Simultaneous Quantification of Five Stereoisomeric Hexoses in Nine Biological Matrices Using Ultrahigh Performance Liquid Chromatography with Tandem Mass Spectrometry

Stereoisomeric hexoses are present in almost all biological organisms in the forms of aldoses and ketoses,with diverse physi-ological and pathophysiological functions.Accurate and simultaneous quantification is vital for understanding their functions individually.However,such analysis remains challenging owing to their highly similar behavior in chromatography and mass spectrometry.By combining the pre-column 3-nitrophenylhydrazine derivatization and ultrahigh performance liquid chroma-tography tandem mass spectrometry(UHPLC-MS/MS),here,we developed a method for simultaneous quantification of five important stereoisomeric hexoses including D-glucose,D-galactose,D-mannose,D-fructose and L-sorbose representing both aldoses and ketoses.The method achieved baseline-separation for all these five derivatized hexoses chromatographically and had high sensitivity(LOD,femtomole on column),excellent linearity(R2>0.995)and efficiency with stable-isotope dilution.With this method,we further quantified these hexoses in nine biological matrices including human biofluids(serum,urine and saliva),human cells,human and mouse feces,rat liver tissue,mung-bean seeds and peach pulp.The results provided quantitative data for these hexoses in multiple biological samples and showed significant concentration diversity for these hexoses in different biological samples,which demonstrated the applicability of the method for simultaneous quantification of these hexose phenotypes of biological systems.

Protective effect of Cardiospermum halicacabum leaf extract on glycoprotein components on STZ-induced hyperglycemic rats

Objective:To investigate the protective role of Cardiospermum halicacabum(C.halicacabum) leaf extract on glycoprotein metabolism in streptozotocin(STZ)-induced diabetic rats.Methods: Diabetes was induced in male albino Wistar rats by intraperitonial administration of STZ.The C.halicacabum leaf extract(CHE) was administered orally to normal and STZ-diabetic rats for 45 days.The effects of C.halicacabum leaf extract(CHE) on plasma and tissue glycoproteins (hexose,hexosamine,fucose and sialic acid) were determined.Results:The levels of plasma and tissues glycoproteins containing hexose,hexosamine and fucose were significantly increased in STZ-induced diabetic rats.In addition,the level of sialic acid significantly increased in plasma and liver while decreased in kidney of STZ-induced diabetic rats.After administration of CHE to diabetic rats,the metabolic alteration of glycoprotein reverted towards normal levels. Conclusions:The present study indicates that the CHE possesses a protective effect on abnormal glycoprotein metabolism in addition to its antihyperglycemic activity.

Carbon utilization profile of a thermophilic fungus, <i>Thermomyces lanuginosus</i>using phenotypic microarray

The thermophilic filamentous fungus, Thermomyces lanuginosus produces the largest amount of xylanase reported. In addition to this, it expresses large amount of other enzymes that have been used industrially or have academic interest. Thus, this fungus has a potential to be applied for biomass conversion to produce biofuel or other applications. In this study, the Biolog system was used to characterize the utilisation and growth of T. lanuginosus on 95 carbon sources. The carbohydrates based compounds, both single sugars and oligosaccharide, showed the best utilisation profile, with the pentose sugar xylose inducing the highest growth, followed by trehelose, raffinose, D-mannose turanose fructose and glucose. Among oligosaccharides, sucrose had the highest mycelium formation followed by stachyose, maltose, maltotriose, glycogen and dextrin. Interestingly the fungus also grew well on cellobiose suggesting that this fungus can produce cellulose hydrolysing proteins. D-alanine was the best amino acid to promote fungal growth while the effect of other amino acids tested was similar to the control. These results demonstrate the ability of this fungus to grow relatively well on most plant based compounds thus making this fungus a possible candidate for plant biomass conversion which can be applied to a number of biotechnological applications including biofuel production.

Immunohistochemical Study of Hexose Transporters GLUT-2 and GLUT-5 in Birds Gastrointestinal Tract

The hexoses glucose, galactose and fructose serve as important dietary energy sources in animals and glucose plays a central role in energy homeostasis within eucaryotic cells. As relatively little is known about patterns of hexose transporters expression in birds gastrointestinal tract, the aim of the study was to examine glucose transporters 2 （GLUT-2） and glucose transporters 5 （GLUT-5） expression in stomach and duodenal epithelium of two different species--broilers （Gallus gallus domesticus） and ostriches （Struthio camelus var. domesticus） chicken. Materials from the two parts of gastrointestinal tract were collected from six 7-day-old female broilers and six 7-day-old female ostriches. Specimens were fixed with 10% formalin, embedded into paraffin, cut into 7 lain thick slices, followed by immunohistochemical staining with polyclonal primary antibodies rabbit anti-GLUT-2 and rabbit anti-GLUT-5; the staining was carried out according to the manufacturers guidelines （IHC kit, Abcam, UK）. The investigation showed that the staining for both antibodies was more intensive in the epithelial cells of stomach and duodenum of the 7-day-old broilers. In duodenal epithelium, goblet cells and brush border membranes were stained in both species, however the signal was stronger for GLUT-5 than GLUT-2. Staining for GLUT-2 and GLUT-5 occurred in different parts of gastrointestinal tract of 7-day-old ostriches, but was weaker compared to 7-day-old broilers, which showed that the gastrointestinal tract of 7-day-old female broilers was more developed for transportation ofhexoses than 7-day-old female ostriches.

Molecular and Kinetic Characterization of the Fermentative Behavior of Saccharomyces cerevisiae Strains Isolated from North Patagonia

At present, Argentina does not count with a production of indigenous yeast strains with suitable technological and oenological features to be used in the regional winery industry. Isolation and molecular characterization of these microorganisms and its fermentation attributes would be relevant to the sustainable development of the activity in the country and to recognize and preserve the biodiversity of the region. Eight strains isolated from grapes and musts from the North Patagonian region and genetically identified as Saccharomyces cerevisiae, were studied for their fermentation behavior, emphasizing in hexose transport through the plasma membrane, which is the limiting step of the process. Thus, sugar consumption profiles were analyzed in different media at laboratory scale, to be subsequently applied to the fermentation of natural musts. Three of the eight initial strains were selected, named NNM10, NIF8 and NMN16 according to their fermentation profiles. The expression of hexose transporters during fermentations revealed interesting differences in the response of each strain to sugar consumption, where transporters HXT2 and HXT5 showed significant changes in expression in Patagonian strains, which are normally associated to endurance to culture stress conditions. The results obtained by combining the characteristics studied, at molecular and physiological level, are extremely encouraging. Native strain NMN16, showed a high potential for application in local winemaking. Assays carried out on a pilot scale will determine the feasibility of applying this strain with promising technological features at industrial scale.

Diverse Functional Roles of Monosaccharide Transporters and their Homologs in Vascular Plants： A Physiological Perspective

Vascular plants contain two gene families that encode monosaccharide transporter proteins. The classical monosaccharide transporter（-like） gene superfamily is large and functionally diverse, while the recently identified SWEET transporter family is smaller and, thus far, only found to transport glucose. These transporters play essential roles at many levels, ranging from organelles to the whole plant. Many family members are essential for cellular homeostasis and reproductive success. Although most transporters do not directly participate in long-distance transport, their indirect roles greatly impact carbon allocation and transport flux to the heterotrophic tissues of the plant. Functional characterization of some members from both gene families has revealed their diverse roles in carbohydrate partitioning, phloem function, resource allocation, plant defense, and sugar signaling. This review highlights the broad impacts and implications of monosaccharide transport by describing some of the functional roles of the monosaccharide transporter（-like） superfamily and the SWEET transporter family.

Functional Characterization and RNAi-Mediated Suppression Reveals Roles for Hexose Transporters in Sugar Accumulation by Tomato Fruit

Hexoses accumulate to high concentrations （-200 mM） in storage parenchyma cells of tomato fruit. Hexoses are sourced from the fruit apoplasm as hydrolysis products of phloem-imported sucrose. Three hexose transporters （LeHT1, LeHT2, LeHT3）, expressed in fruit storage parenchyma ceils, may contribute to hexose uptake by these cells. An analysis of their full-length sequences demonstrated that all three transporters belong to the STP subfamily of monosaccharide transporters that localize to plasma membranes. Heterologous expression of LeHT1 （and previously LeHT2, Gear et al., 2000）, but not LeHT3, rescued a hexose transportimpaired yeast mutant when raised on glucose or fructose as the sole carbon source. Biochemically, LeHT1, similarly to LeHT2, exhibited transport properties consistent with a high- affinity glucose/H^＋ symporter. Significantly, LeHT1 and LeHT2 also functioned as low-affinity fructose/H^＋ symporters with apparent Km values commensurate with those of fruit tissues. A substantial reduction （80-90%） in fruit expression levels of all LeHT genes by RNAi-mediated knockdown caused a 55% decrease in fruit hexose accumulation. In contrast, photoassimilate production by source leaves and phloem transport capacity to fruit were unaffected by transporter knockdown. Collectively, these findings demonstrate that LeHTs play key roles in driving accumulation of hexoses into storage parenchyma cells during tomato fruit development.

Post-translational inhibitory regulation of acid invertase induced by fructose and glucose in developing apple fruit

Acid invertase (EC 3.2.1.26) is one of the key enzymes involved in the carbohydrate sink-organ development and the sink strength modulation in crops. The experiment conducted with 'Starkrimson' apple (Malus domestica Borkh) fruit showed that, during the fruit development, the activity of acid invertase gradually declined concomitantly with the progressive accumulation of fructose, glucose and sucrose, while Western blotting assay of acid invertase detected a 30 ku peptide of which the immuno-signal intensity increased during the fruit development. The immuno-localization via immunogold electron microscopy showed that, on the one hand, acid invertase was mainly located on the flesh cell wall with numbers of the immunosignals present in the vacuole at the late stage of fruit development; and on the other hand, the amount of acid invertase increased during fruit development, which was consistent with the results of Western blotting. The in vivo pre-incubation of fruit discs with soluble sugars showed that the activity of extractible acid invertase was inhibited by fructose or glucose, while Western blotting did not detect any changes in apparent quantity of the enzyme nor other peptides than 30 ku one. So it is considered that fructose and glucose induced the post-translational or translocational inhibitory regulation of acid invertase in developing apple fruit. The mechanism of the post-translational inhibition was shown different from both the two previously reported ones that proposed either the inhibition by hexose products in the in vitro chemical reaction equilibrium system or the inhibition by the proteinaceous inhibitors. It was hypothesized that fructose and glucose might induce acid invertase inhibition by modulating the expression of some inhibition-related genes or some structural modification of acid invertase.

NEW WAVE-DEPENDENT FORMULAE FOR SEA SPRAY FLUX AT AIR-SEA INTERFACE

A bubble-derived Sea Spray Generation Function （SSGF） was deduced, which was integrated into the new wave-dependent SSGF. The effect of sea spray feedback was parameterized by the feedback coefficients A and B. The relationship of A（B） and the wind-sea Reynolds number was derived, which shows that the feedback effect of sea spray is decreasing with the wave developing. New sea spray flux formulae, including the new SSGF and new feedback coefficients, were proposed, which can reveal clearly the influence of wave state on sea spray fluxes. Spray fluxes in the Humidity Exchange Over the Sea （HEXOS） experiment were simulated again, and results show that spray momentum flux is small in this experiment, but spray heat flux is large. Comparisons of the sea spray fluxes calculated by two kinds of formulae in different wave states show that previous wave-independent spray flux formulae are the special cases of the presented new wave dependent formulae for sea spray flnx.