends with the formation of UDP-N-GlcNAc after four enzymatic steps that start with fructose-6-phosphate and L-glutamine in a short deviation of glycolysis. This activated aminosugar is used for the synthesis of a large variety of biomacromolecules in a vast number of organisms including bacteria, fungi, insects, crustaceans and mammalian cells. The first reaction of the HBP is catalyzed by GlcN-6-P synthase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16), a critical enzyme that has been considered as a potential target for antifungals. The enzyme regulates the amount of cell UDP-N-GlcNAc and in eukaryotes is feedback inhibited by the activated aminosugar and other factors. The native and recombinant forms of GlcN-6-P synthase has been purified and characterized from both prokaryotic and eukaryotic organisms and demonstrated its critical role in CW remodeling and morphogenesis after exposure of some fungi to agents that stress the cell surface by interacting with wall polymers. This review deals with some of the cell compensatory responses of fungi to wall damage induced by Congo Red and Calcofluor White., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ortiz-Ramírez, Cuéllar-Cruz and López-Romero.)

The present study investigated the therapeutic effect of curcumin on bleomycin (BLM)-induced alterations in glycoprotein components in the fibrotic lungs. Analysis of the bronchoalveolar lavage fluid (BALF) demonstrated increased fibronectin content at 3, 5, 7, and 14 days after BLM administration. Similarly, lung tissue fibronectin content revealed a progressive increase at various times (days 3, 5, 7, 14, and 28) during the development of lung fibrosis. In addition, alveolar macrophage release of fibronectin was also elevated in BLM-treated rats. Analysis of carbohydrate moieties of glycoproteins revealed an increase in total hexose, fucose, sialic acid and hexosamine levels at 7, 14, and 28 days after BLM treatment. Furthermore, the activities of lung glycosidases such as N-acetyl-β-D-glucosaminidase, β-glucosidase, β-galactosidase, and β-fucosidase in the fibrotic rats were elevated. Importantly, curcumin significantly inhibited the BLM-induced increases in BALF and lung fibronectin levels. Treatment of BLM rats with curcumin dramatically suppressed alveolar macrophage release of fibronectin. Curcumin also inhibited the increases in complex carbohydrates and glycosidases in the fibrotic lungs. These findings suggest that BLM-induced lung fibrosis is associated with accumulation of glycoproteins, and curcumin has the ability to suppress the enhanced deposition of glycoproteins in the fibrotic lung.

To detect and characterise compounds in complex matrices, it is often necessary to separate the compound of interest from the matrix before analysis. In our previous work, we have developed the coupling of supercritical fluid chromatography (SFC) with nuclear magnetic resonance (NMR) spectroscopy for the analysis of nonpolar samples [Van Zelst et al., Anal. Chem., 2018, 90, 10457]. In this work, the SFC-NMR setup was successfully adapted to analyse polar samples in complex matrices. In-line SFC-NMR analysis of two N-acetylhexosamine stereoisomers was demonstrated, namely N-acetyl-mannosamine (ManNAc) and N-acetyl-glucosamine (GlcNAc). ManNAc is a metabolite that is present at elevated concentrations in patients suffering from NANS-mediated disease. With our SFC-NMR setup it was possible to distinguish between the polar stereoisomers. Until now, this was not possible with the standard mass-based analysis techniques. The concentrations that are needed in the SFC-NMR setup are currently too high to be able to detect ManNAc in patient samples (1.7 mM vs. 0.7 mM). However, several adaptations to the current setup will make this possible in the future.

Lactobacillus farciminis CIP 103136 is a bacterial strain with recognized probiotic properties. However, the mechanisms underlying such properties have only been partially elucidated. In this study, we isolated and purified a cell-wall associated polysaccharide (CWPS), and evaluated its biological role in vitro. The structure of CWPS and responses from stimulation of (i) human macrophage-like THP-1 cells, (ii) human embryonal kidney (HEK293) cells stably transfected with Toll-like receptors (TLR2 or TLR4) and (iii) human colonocyte-like T84 intestinal epithelial cells, upon exposure to CWPS were studied. The structure of the purified CWPS from L. farciminis CIP 103136 was analyzed by nuclear magnetic resonance (NMR), MALDI-TOF-TOF MS, and methylation analyses in its native form and following Smith degradation. It was shown to be a novel branched polysaccharide, composed of linear backbone of trisaccharide repeating units of: [→6αGlcpNAc1 → 4βManpNAc1 → 4βGlcpNAc1→] highly substituted with single residues of αGlcp, αGalp and αGlcpNAc. Subsequently, the lack of pro- or anti-inflammatory properties of CWPS was established on macrophage-like THP-1 cells. In addition, CWPS failed to modulate cell signaling pathways dependent of TLR2 and TLR4 in transfected HEK-cells. Finally, in T84 cells, CWPS neither influenced intestinal barrier integrity under basal conditions nor prevented TNF-α/IFN-γ cytokine-mediated epithelium impairment.

Valienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs. It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer. A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux. The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008. Valienone was derivatized by 2, 4-dinitrophenylhydrazine (DNPH) in 10 mmol·L -1 H 3 PO 4 at 37 °C for 45 min and the derivatives were separated on Eclipse XDB-C 18 (5 μm, 4.6 mm × 150 mm) column at 30 °C eluted with 50% acetonitrile for 18 min. The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by computational studies. The method was shown to be effective, sensitive, and reliable. Good linearity was found in the range of 5-2 000 μg·mL -1 . The intra- and inter-day precisions were 1.1%-2.7% and 1.7%-2.2%, respectively. The absolute recovery of the spiked samples was 97.2%-102.6%. To date, this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium. This method successfully helped evaluate the valienone production capability of the engineered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods., (Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Antibiotic industrial dregs, generated from the production of antibiotics by fermentation, are banned in China as animal feed additives. Official monitoring programs require the analysis of feeds for possible illegal use of the dregs. A rapid and sensitive method was developed for the simultaneous determination of streptomycin and streptidine as markers for streptomycin industrial dregs in pig and poultry compound feeds. After extraction with 20% aqueous trichloroacetic acid and pH adjustment, sample cleanup was performed by weak cation-exchange solid-phase extraction. UPLC-ESI-MS/MS was carried out using a hydrophilic interaction chromatography(HILIC)column to achieve separation. Quantification required matrix-matched calibrations in a linear range of 50-1000μgkg -1 ; the calibration curves were linear in this range with coefficients of determination of 0.991 and 0.994 for streptomycin and streptidine, respectively. The method validity parameters-LODs (20μgkg -1 ) and LOQs (50μgkg -1 ), recoveries (71-78% and 75-84%, respectively), and relative reproducibility (5.4-9.6%)-satisfy the requirements of routine analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Metabolic sugar labeling followed by the use of reagent-free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Herein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N-azidoacetylmannosamine (Ac4 ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac4 ManAz-loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor-localized ultrasound pulses. The released Ac4 ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac4 ManAz-loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A sensitive and efficient method of high performance liquid chromatography using 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) as pre-column derivatization reagent coupled with UV detection (HPLC-UV) and online mass spectrometry identification was established for determination of the most common N-Acetylhexosamines (N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-galactosamine (GalNAc)) and N-acetylneuraminic acid (Neu5Ac). In order to obtain the highest liberation level of the three monosaccharides without destruction of Neu5Ac or conversion of GlcNAc/GalNAc to GlcN/GalN in the hydrolysis procedure, the pivotal parameters affecting the liberation of N-acetylhexosamines/Neu5Ac from sample were investigated with response surface methodology (RSM). Under the optimized condition, maximum yield was obtained. The effects of key parameters on derivatization, separation and detection were also investigated. At optimized conditions, three monosaccharides were labeled fast and entirely, and all derivatives exhibited a good baseline resolution and high detection sensitivity. The developed method was linear over the calibration range 0.25-12μM, with R(2)>0.9991. The detection limits of the method were between 0.48 and 2.01pmol. Intra- and inter-day precisions for the three monosaccharides (GlcNAc, GalNAc and Neu5Ac) were found to be in the range of 3.07-4.02% and 3.69-4.67%, respectively. Individual monosaccharide recovery from spiked milk was in the range of 81%-97%. The sensitivity of the method, the facility of the derivatization procedure and the reliability of the hydrolysis conditions suggest the proposed method has a high potential for utilization in routine trace N-acetylhexosamines and Neu5Ac analysis in biological samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Elevated levels of the second messenger c-di-GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food-borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface-bound. Secreted carbohydrates represent exclusively cell-wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a β-1,4-linked N-acetylmannosamine chain decorated with terminal α-1,6-linked galactose. All genes of the pssA-E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS-specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS-mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c-di-GMP-dependent EPS production., (© 2015 John Wiley & Sons Ltd.)

Metabolomics is the identification and quantitation of small bio-molecules (metabolites) in biological samples under various environmental and genetic conditions. Mass spectrometry provides the unique opportunity for targeted identification and quantification of known metabolites by selective reaction monitoring (SRM). However, reproducibility of this approach depends on careful consideration of sample preparation, chemical classes, and stability of metabolites to be evaluated. Herein, we introduce and validate a targeted metabolite profiling workflow for cultured cells and tissues by liquid chromatography-triple quadrupole tandem mass spectrometry. The method requires a one-step extraction of water-soluble metabolites and targeted analysis of central metabolites that include glycolysis, amino acids, nucleotides, citric acid cycle, and the hexosamine biosynthetic pathway. The sensitivity, reproducibility and molecular stability of each targeted metabolite were assessed under experimental conditions. Quantitation of metabolites by peak area ratio was linear with a dilution over a 4 fold dynamic range with minimal deviation R(2)=0.98. Inter- and intra-day precision with cells and tissues had an average coefficient of variation <15% for cultured cell lines, and somewhat higher for mouse liver tissues. The method applied in triplicate measurements readily distinguished immortalized cells from malignant cells, as well as mouse littermates based on their hepatic metabolic profiles., (Copyright © 2014. Published by Elsevier B.V.)

Aims: Exopolysaccharides (EPS) are industrially valuable molecules with numerous useful properties. This study describes the techniques used for the identification of a novel Vibrio bacterium and preliminary characterization of its EPS., Methods and Results: Bioprospection in marine intertidal areas of New Caledonia followed by screening for EPS producing brought to selection of the isolate NC470. Phylogenetic analysis (biochemical tests, gene sequencing and DNA-DNA relatedness) permitted to identify NC470 as a new member of the Vibrio genus. The EPS was produced in batch fermentation, purified using the ultrafiltration process and analysed by colorimetry, Fourier Transform Infrared spectroscopy, gas chromatography, Nuclear Magnetic Resonance and HPLC-size exclusion chromatography. This EPS exhibits a high N-acetyl-hexosamines and uronic acid content with a low amount of neutral sugar. The molecular mass was 672 × 10(3)  Da. These data are relevant for possible technological exploitation., Conclusions: We propose the name Vibrio neocaledonicus sp. nov for this isolate NC470, producing an EPS with an unusual sugar composition. Comparison with other known polymers permitted to select applications for this polymer., Significance and Impact of the Study: This study contributes to evaluate the marine biodiversity of New Caledonia. It also highlights the biotechnological potential of New Caledonia marine bacteria., (© 2013 The Society for Applied Microbiology.)

Sugar coated: We recently developed methylcyclopropenes as low-molecular-weight tetrazine coupling partners. Here, we demonstrate that methylcyclopropenes can meet the stringent steric demands required for metabolic imaging of unnatural mannosamines on live cells. Using sequential azide-alkyne chemistry, we also demonstrate multicolor imaging of two different metabolically incorporated unnatural sugars., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Streptomycin, the classical aminoglycoside antibiotic, generally considered the most basic drug compound was characterized in terms of protonation macro- and microconstants. ¹H NMR-pH and ¹H-¹³C HSQC-pH titrations were carried out on streptomycin and streptidine, a symmetrical constituent compound of reduced complexity to monitor the proton-binding processes of the basic sites. Accurate, undistorted, electrodeless pH measurement was ensured by a new set of in tube indicators. The microscopic protonation constants of the two guanidino groups of streptomycin were calculated by evaluating the various NMR-pH data and transferring the pair-interactivity parameter from streptidine to streptomycin. Inherent guanidino basicities fall in the range of 13.03-13.39 log k units, which drop to 12.48-12.85 upon protonation of the other site. pH-dependent distribution of the major microspecies and charge-related biological consequences are provided., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

A simple and rapid CZE method was established for the simultaneous determination of valienamine, acarbose and validamycin A, using a 20-kV CZE with the detection wavelength of 193 nm and 50 mM phosphoric acid-20 mM Tris (pH 5.3) as a running buffer. The calibration curves of valienamine, acarbose, and validamycin A showed a good linear relationship at a concentration range of 5-1000 microg/mL. The detection limits of valienamine, acarbose, and validamycin A were 0.3, 0.6, and 0.6 microg/mL, respectively, and the average recoveries of each of the above were 99.9, 99.5, and 100.3%. The method has been successfully applied for simultaneous determination of substrate and product in the process of preparation of valienamine.

Oligonol is a polyphenol formulation enriched with catechin-type oligomers. As an initial approach to assess the chemopreventive potential of Oligonol, the study investigated the effects of Oligonol on the inhibition of lung metastasis induced by B16F-10 melanoma cells in C57BL/6 mice. Oligonol, which is abundantly found in plants, vegetables and fruits, was found to possess antimetastatic activity against B16F-10 melanoma cells. Continued consumption of Oligonol, even at high doses, for long periods does not seem to cause any toxic symptoms because excess Oligonol is stored in adipose tissue rather than in the liver. However, the mechanism by which Oligonol exerts its antimetastatic activity remains unclear. Further investigations are required to clarify the exact role of Oligonol in such B16F-10 melanoma regulation., (Copyright 2009 John Wiley & Sons, Ltd.)

The objective of this investigation was to determine the efficacy of crocetin in preventing lung tumorigenesis in mice. We evaluated crocetin in Swiss albino mice treated with the tobacco-specific carcinogen benzo(a)pyrene [B(a)P] for their ability to inhibit pulmonary adenoma formation and growth. Male Swiss albino mice (7 weeks old) were given 100 mg/kg B(a)P by i.p. injection, and 4 or 14 weeks later, they were given crocetin 50 mg/kg by i.p. injection 3 days/week. Crocetin (50 mg/kg body weight) reduced proliferating cells by 68% and 45% in 18 and 8 weeks of treatment respectively. The levels of glycoproteins and polyamines were significantly altered in the B(a)P-induced animals than in crocetin treatment groups. The activity of crocetin was more pronounced in the cancer. Taken together, these results indicate that crocetin was capable of inhibiting proliferation cells by inhibiting proliferating cells, glycoprotein and polyamine synthesis., ((c) 2008 John Wiley & Sons, Ltd.)

The effects of oral and topical application of Calendula officinalis flower extract on excision wounds made in rats were checked. The parameters assessed were the days needed for re-epithelization and percentage of wound closure. The hydroxy proline and hexosamine content in the granuloma tissue of the wound was also measured. The percentage of wound closure was 90.0% in the extract-treated group, whereas the control group showed only 51.1% on the eighth day of wounding (p < .01). The days needed for re-epithelization were 17.7 for the control animals; extract treatment at a dose of 20 or 100 mg/kg b.wt reduced the period to 14 and 13 days, respectively. A significant increase was observed in the hydroxy proline and hexosamine content in the extract-treated group compared with the untreated animals. The data indicate potent wound healing activity ofC. officinalis extract.

Recent investigations have implicated aberrant glycosylations in various malignancies, including epithelial ovarian cancer (EOC). The protocol here identifies O-linked carbohydrate patterns in EOC plasma glycoproteins through chemical cleavage and purification of these glycans. Dialyzed plasma is subjected to reductive beta-elimination with alkaline borohydride to release O-linked oligosaccharides from glycoproteins. Enrichment of released glycans, as well as removal of peptide and other contaminants, is followed by carbohydrate pattern analysis with MALDI-FT-ICR-MS.

The official method for the determination of the composition and related substances of gentamicin prescribed by the European Pharmacopoeia (Ph. Eur.) is liquid chromatography combined with pulsed electrochemical detection (LC-PED). However, this method utilizes a polymer stationary phase which shows rather low efficiency towards the separation of the main gentamicin components. Moreover, the mobile phase contains a lot of non volatile salts: sodium sulphate and sodium octanesulphonate. Following a comparative study, the most performant LC-PED method has been evaluated and validated using a reversed phase C18 column (C18, 250 x 4.6mm ID, 110 A, 5 microm) kept at 35 degrees C with a mobile phase containing volatile ion pairing agents: trifluoroacetic acetic acid (TFA) and pentafluoropropionic acid (PFPA). In addition to the selectivity of the main gentamicin components and its related substances, the method is repeatable, linear and proves to be robust. It is also applicable to a wider number of C18 columns.

This study was an investigation of the antimetastatic activity of amentoflavone using B16F-10 melanoma-induced experimental lung metastasis in C57BL/6 mice. Amentoflavone treatment significantly reduced tumor nodule formation accompanied by reduced lung collagen hydroxyproline, hexosamine, and uronic acid levels. Serum sialic acid and gammaglutamyl transpeptidase levels were also significantly inhibited after amentoflavone treatment. Amentoflavone treatment up-regulated the lung tissue inhibitor of metalloprotease-1 and tissue inhibitor of metalloprotease-2 expression. The cytokine profile and growth factors such as interleukin-1beta , interleukin-6, tumor necrosis factor-alpha, granulocyte monocyte- colony stimulating factor, vascular endothelial growth factor, interleukin-2, and tissue inhibitor of metalloprotease-1 in the serum of these animals were markedly altered after amentoflavone treatment. This altered level of cytokines after amentoflavone treatment was also accompanied by enhanced natural killer cell antibody-dependent cellular cytotoxicity. The study reveals that amentoflavone treatment could alter proinflammatory cytokine production and could inhibit the activation and nuclear translocation of p65, p50, c-Rel subunits of nuclear factor-kappaB, and other transcription factors such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response element-binding protein in B16F-10 melanoma cells.

The purpose of the study was to investigate the effects of L-carnitine (CA) on the susceptibility of erythrocyte (RBC) to peroxide-induced lipid oxidation, RBC membrane composition, ATPases activity and oxidative stress in fructose-fed hyperinsulinemic rats. The rats were subjected to experimental hyperinsulinemia and hyperglycemia by feeding a high fructose diet (60 g/100 g) for 6 weeks. The rats showed significant alterations in the RBC membrane composition. The protein content was lower than control animals, while cholesterol, phospholipids and free fatty acids were higher in fructose-fed animals. Significant differences in the total carbohydrate and relative proportions of hexose, hexosamine, sialic acid and fucose of membranes were observed. In these rats, membrane-bound ATPases (total ATPase, Na+, K+ ATPase, Mg2+ and Ca2+ ATPases) were significantly lower while thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LHP) in RBC membrane were significantly higher than those of control rats. The red cells were more susceptible to peroxide-induced oxidative stress that correlated with reduced levels of vitamin E found RBC membrane. When fructose-diet fed rats were treated simultaneously with CA (300 mg/kg b.w/day, i.p.), such alterations in membrane composition and enzyme activities did not occur. Effects of fructose loading on lipid peroxidation was also alleviated by CA. These findings suggest that high levels of dietary fructose is detrimental to RBC membrane integrity and that CA may have membrane stabilizing effects in this diet-induced model of type 2-diabetes.

A simple and valid thin-layer chromatographic method for the separation and quantitative determination of valienamine and validamine is described. The two compounds are separated using a Silica gel G plate as the stationary phase and a mixture of 1-PrOH-AcOH-H2O (4:1:1, v/v/v) as the mobile phase. The plate is developed for 1 h at 25 degrees C and dried by a hairdrier, then immersed in 0.1% ninhydrin aqueous solution and heated for 5 min at 121 degrees C. The reacted spots are scanned with a single wavelength at 420 nm in the measurement mode of absorption. The limits of detection of the two compounds are both 0.4 microg. The responses of the densitometry are highly correlated with the amounts of valienamine and validamine in the range of 0.4-2.8 pg. Moreover, the method shows good accuracy and high precision.

Carbohydrate residues on membrane proteins from sperm are important in gamete interaction. In recent years, Arylsulfatase A (AS-A) has been acquiring an important role from the various putative gamete interaction responsibles in sperm. The aim of this study was to determine if the capacitated boar sperm Arylsulfatase-A (AS-A), contains D-mannose, N-acetylglucosamine and/or sialic acid residues by its purification using affinity chromatography with Concanavalia ensiformis Agglutinin(Con-A) or Wheat Germ Agglutinin (WGA) as ligands. Sperm samples were capacitated in TALP-HEPES medium. Protein extract was added to the affinity columns. Sequencing of retained proteins was done after SDS-PAGE. Total capacitated sperm proteins electrophoresis showed molecular masses between 14 kDa and 102 kDa. A major band of 68 kDa, and 2 minor bands of 52 kDa and 47 kDa were observed. They were AS-A, hyaluronidase and lactadherin, respectively. The Con-A-retained proteins (RP) pattern showed bands from 14 to 98 kDa. After sequencing and BLAST analysis, the 62 kDa band corresponded to Arylsulfatase-A. The WGA RP fraction showed bands from 14 to 100 kDa. The 65 kDa band corresponded to AS-A. This study showed that AS-A has mannose, N-acetylglucosamine and/or sialic acid residues as part of its glycosilation. In this study AS-A was isolated from boar capacitated sperm by affinity chromatography using separately Con-A and WGA, indicating that there are mannose, N-acetylglucosamine and/or sialic acid residues in its glycosilation. AS-A is a membrane protein of capacitated sperm. Further investigation is needed to fully characterize the glycosidic residues bore by AS-A and to determine its function.

The present investigation was undertaken to determine the efficacy of topical administration of flavone of sea buckthorn (Hippophae rhamnoides L.) on cutaneous wound healing in rats. Four full-thickness excision wounds were created on the back of rat and 1.0% w/v flavone prepared in propylene glycol was applied topically. Control animals received the vehicle alone in an identical manner. The healing of the wound was assessed by the rate of wound contraction, period of epithelialization, hydroxyproline, hexosamine, antioxidants estimation and histopathology of the granulation tissue. The sea buckthorn flavone promoted the wound healing activity as indicated by improved rate of wound contraction, decreased time taken for epithelialization (16.3 days versus 24.8 days in controls) and significant increase in hydroxyproline (26.0%) and hexosamine (30.0%) content. These findings were also confirmed by histopathological examinations. In addition, it was observed that sea buckthorn flavone possesses potent antioxidant properties as evidenced by significant increase in reduced glutathione (55.0%), vitamin C (70.0%) and catalase (20.0%) activities in wound granulation tissue. The flavone treatment also resulted in significant decrease in lipid peroxide levels (39.0%). The results suggest that the sea buckthorn flavone promotes wound healing activity.

Lung cancer is one of the leading causes of cancer death in the world and is notoriously difficult to treat effectively. In the present study, male Swiss albino mice were divided into five groups of six animals each: group I animals received corn oil orally and served as a control; group II cancer-induced animals received benzo(a)pyrene (50 mg/kg bodyweight dissolved in corn oil, orally) twice weekly for four successive weeks; group III cancer-bearing animals (after 12 weeks of induction) were treated with paclitaxel (33 mg/kg bodyweight, i.p.) once weekly for 4 weeks; group IV cancer-bearing animals were treated with paclitaxel along with Withania somnifera (400 mg/kg bodyweight) orally once weekly for 4 weeks; and group V animals constituted the drug control treated with paclitaxel along with W. somnifera. The serum, lung and liver were investigated biochemically for aryl hydrocarbon hydroxylase, gamma-glutamyl transpeptidase, 5'-nucleotidase, lactate dehydrogenase and protein-bound carbohydrate components (hexose, hexosamine and sialic acid). These enzyme activities were increased significantly in cancer-bearing animals compared with control animals. The elevation of these in cancer-bearing animals was indicative of the persistent deteriorating effect of benzo(a)pyrene in cancer-bearing animals. Our data suggest that paclitaxel, administered with W. somnifera, may extend its chemotherapeutic effect through modulating protein-bound carbohydrate levels and marker enzymes, as they are indicators of cancer. The combination of paclitaxel with W. somnifera could effectively treat the benzo(a)pyrene-induced lung cancer in mice by offering protection from reactive oxygen species damage and also by suppressing cell proliferation.

Total glycolipid content of Saccharomyces cerevisiae cells increased in ethanol-treated yeast cells. Sialic acid and hexosamine contents of glycolipids from ethanol-treated cells decreased, whereas those of hexoses increased. Increased sialidase activity in the presence of ethanol may be responsible for the decrease in sialic acid content of glycolipids. The saccharide moieties of glycolipids of S. cerevisiae consisted of fucose, mannose, galactose, and glucose. Ethanol treatment of yeast cells caused an increase in glucose and a decrease in galactose content of glycolipids. The changes in glucose content can be related to changes in beta-glucosidase activity under alcohol stress. The content of cerebrosides, sulfatides, and monoglucosyldiglycerides was enhanced following ethanol treatment. An increase in cerebroside as well as in sulfatide content during alcohol stress might play an important role in stabilizing the membrane both physically and structurally. Such variations in glycolipid content and composition of S. cerevisiae cells may represent an adaptive response to ethanol stress.

A reversed-phase high-performance liquid chromatography method for the quantitative analysis of valienamine in the microbial degradation of validamycin A, using a procedure for pre-column derivatization of valienamine with p-nitrofluorobenzene is described. Valienamine in the broth was first isolated with the ion-exchange method. The optimized conditions for the derivatization were the reaction time 30 min and reaction temperature 100 degrees C. With the mobile phases consisting of acetonitrile-water (12:88) (eluent A) and methanol (eluent B), the gradient was carried out with 100% of A for 15 min and then 100% of B for another 10 min. The parameters in the process were the flow rate of the mobile phase 1.0 ml/min, the injection volume 20 microl, the column temperature 40 degrees C and wavelength of ultraviolet detection 398 nm in all runs. A good linearity was found in the range of 0.5-150.0 microg/ml. Both intra- and inter-day precisions of valienamine, expressed as the relative standard deviation, were less than 9.4%. Accuracy, expressed as the relative error, range from -0.5 to 2.7%. The mean absolute recovery of valienamine at three different concentrations was 94.2%. The method was proved suitable for the study on the process of microbial degradation of validamycin A to produce valienamine.

An efficient synthesis of valienamine is described. Valienamine was synthesized starting from commercially available 2,3,4,6-tetra-O-benzyl-D-glucose in nine steps, using ring-closing metathesis of (4S,5S,6S)-4,5,6-tribenzyloxy-7-(benzyloxymethyl)octa-1,7-dien-3-ol as a key step.

During the hydrolysis of an exopolysaccharide (EPS) produced by Citrobacter sp., the maximum liberation of hexosamine was obtained with 6 M HCl at 115 degrees 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.

This review provides a chronological survey of over fifty fluorescent chemosensors for carbohydrates from the period between 1992 to the present. The survey contains only those sensors that are synthetic or chemosensory, utilize boronic acids and display a fluorescence response in the form of intensity changes or shifts in wavelength. With each compound listed, a description of the saccharide probe is given with regard to concentration, excitation and emission wavelengths, pH and solvent mixture proportions. In addition, the selectivity of each chemosensor is provided as well as the trends in binding constants. Where possible, a description of the fluorescence signaling mechanism is given as well as commentary on the probe's unique features within this class of sensors.

A simple method to differentiate underivatized diastereomeric hexosamine monosaccharides, glucosamine, galactosamine, and mannosamine is reported by applying the kinetic method using N-acetylhexosamines or naturally occurring amino acids as reference bases under electrospray ionization conditions. The observed differences to distinguish the diastereomeric hexosamines are found mainly due to the proton affinity (PA) differences between the analyte and the reference base. The PA values of the hexosamines are not available in the literature, and hence, we estimated them by the kinetic method using N-acetylhexosamines as reference bases. The determined PA values are 223.97 kcal/mol for glucosamine, 224.99 kcal/mol for mannosamine, and 224.71 kcal/mol for galactosamine. The similar PA values were also obtained by using amino acids as reference bases. We have applied the same methodology to quantify these hexosamines in a mixture following the three-point calibration method suggested in the literature.

The objective of this study was to find the changes in glycoprotein composition in both diabetic and non-diabetic patients with and without cardiovascular complications. The study was carried out in Ziauddin Medical University Karachi, Pakistan. Eighty-three patients and control subjects were selected. Among them twenty-one were diabetic patients without any clinical evidence of chronic diabetic complications, twenty-one were diabetic patients with cardiovascular complications, twenty were non-diabetic patients with cardiovascular complications and twenty-one apparently normal, age, sex and weight matched control subjects were investigated. All these patients were selected on clinical grounds from National Institute of Cardiovascular Disease, Karachi. Fasting plasma glucose was increased in all diabetic patients and correlated significantly with and without cardiovascular complications. Fasting plasma glucose, glycosylated haemoglobin, glycosylated plasma proteins, serum fructosamine, sialic acid, hexosamine and total serum protein and its fractions were increased in diabetic patients with and without cardiovascular complications. Fasting plasma glucose, glycosylated haemoglobin, glycosylated plasma proteins, serum fructosamine, sialic acid and hexosamine were not different in diabetic patients with cardiovascular complications and diabetic patients without chronic complications as compared with control subjects. In conclusion, fasting plasma glucose, glycosylated haemoglobin, glycosylated plasma proteins, serum fructosamine, sialic acid, hexosamine and total serum proteins and its fractions were increased in diabetic patients with and without complications, but these parameters remained within normal limits in non-diabetic patients with cardiovascular complications.

Human vitreous gel is a special type of extracellular matrix, in which interpenetrating networks of collagen fibrils and hyaluronan are found. In this study, we report that apart from significant amounts of collagen, hyaluronan and sialylated glycoproteins, it was found that the human vitreous gel also contained low amounts of versican-like proteoglycan. The concentration of versican-like proteoglycan in the whole vitreous is 0.06 mg protein/ml of vitreous gel and represents a small percentage (about 5%) of the total protein content. The versican-like proteoglycan has a molecular mass of 380 kDa, as estimated by gel chromatography. Its core protein is substituted by chondroitin sulphate side chains (average molecular weight 37 kDa), in which 6-sulphated disaccharides predominated. According to the physicochemical data, the number of chondroitin sulphate chains is likely to be 5-7 per molecule. These proteoglycan monomers form large aggregates with endogenous hyaluronan. Versican, which is able to bind lectins via its C-terminal region, may bridge or interconnect various constituents of the extracellular matrix via its terminal domains in order to stabilize large supramolecular complexes at the vitreous, contributing towards the integrity and specific properties of the tissue.

The aim of this study was to assess the influence of epidermal growth factor (EGF) on glycoprotein biosynthesis in portal hypertensive (PHT) gastric mucosa. Portal-vein ligation (PVL) for a period of 4 weeks was applied to 40 male Wistar rats to produce experimental portal hypertension. The rats were subdivided into four groups. Human EGF was administrated to these four groups of animals at a does of 0, 10, 25, and 50 microg/kg/day for 7 days. An additional group of 10 rats without PVL and EGF pretreatment was employed as a control. The severity of gross gastric mucosal lesions was evaluated macroscopically by a gross ulcer index. Glycoprotein biosynthesis of the gastric mucosa was determined by the incorporation rate of [(3)H]glucosamine. Quantitative changes of gastric mucosal hexosamines were also used for mucosal glycoproteins analyses. The gross mucosal damage was considerably greater in the PVL group without EGF pretreatment than in the EGF-pretreated groups (p <.05). The incorporation rate of [(3)H]glucosamine was significantly higher in the control group and the EGF-pretreated groups than in the PVL group without EGF pretreatment (p <.05). Moreover, the incorporation rate of [(3)H]glucosamine and the gastric mucosal hexosamine content were closely relevant to administration does of human EGF (p <.001). In addition, the reduction of glycoprotein biosynthesis was closely related to the increase in portal pressure (p =.001) and the severity of portal hypertensive gastropathy (p <.001). Our current study shows that the rate of incorporation of glucosamine is decreased in the PHT gastric mucosa and that EGF significantly stimulated glycoprotein synthesis in the PHT gastric mucosa. Accordingly, these findings may be helpful to explain the protective effect of EGF on the PHT gastric mucosa via increased glycoprotein biosynthesis in the stomach.

Oro-maxillofacial diseases may influence structure and function of salivary glands. In this study, 32 hamsters were treated with topical application of 7,12-dimethylbenzanthracene (DMBA) on the buccal pouch. After 16 weeks, the animals were killed and the major salivary glands extracted. The activities of some lysosomal glycosidases and their natural substrates were measured to understand how the carcinogenetic stress and the inflammatory reaction could influence the physiology of the salivary glands. Large differences were observed in lysosomal activities among treated and untreated animals. Similarly, large differences were shown in the concentration of natural substrates, including sialic acids. These results suggest that inflammation and/or tumors induce profound changes in the biology of the salivary glands.

This study indicated that human normal rectum (HNR) and human rectum carcinoma (HRC) contained three populations of proteoglycans (PGs). About 63% of the HNR PGs, in terms of uronic acid, were heparan sulfate proteoglycans (HSPGs) of M(r) 500,000 with HS side-chains of M(r) 35,000. The other two populations were versican (29%) and decorin (8%) of M(r) 715,000 and 90,000, respectively, bearing mainly dermatan sulfate (DS) (73%) and chondroitin sulfate (CS) (27%) chains of M(r) 24-26,000 and 20-22,000, respectively. In contrast, in terms of uronic acid, HRC contained 2-fold amounts of PGs. The majority of these PGs (87%) were versican and decorin of lower hydrodynamic size (500,000 and 70,000, respectively) than in HNR, with CS as prominent GAG (70%) in both types of PG. The M(r) of CS and DS chains in these PGs was 12-14,000 and 14-16,000, respectively. The remaining portion (13%) of PG was HSPGs of lower hydrodynamic size (300,000) with smaller HS chains (29,000) than HSPGs of HNR. Moreover, the molar concentrations of versican and decorin estimated from PG-derived protein contents represented a significant, but disproportionate increase, about 5-fold and 8-fold, respectively. The sulfation pattern of rectum carcinoma-associated versican and decorin was significantly altered mainly in containing (62%) 6-sulfated disaccharides and a significant proportion (10%) of non-sulfated disaccharides. DS chains of the tumor-associated versican and decorin contained decreased amounts of iduronic acid. On the metabolic level, the abnormally high production of versican and decorin in this malignant tumor suggests a dramatic modification in their biosynthetic steps at both translational and posttranslational levels.

Renal involvement in systemic lupus erythematosus (SLE) affects the disease outcome. In order to advance the diagnosis and the initiation of therapy, non-invasive diagnostic techniques are required. In this study, urinary glycosaminoglycans (GAG) and heparan sulphate (HS) were measured in 26 patients with biopsy-proven lupus nephritis and compared to 16 healthy controls. Uronic acid as a representative of GAGs in urine was determined spectrophotometrically with the meta-hydroxydiphenyl, following acid treatment. HS was determined as hexosamine by the method of Smith and Gilkerson. The median values of GAG (3.99 mg/g crea./day) and HS (2.41 mg/g crea./day) in patients were significantly ( P = 0.001) higher than in the control group (1.98 and 0.87, respectively). There was a positive correlation between GAG and HS values ( P = 0.000, r = 0.924) in SLE patients. There were no differences in HS excretion, microalbuminuria and SLE-DAI scores between different classes of lupus nephritis. However, GAG values in class 3 nephritis were significantly ( P = 0.033) higher than from both class 2 and class 4 lupus nephritis. There were no differences in all the measured parameters between normoalbuminuric, microalbuminuric and macroproteinuric patients. Furthermore, there were no correlations between GAG, HS excretions and SLE-DAI scores or microalbuminuria. These results suggest that urinary GAG and HS may serve as useful, independent and non-invasive markers of lupus nephritis.

Background/aims: Studies of the most important defensive factor, gastric mucus, in the treatment of gastric ulcers in elderly patients have been lacking. Therefore we focused on the changes in gastric mucus during the ulcer-healing process in elderly patients., Methodology: Twenty elderly patients (> or = 65 years old), and 20 younger patients (< 65 years old) with gastric ulcers were administered antisecretory agents for 24 weeks. Biopsies were taken from the antrum and body of the stomach, and the levels of gastric mucosal hexosamine and periodic acid-Schiff-positive gastric mucus were measured., Results: In both groups, the hexosamine levels in the specimens from the body of the stomach declined during the healing process. The decrease was more marked in the elderly, and the recovery of this level was also slower than in the younger group. The periodic acid-Schiff-positive mucosal index was also lower in the elderly. A decrease in body periodic acid-Schiff-positive mucus was seen with treatment in both groups, but recovery was slower in the elderly group., Conclusions: A decrease in gastric mucus, as a gastric mucosal defensive factor, was seen in gastric ulcers in elderly patients. The potential usefulness of the administration of mucosal protective agents for elderly patients with gastric ulcers was suggested.

Under High Aspect Ratio Vessel (HARV) bioreactor culture conditions designed to simulate the microgravity of orbital space flight, insect tissue culture cells infected with a baculovirus expression vector produced a human glycoprotein with tri- and tetra-antennary complex N-linked oligosaccharides containing terminal sialic acid residues. The oligosaccharide structures were similar to those produced in human placental cells. Insect cells cultured in T-flasks only performed incomplete oligosaccharide processing. The mechanism of HARV-mediated changes in the eukaryotic N-linked glycosylation pathway was investigated and could be mimicked under T-flask growth conditions with the addition of N-acetylmannosamine to the culture medium. The significance of these investigations is discussed with respect to the production of recombinant therapeutic glycoproteins, insect physiology, and orbital space flight.

Pretreatment with the butanol extract of the water fraction of Phyllanthus emblica fruits at the dose of 100 mg/kg body-weight, orally administered to rats for 10 consecutive days, was found to enhance secretion of gastric mucus and hexosamine (P<0.001) in the indomethacin induced ulceration of rats. The morphological observations also supported a protective effect of the stomach wall from lesion. The indomethacin treatment of the premedicated animals with the drug hardly affected either the malondialdehyde (MDA) or superoxide dismutase (SOD) level in gastric tissue while the ulcerative agent itself significantly enhanced both the levels. An antioxidant property appears to be predominantly responsible for this cytoprotective action of the drug.

The glycocalyx of the mucosal surface of urinary bladder acts as an effective barrier against invasion by pathogenic microorganisms and injury from toxic substances in the urine. Defects in these bladder mucosal components could thus be important factors in the development of diseases such as interstitial cystitis and lower urinary tract infections. However, information on the nature of glycoconjugates of mammalian bladder mucosa is very limited. In this study, the glycoconjugates of rabbit bladder were examined histochemically using biotinylated lectins with specificities for a variety of carbohydrate moieties. Three [Artocarpus integrifolia (Jacalin), Datura stramonium (DSL), and Maackia amurensis II (MAL-II)] of the lectins bound predominantly to the luminal cell layer, with decreased binding to the basal layers of the epithelium. In contrast, Ricinus communis I and Sambucus nigra lectins did not bind to the cells in the epithelium but strongly interacted with the subepithelial layers, especially the lamina propria. The intensity of the staining by Jacalin and MAL-II was significantly reduced by prior treatment of the bladder sections with O-sialoglycoprotein endopeptidase, indicating that the ligands of these lectins are primarily mucin glycoproteins. In parallel biochemical studies, a high-molecular-weight glycoprotein with characteristics typical of epithelial mucins was purified from the mucosa of rabbit bladder explant cultures metabolically labeled with [(3)H]glucosamine. Quantitative analysis of the sialic acid, uronic acid, and hexosamine contents of delipidated rabbit bladder mucosa revealed a larger proportion of sialoglycoproteins compared with glycosaminoglycans. Taken together, the results of histochemical and biochemical analyses indicate that glycoproteins rather than glycosaminoglycans are the major components of the bladder epithelium, and that the former include a mucin., (Copyright 2000 Academic Press.)

A Gram-positive, cold-adapted, aerobic, spherical actinobacterium (strain T2T) with a quite low cardinal growth temperature was isolated from Chinese Great-Wall station in Antarctica. Sequence comparisons of the 16S rDNA indicated the isolate to be a phylogenetic member of the genus Micrococcus, family Micrococcaceae, in which it represents a novel lineage. The phylogenetic distinctness of the isolate with respect to the type strains Micrococcus luteus and Micrococcus lylae was supported by DNA-DNA similarity values of less than 40%. Chemotaxonomic properties supported the placement of the isolate in the genus Micrococcus. The diagnostic diamino acid of the cell-wall peptidoglycan is lysine. The predominant menaquinones are MK-8 and MK-8(H2). The G + C content of the DNA of the isolate is 66.4 mol%. Genotypic, morphological and physiological characteristics were used to describe a new species of Micrococcus, for which the name Micrococcus antarcticus is proposed. The type strain is T2T (= AS 1.2372T).

A tandem electrospray mass spectrometric (MS(n)) technique for the analysis of N-acetylated hexose carbohydrates using ferrocene boronate (F(c)Bor) derivatization was developed. The biologically important N-acetyl hexosamines can be readily distinguished utilizing this technique. The analysis is made possible by utilizing the inherent electrochemical properties of the electrospray device to produce oxidized, pre-formed single-electron ferrocenyl ions in a non-aqueous solvent system. The electrospray device was modified using a custom built cell consisting of concentric stainless steel tubes, which produced an enhanced signal for the molecular ion of each analyte species. The MS(2) spectra derived from isomeric populations of ferrocenyl boronic esters of these carbohydrates when generated under the same conditions possessed features unique to each sugar allowing easy differentiation between a number of N-acetyl hexosamine isomers., (Copyright 2000 John Wiley & Sons, Ltd.)

In Heteropneustes fossilis, significant annual variations were observed in seminal vesicle-somatic index (SVSI), gonadosomatic index (GSI), concentrations of total proteins, hexosamines, fructose and glucose in both SV and testis, and in plasma testosterone with high values in late prespawning-early spawning phases (June-July) and low or undetectable levels in resting phase (December-January) except for glucose. There is an inverse relationship between the annual patterns of fructose and glucose with fructose dominant in the prespawning and early spawning phases (June-July), and glucose in the resting phase (November-January). The increase in the concentrations of SV and testicular protein, hexosamine and fructose can be correlated with the increase in testosterone concentration on one hand and with the increase of SVSI and GSI, on the other. The decrease in glucose level in the recrudescent phase may be due to its increased conversion into fructose, the main seminal sugar in this species.

Background and Aims: Portal hypertensive gastropathy (PHG) is now recognized as a distinct entity; however, the angiogenesis in the portal hypertensive gastric mucosa has yet to be elucidated. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in both physiological and pathological angiogenesis. The aim of this study was thus to examine the function of VEGF in the portal hypertensive and non-portal hypertensive gastric mucosa., Method: Forty-five cirrhotic patients were divided into 3 groups as follows. Group I included 15 patients without PHG who were treated with 1.5 g teprenone/day for 8 weeks: PHG(-)-t. Group II included 15 patients with PHG who were not treated with teprenone: PHG(+)-n. Group III included 15 patients with PHG who were treated with teprenone for 8 weeks: PGH(+)-t. The gastric mucosal blood flow (GMBF), the concentration of gastric mucosal VEGF and hexosamine and the endoscopic findings were studied both before and after medication., Results: Before teprenone treatment, the GMBF in the antrum, fundus, fornix were significantly higher in PHG(+)-n than PHG(-)-t. After treatment, the GMBF in the fundus and fornix significantly decreased more than before treatment in the PHG(+)-t. After treatment, the GMBF in the antrum increased significantly more than before treatment in PHG(-)-t. The gastric VEGF and hexoxamine concentration in the antrum were significantly higher in PHG(+)-n than in PHG(-)-t. After treatment, the gastric VEGF and hexosamine concentration in the antrum significantly decreased in PHG(+)-t while no change in concentration was recognized in PHG(+)-n. In the endoscopic findings, a decrease in the PHG score was recognized in 2 patients in PHG(+)-t., Conclusion: Portal hypertensive gastric mucosal change was thus found to trigger a high concentration of VEGF and hexosamine. Such increased activity of VEGF and hexosamine may thus account for the presence of active congestion in PHG., (Copyright 2000 S. Karger AG, Basel)

The administration of methallibure (2 microg/g BW, daily for 15 days) in Clarias batrachus in prespawning phase (May-June) resulted in decreased weights of seminal vesicle (SV) and testis, and reductions in the concentrations of total proteins, fructose, hexosamines, and sialic acid in SV and testis. The inhibitory changes can be attributed to impairment of steroidogenesis, serum levels of testosterone and estradiol -17beta decreased significantly. Withdrawal of methallibure treatment for 7 and 15 days resulted in gradual recovery and restoration of all the above parameters except the sialic acid levels in the SV and testis, and fructose level in the SV. The methallibure induced regressive changes in the SV and testis were discussed in the light of its GTH inhibiting property.

A rapid means of stereochemical differentiation and quantification for the hexosamine monosaccharides was achieved using electrospray ionization quadrupole ion trap mass spectrometry. The hexosamine monosaccharides, glucosamine, galactosamine, and mannosamine, were derivatized with [Co(DAP)2Cl2]Cl, and the complex [Co(DAP)2(HexNH2)]Cl was generated. Subjecting this complex to collision-induced dissociation provided a unique product ion spectrum for each of the diastereomeric monosaccharide complexes, thus differentiating the stereoisomers. Furthermore, the stereoisomers were quantified. This was achieved by using the relative abundances of product ions from pure standards and using these values to determine the ratio of isomeric products in a mixture. The utility of this quantification method was demonstrated by successfully determining the composition of two- and three-component mixtures of the hexosamines.