Sugar Assay Research Articles

SCARECROW, SCR‐LIKE 23 and SHORT‐ROOT control bundle sheath cell fate and function in Arabidopsis thaliana

Bundle sheath (BS) cells form a single cell layer surrounding the vascular tissue in leaves. In C3 plants, photosynthesis occurs in both the BS and mesophyll cells, but the BS cells are the major sites of photosynthesis in C4 plants, whereas the mesophyll cells are only involved in CO2 fixation. Because C4 plants are more efficient photosynthetically, introduction of the C4 mechanism into C3 plants is considered a key strategy to improve crop yield. One prerequisite for such C3-to-C4 engineering is the ability to manipulate the number and physiology of the BS cells, but the molecular basis of BS cell-fate specification remains unclear. Here we report that mutations in three GRAS family transcription factors, SHORT-ROOT (SHR), SCARECROW (SCR) and SCARECROW-LIKE 23 (SCL23), affect BS cell fate in Arabidopsis thaliana. SCR and SCL23 are expressed specifically in the BS cells and act redundantly in BS cell-fate specification, but their expression pattern and function diverge at later stages of leaf development. Using ChIP-chip experiments and sugar assays, we show that SCR is primarily involved in sugar transport whereas SCL23 functions in mineral transport. SHR is also essential for BS cell-fate specification, but it is expressed in the central vascular tissue. However, the SHR protein moves into the BS cells, where it directly regulates SCR and SCL23 expression. SHR, SCR and SCL23 homologs are present in many plant species, suggesting that this developmental pathway for BS cell-fate specification is likely to be evolutionarily conserved.

The effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce somatic embryos

Somatic embryogenesis (SE) represents a useful experimental system for studying the regulatory mechanisms of embryo development. In this study, the effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce [Picea abies (L.) Karst] somatic embryos were investigated. Using time lapse photography, we monitored development from proliferation of proembryogenic masses (PEMs) to maturation of somatic embryos in two P. abies cell lines cultured on two maturation treatments. A combination of sugar assays, metabolic and proteomic analyses were used to quantify storage reserves in the mature somatic embryos. The maturation treatment containing a nonpermeating osmoticum, polyethylene glycol (PEG, 7.5%) and maltose (3%) as the carbohydrate gave significantly high maturation and low germination frequencies of somatic embryos compared to the treatment with only 3% sucrose. Somatic embryos treated with 3% sucrose contained high levels of sucrose, raffinose and late embryogenesis abundant (LEA) proteins. These compounds are known to be involved in the acquisition of desiccation tolerance during seed development and maturation. In addition the sucrose treatment significantly increased the content of starch in the somatic embryos while the maltose and PEG treatment resulted in somatic embryos with a high content of storage proteins. The high levels of sucrose, raffinose and LEA proteins in the embryos treated with 3% sucrose suggest that sucrose may improve the germination of somatic embryos by promoting the acquisition of desiccation tolerance.

Measurement of the distribution of non-structural carbohydrate composition in onion populations by a high-throughput microplate enzymatic assay

Non-structural carbohydrate (NSC; glucose, fructose, sucrose and fructan) composition of onions (Allium cepa L.) varies widely and is a key determinant of market usage. To analyse the physiology and genetics of onion carbohydrate metabolism and to enable selective breeding, an inexpensive, reliable and practicable sugar assay is required to phenotype large numbers of samples. A rapid, reliable and cost-effective microplate-based assay was developed for NSC analysis in onions and used to characterise variation in tissue hexose, sucrose and fructan content in open-pollinated breeding populations and in mapping populations developed from a wide onion cross. Sucrose measured in microplates employing maltase as a hydrolytic enzyme was in agreement with HPLC-PAD results. The method revealed significant variation in bulb fructan content within open-pollinated 'Pukekohe Longkeeper' breeding populations over a threefold range. Very wide segregation from 80 to 600 g kg(-1) in fructan content was observed in bulbs of F2 genetic mapping populations from the wide onion cross 'Nasik Red × CUDH2150'. The microplate enzymatic assay is a reliable and practicable method for onion sugar analysis for genetics, breeding and food technology. Open-pollinated onion populations may harbour extensive within-population variability in carbohydrate content, which may be quantified and exploited using this method. The phenotypic data obtained from genetic mapping populations show that the method is well suited to detailed genetic and physiological analysis.

An Extra Peptide within the Catalytic Module of a β-Agarase Affects the Agarose Degradation Pattern

Agarase hydrolyzes agarose into a series of oligosaccharides with repeating disaccharide units. The glycoside hydrolase (GH) module of agarase is known to be responsible for its catalytic activity. However, variations in the composition of the GH module and its effects on enzymatic functions have been minimally elucidated. The agaG4 gene, cloned from the genome of the agarolytic Flammeovirga strain MY04, encodes a 503-amino acid protein, AgaG4. Compared with elucidated agarases, AgaG4 contains an extra peptide (Asn(246)-Gly(302)) within its GH module. Heterologously expressed AgaG4 (recombinant AgaG4; rAgaG4) was determined to be an endo-type β-agarase. The protein degraded agarose into neoagarotetraose and neoagarohexaose at a final molar ratio of 1.5:1. Neoagarooctaose was the smallest substrate for rAgaG4, whereas neoagarotetraose was the minimal degradation product. Removing the extra fragment from the GH module led to the inability of the mutant (rAgaG4-T57) to degrade neoagarooctaose, and the final degradation products of agarose by the truncated protein were neoagarotetraose, neoagarohexaose, and neoagarooctaose at a final molar ratio of 2.7:2.8:1. The optimal temperature for agarose degradation also decreased to 40 °C for this mutant. Bioinformatic analysis suggested that tyrosine 276 within the extra fragment was a candidate active site residue for the enzymatic activity. Site-swapping experiments of Tyr(276) to 19 various other amino acids demonstrated that the characteristics of this residue were crucial for the AgaG4 degradation of agarose and the cleavage pattern of substrate.

C-3. ピリドキサ-ル・亜鉛イオンによるアミノ糖およびアミノ酸の蛍光分析

C-3. ピリドキサ-ル・亜鉛イオンによるアミノ糖およびアミノ酸の蛍光分析

Identification and assay of carbohydrates in olive drupes by cesium attachment electrospray tandem mass spectrometry (ESI‐MS/MS)

Identification and assay of sugars in olive drupes during their ripening phase is evaluated by tandem mass spectrometry. Recent achievements have shown that the quality of an extra virgin olive oil can be directly correlated to the ripening degree of the drupes that is likely linked to their saccharides content. An innovation in this peculiar food chain is now proposed that considers a high throughput assessment of the carbohydrate content by a recently introduced protocol based on the gas-phase chemistry of sugar-cesium adducts by ESI-MS/MS.

Polyethylene glycol versus dual sugar assay for gastrointestinal permeability analysis: is it time to choose?

BackgroundIncreased intestinal permeability is an important measure of disease activity and prognosis. Currently, many permeability tests are available and no consensus has been reached as to which test is most suitable. The aim of this study was to compare urinary probe excretion and accuracy of a polyethylene glycol (PEG) assay and dual sugar assay in a double-blinded crossover study to evaluate probe excretion and the accuracy of both tests.MethodsGastrointestinal permeability was measured in nine volunteers using PEG 400, PEG 1500, and PEG 3350 or lactulose-rhamnose. On 4 separate days, permeability was analyzed after oral intake of placebo or indomethacin, a drug known to increase intestinal permeability. Plasma intestinal fatty acid binding protein and calprotectin levels were determined to verify compromised intestinal integrity after indomethacin consumption. Urinary samples were collected at baseline, hourly up to 5 hours after probe intake, and between 5 and 24 hours. Urinary excretion of PEG and sugars was determined using high-pressure liquid chromatography-evaporative light scattering detection and liquid chromatography-mass spectrometry, respectively.ResultsIntake of indomethacin increased plasma intestinal fatty acid-binding protein and calprotectin levels, reflecting loss of intestinal integrity and inflammation. In this state of indomethacin-induced gastrointestinal compromise, urinary excretion of the three PEG probes and lactulose increased compared with placebo. Urinary PEG 400 excretion, the PEG 3350/PEG 400 ratio, and the lactulose/rhamnose ratio could accurately detect indomethacin-induced increases in gastrointestinal permeability, especially within 2 hours of probe intake.ConclusionHourly urinary excretion and diagnostic accuracy of PEG and sugar probes show high concordance for detection of indomethacin-induced increases in gastrointestinal permeability. This comparative study improves our knowledge of permeability analysis in man by providing a clear overview of both tests and demonstrates equivalent performance in the current setting.

Silver nanoparticles: a potential nanocatalyst for the rapid degradation of starch hydrolysis by α-amylase

Silver nanoparticles (AgNPs) are proven to be an effective catalytic material for various applications due to their excellent optical and electronic properties. In this paper, we describe a novel approach for the degradation of starch using the catalytic behaviour of AgNPs in an enzyme catalysed reaction of starch hydrolysis by α-amylase. AgNPs were synthesized by soluble starch reducing silver nitrate to silver atoms. An increase of 4.7-fold in reducing sugar formation and 1.5 times faster enzyme activity confirmed the catalytic activity of AgNPs as a nanocatalyst. Surprisingly, starch degradation tests revealed that 9.9mg of starch was hydrolysed within 5min, which corroborates with the reducing sugar assay. In short, the present study paves way for the faster degradation of starch by immobilizing the enzyme onto the surface of the AgNP, which could be a promising application in the food industry.

Lateral Laparoscopic Approach to Pancreatic Tail Insulinomas

ABSTRACT Pancreatic endocrine tumors are relatively rare lesions and laparoscopic surgery is being increasingly used, especially for insulinomas because of their relatively small size and low incidence of malignancy. Laparoscopic approach to pancreatic tumors has been described in the supine position, transomentally via the lesser sac with anterior stomach retraction. We propose a simplified lateral laparoscopic approach to insulinomas localized preoperatively to the tail or distal body of pancreas. Four patients with pancreatic tail insulinomas underwent laparoscopic surgery between November 2006 and February 2008. Diagnosis was confirmed by fasting sugar, insulin and proinsulin assays. Lesions were localized by multiphasic CT scan/MRI scan and endoscopic ultrasound. All these cases had definitely identifiable enhancing lesions in the distal body/tail in relation to the splenic hilum that appeared accessible by a lateral approach. Except for the first case which was done through the traditional supine approach, the other cases were done by the lateral approach. The patients were positioned right lateral with a kidney bridge. Four subcostal ports were placed and the left colon and spleen with pancreatic tail were mobilised in the same fashion as for splenectomy or adrenalectomy. Tumors were easily identifiable corresponding to the imaging studies. Laparoscopic enucleation was successfully completed in all four patients with lesions in the tail of pancreas, one by the traditional approach and other three by the proposed lateral approach. One patient had associated splenectomy because of the proximity of the lesion to the splenic vessels. Two patients had minor pancreatic leak managed conservatively. The left lateral transperitoneal laparoscopic approach to insulinomas located in the tail of pancreas is feasible and safe. The procedure can be done with ease by surgeons who are familiar with adrenalectomy and splenectomy. How to cite this article Thomas JK, Abraham D, Joseph P, Paul MJ. Lateral Laparoscopic Approach to Pancreatic Tail Insulinomas. World J Endocr Surg 2012;4(1):3-7.

In vitro assessment of three fibrolytic enzyme preparations as potential feed additives in equine diets

A series of in vitro experiments were conducted to assess three fibrolytic enzyme preparations as potential feed additives in equine diets. The three fibrolytic enzyme preparations were a concentrated cellulase (E1), an acid cellulase (E2) and a concentrated xylanase (E3). The enzymes were evaluated on their ability to modify the cell wall fraction of high-temperature dried lucerne (HTL) under various experimental conditions including differences in temperature, pH, incubation period, substrate levels and particle size to enable selection of the enzyme preparation most effective in the hydrolysis of lucerne. Results showed enzyme activities (as measured by reducing sugar assays) to be greatest at 50°C, pH 5 and over an incubation period of greater than 20h. E1 exhibited the greatest effect on total monosaccharide release from the HTL compared to E2 and E3. Moreover, dry matter (DM) and total non-starch polysaccharide (TNSP) losses were also greater in HTL treated with E1 compared to E2 and E3. Therefore, since the cell wall fraction of HTL contained substantial amounts of cellulose, the enzyme with the highest cellulase activity (Enzyme 1) was most effective in hydrolysing the cell walls of HTL. Consequently, it would appear that the application of exogenous fibrolytic enzyme preparations to forages requires the chemical characterisation of the target forage to enable selection of enzymes that are (a) most suitable to degrade the cell wall components of the candidate forage and (b) effective under field conditions.

Grafting cyclodextrins to calcium phosphate ceramics for biomedical applications

The grafting of hydroxyapatite/beta-tricalcium phosphate with β-cyclodextrin was achieved using a two step reaction with (3-glycidyloxypropyl)trimethoxysilane as a linker. Firstly, the silane group was brought to react with the hydroxyl groups at the surface of the hydroxyapatite/beta-tricalcium phosphate and secondly, the epoxy group was brought to react with the β-cyclodextrin. The amount and distribution of covalently bound β-cyclodextrin were investigated by thermogravimetric analysis, colorimetric total sugar assay, fluorescence spectroscopy and confocal laser scanning microscopy. The concentration of grafted β-cyclodextrin was found to be approximately 3.5 μmol per gram of material corresponding to an almost complete surface coverage, when assuming a closed hexagonal packed monolayer.

An Easy and Practical Method for Detection and Estimation of Microcrystalline Cellulose in Pasteurized Milk

Microcrystalline cellulose (MCC) is suspected to be a new adulteration in pasteurized milk in China, yet an efficient method for MCC detection in dairy has not been established. This study presents a novel procedure to detect and estimate MCC in pasteurized milk using dialysis, cellulase hydrolysis, and a reducing sugar assay. The background value of reducing sugar was eliminated by dialysis, and cellulase activity toward MCC was stable in dialyzed milk. A criterion for MCC detection and an empirical formula for MCC estimation were summarized based on the reducing sugar variation after hydrolysis. The detection sensitivity was below 0.5 g/L. Reducing sugar distribution after cellulase-catalyzed hydrolysis was examined by HPLC, and revealed that most of the detected sugar was glucose. This paper describes a practical method for detection of MCC in pasteurized milk that might benefit dairy QC.

Identification of plant tissues from the gut of Phlebotomus papatasi by DNA analysis

Sugar and cellulose assays can determine if an insect has fed on sugar and plant tissue but they cannot identify the species of source plant. In this study, we used DNA analysis together with traditional cellulose and sugar assays to examine the plant feeding habits of wild Phlebotomus papatasi during the dry season in the Lower Jordan Valley, Israel. About 37% of the sand flies were positive for sugar, 45% were positive for cellulose in the gut, and 41% contained amplifiable plant DNA. All of the plant DNA fragments sequenced from both males and females were identified as Suaeda asphaltica.

Feeding activity pattern in a parasitic wasp when foraging in the field

AbstractIndividual decision‐making and behavioral plasticity, and hence reproductive success, depend on nutritional state. Despite the importance of food for life‐history functions, when and how often parasitoids encounter and consume food in natural settings remain largely unknown. In this study, we aimed at determining the food intake history of the Venturia canescens parasitoids in field conditions. To fulfill this objective, we compared the nutritional state of lab individuals of known feeding history and of field‐released individuals allowed to freely forage under natural conditions. Nutritional state and feeding history were determined by using a combination of physiological parameters based on sugar assays. Field‐released wasps were caught during periods starting either 5 or 24 h after release. Our results show that in the field, the wasps search actively for carbohydrate food sources. Our work also indicates that feeding activity of wasps in the field can be better understood by using the combination of key physiological parameters. The adaptive value of the feeding pattern and the relevance of the approach used are discussed.

A multifaceted hemolymph defense against predation in Diabrotica virgifera virgifera larvae

The physical and chemical aspects of Diabrotica virgifera virgifera larval hemolymph were quantitatively assessed against two predatory beetle species in the laboratory. Adult Poecilus cupreus and Harpalus pensylvanicus (Coleoptera: Carabidae) were fed pupae, second or third instar D. v. virgifera or a palatable surrogate prey, i.e., Calliphora vicina or Sarcophaga bullata larvae (Diptera: Calliphoridae, Sarcophagidae, respectively) of equivalent size. The ethanol-soluble fraction of third instar D. v. virgifera hemolymph was extracted and suspended in a 0.24 M sucrose solution and offered to H. pensylvanicus (using a sucrose only control for comparison). The mean duration until first consumption was recorded for each predator, as was the amount of time spent eating, cleaning, resting, or walking for 2 min post-attack (or 5 min for the sugar assay). Maggots and D. virgifera larvae and pupae were attacked equally by both predators. But upon attack, D. v. virgifera larval hemolymph coagulated onto the mouthparts of the predators, which they began vigorously cleaning. Predators ate the sucrose solution for significantly longer than hemolymph + sucrose solution, indicating the presence of deterrent chemicals in the hemolymph. This research suggests that D. v. virgifera larvae are defended from predation by sticky and repellent hemolymph. We hypothesize that this defense partially explains the widespread success of D. v. virgifera as an invasive pest.

Assay and heterologous expression in Pichia pastoris of plant cell wall type-II membrane anchored glycosyltransferases

Two Arabidopsis xylosyltransferases, designated RGXT1 and RGXT2, were recently expressed in Baculovirus transfected insect cells and by use of the free sugar assay shown to catalyse transfer of D-xylose from UDP-α-D-xylose to L-fucose and derivatives hereof. We have now examined expression of RGXT1 and RGXT2 in Pichia pastoris and compared the two expression systems. Pichia transformants, expressing soluble, secreted forms of RGXT1 and RGXT2 with an N- or C-terminal Flag-tag, accumulated recombinant, hyper-glycosylated proteins at levels between 6 and 16 mg protein • L-1 in the media fractions. When incubated with 0.5 M L-fucose and UDP-D-xylose all four RGXT1 and RGXT2 variants catalyzed transfer of D-xylose onto L-fucose with estimated turnover numbers between 0.15 and 0.3 sec-1, thus demonstrating that a free C-terminus is not required for activity. N- and O-glycanase treatment resulted in deglycosylation of all four proteins, and this caused a loss of xylosyltransferase activity for the C-terminally but not the N-terminally Flag-tagged proteins. The RGXT1 and RGXT2 proteins displayed an absolute requirement for Mn2+ and were active over a broad pH range. Simple dialysis of media fractions or purification on phenyl Sepharose columns increased enzyme activities 2-8 fold enabling direct verification of the product formed in crude assay mixtures using electrospray ionization mass spectrometry. Pichia expressed and dialysed RGXT variants yielded activities within the range 0.011 to 0.013 U (1 U = 1 nmol conversion of substrate • min-1 • µl medium-1) similar to those of RGXT1 and RGXT2 expressed in Baculovirus transfected insect Sf9 cells. In summary, the data presented suggest that Pichia is an attractive host candidate for expression of plant glycosyltransferases.

Aminoglycoside antibiotics may interfere with microbial amino sugar analysis

The amino sugars (e.g., glucosamine, galactosamine, mannosamine, muramic acid) in soils are frequently employed as biomarkers of microbial residues. The analysis of amino sugars in environmental matrices, however, is expected to be more complicated than their determination in isolated microbial cells. In this study, we employed a widely used protocol for amino sugar analysis, and found that some aminoglycoside antibiotics interfere with amino sugar quantification in vitro. The method converts the aminoglycosides to compounds that coelute with the aldononitrile acetate derivatives of the amino sugars. Specifically, streptomycin significantly interferes with muramic acid analysis, and kanamycin, tobramycin and amikacin hamper glucosamine measurement. Mass spectrometry confirmed that the interfering compounds from aminoglycosides are not actually genuine microbial amino sugar monomers (bacterial muramic acid or fungal glucosamine), and are most likely to be N-methyl glucosamine or 3-amino-3-deoxy-glucopyranose. In contrast to their effects on muramic acid and glucosamine analyses, aminoglycosides do not interfere with galactosamine and mannosamine quantification. The few data that exist on the environmental occurrence of aminoglycoside antibiotics suggest they occur at only trace levels. Our findings may have implications for the qualitative and quantitative validity of results from amino sugar assays in some context. Application of the aldononitrile acetate derivatization method to samples (especially in selective microbial cultures using aminoglycosides as inhibitors) requires that potential interference be evaluated.

Influence of Microgravity Environment on Root Growth, Soluble Sugars, and Starch Concentration of Sweetpotato Stem Cuttings

Because sweetpotato [Ipomoea batatas (L.) Lam.] stem cuttings regenerate very easily and quickly, a study of their early growth and development in microgravity could be useful to an understanding of morphological changes that might occur under such conditions for crops that are propagated vegetatively. An experiment was conducted aboard a U.S. Space Shuttle to investigate the impact of microgravity on root growth, distribution of amyloplasts in the root cells, and on the concentration of soluble sugars and starch in the stems of sweetpotatoes. Twelve stem cuttings of 'Whatley/Loretan' sweetpotato (5 cm long) with three to four nodes were grown in each of two plant growth units filled with a nutrient agarose medium impregnated with a half-strength Hoagland solution. One plant growth unit was flown on Space Shuttle Colombia for 5 days, whereas the other remained on the ground as a control. The cuttings were received within 2 h postflight and, along with ground controls, processed in approximately 45 min. Adventitious roots were counted, measured, and fixed for electron microscopy and stems frozen for starch and sugar assays. Air samples were collected from the headspace of each plant growth unit for postflight determination of carbon dioxide, oxygen, and ethylene levels. All stem cuttings produced adventitious roots and growth was quite vigorous in both ground-based and flight samples and, except for a slight browning of some root tips in the flight samples, all stem cuttings appeared normal. The roots on the flight cuttings tended to grow in random directions. Also, stem cuttings grown in microgravity had more roots and greater total root length than ground-based controls. Amyloplasts in root cap cells of ground-based controls were evenly sedimented toward one end compared with a more random distribution in the flight samples. The concentration of soluble sugars, glucose, fructose, and sucrose and total starch concentration were all substantially greater in the stems of flight samples than those found in the ground-based samples. Carbon dioxide levels were 50% greater and oxygen marginally lower in the flight plants, whereas ethylene levels were similar and averaged less than 10 nL.L (-1). Despite the greater accumulation of carbohydrates in the stems, and greater root growth in the flight cuttings, overall results showed minimal differences in cell development between space flight and ground-based tissues. This suggests that the space flight environment did not adversely impact sweetpotato metabolism and that vegetative cuttings should be an acceptable approach for propagating sweetpotato plants for space applications.

ORIGINAL RESEARCH: Endocellulolytic activity of theClostridium thermocellumCel9C (formerly CbhA) catalytic domain

CbhA and CelK are family-9 glycosyl hydrolases (β-1,4-glucanase) isolated from the bacterium Clostridium thermocellum.1 The members of the glycosyl hydrolase (GH) family-9 have been almost exclusively endocellulolytic, with a few being processive endoglucanses (enzymes demonstrating both endo- and exocellulolytic activity).2 CbhA and CelK are the only members of the family reported to be solely exohydrolytic. We report activity assays on various substrates, soluble to insoluble reducing sugar assays, viscosity measurements, and thinlayer chromatography product analysis, which all showed that the truncated CbhA, expressed in E. coli, acts as an endocellulase, like most other family-9 glycosyl hydrolases.

Blood glucose monitor: an alternative off-line method to measure glucose concentration during fermentations with Trichoderma reesei

Two home, blood-glucose monitoring meters, OneTouch Ultra and Ascensia Contour, were used to determine the glucose concentration during fermentations of Trichoderma reesei in both flasks and bioreactors. The results, when compared to those given by the 3,5-dinitrosalicylic acid reducing sugar assay, HPLC and YSI 2700 SELECT Biochemistry analyzer, showed that the glucose meters are a quick, reliable and economical alternative method for frequent glucose concentration measurement during fermentation. For T. reesei fermentations, the OneTouch meter was the more suitable.