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Role For Sucrose Phosphate Synthase In Intrafiber Cellulose Synthesis

Data suggest that synthesis of sucrose from hexoses within the fiber involves SPS acting coordinately with sucrose phosphate phosphatase. SPS catalyzes the irreversible reaction, Fru-6-P + UDP-Glc Suc-P + UDP, which is the first committed step of the Suc synthesis pathway. SPS exerts a high level of control over this process in leaves (Lunn and MacRae 2003), which is facilitated by extensive regulatory mechanisms (G6P activation, Pi inhibition, phophorylation at multiple sites, and interaction with 14-3-3 proteins) that allow fine control of flux through Suc (Huber and Huber 1996 Winter and Huber 2000). It has recently been shown that SPS is active during high rate cellulose synthesis for secondary wall deposition in heterotrophic cotton fibers and tracheary elements. The in vitro tracheary element differentiation system was particularly useful in showing a strong correlation between increasing SPS activity and episodic highrate secondary wall cellulose synthesis even though the...

Fractionation of Oligonucleosomes on Sucrose Gradients

Prepare sucrose solutions for the linear sucrose gradient Sucrose, 75 12 sucrose solution 50 sucrose solution 2. Prepare linear 12-50 sucrose gradient in 6 polyallomer tubes for a Beckman SW-28 rotor. Fill the chambers of the gradient mixer with 19 mL each of 12 and 50 sucrose solution and dispense into one tube, repeating the procedure six times, or alternatively with 114 mL each sucrose solution and dispense into six tubes simultaneously using a multichannel peristaltic pump.

Production of 3Ketosucrose in a Two Step Reaction

3-Keto-sugars are excreted into the culture medium in batch fermentations. Therefore, the production of 3-keto-disaccharides can be performed directly by isolation of 3-keto-sugars from the culture medium. As they are not easily separated from this medium, a two-step procedure with the use of resting cells was preferred. Resting cells with a high enzyme activity provide the advantage of achieving higher product yields via use of higher cell densities in the reaction mixture. Although different new 3-keto-derivatives were produced by Agrobacterium tumefaciens, we will exemplify the production of 3-keto-disaccharide with sucrose as the substrate investigated most fully.

Sucrose Medium

This solid medium is used for culturing L. mesenteroides ATCC 12291. The medium consists of 10 g L tryptone, 10 g L yeast extract, 10 g L K,HPO4, 20 g L sucrose, 10 mL L vitamin mineral solution, and 20 g L agar at pH 7.5. The pH of the solution is corrected at pH 7.5 by addition of NaOH or HCl prior to sterilization. Sterilization is performed at 121 C for 15 min, after which the filter-sterilized vitamin mineral solution (see Subheading 2.2.2.) is added and Petri dishes are poured with the medium.

Vegetables and Fruits

How nutritious are canned and frozen vegetables and fruits Most frozen produce is processed without cooking, so most of the micronutrient content is conserved. But canned vegetables and fruits undergo a heating process that destroys much of the vitamin C and B vitamins.8 Also, minerals leach out of canned food into the water, and unless the liquid in the can is used in food preparation, the minerals will be lost. Large amounts of sodium are added during the processing and canning of vegetables. Canned fruit is often conserved in heavily sugared water. A fresh peach has about 70 calories a canned peach, with the added sugar, contains about 180 calories. When available, fruit that is conserved in its own juice is preferable.

Effect of Formulation

Sugars are the second major component, after flours, in the formulation of pre-sweetened cereals. In directly expanded products, the total concentration of sugar added during extrusion ranges from 0 to 28 (75). Sugars are added to RTE cereals principally for flavor. However, sugars also contribute to the color, structure, and texture of extruded products (76-79). At the high temperatures of extrusion, reducing sugars, such as glucose, fructose, maltose, and lactose, take part in Maillard browning reactions with the amino groups of proteins and peptides. Depending on the balance of reactants and the conditions of processing, Maillard reactions contribute to the desirable color and flavor of products such as breakfast cereals. Sucrose is the most commonly used and investigated sugar. Replacement of a fraction of the starch-containing material by sucrose changes the extrusion process parameters, such as SME and die pressure, which in turn leads to changes in the structure of the...

Atpdependent Chromatin Remodelling

Abbreviations ATP Adenosine triphosphate ATR-X Alpha-thalassemia mental retardation syndrome, X-Linked BRG1 Brahma-Related Gene 1 CHD Chromodomain, Helicase, DNA-binding ISWI Imitation Switch HDAC Histone deacetylase PCNA Proliferating Cell Nuclear Antigen RSC Remodels the Structure of Chromatin PHD Plant Homeodomain SWI Switch SNF Sucrose Non-Fermenting SNF2H Sucrose Non-fermenting homologue

The Ontogeny of Pain Neurobiology

Models, with the staged development of the rat pup as the accepted model. A rat pup at P7 (day 7 of life) is comparable to a full-term infant and at P21 is comparable to a human adult (23,24). Conflicting research on the induction of endogenous opioid system activity from nutritive and nonnutritive suckling exists. Early studies suggested that orogustatory response to sucrose water caused release of endogenous opioids. Recent studies found no change in measured opioid ligand concentrations after suckling. Furthermore, the ontogeny of opioid receptors and their ligands is not uniform. and k receptors appear much earlier than 5 receptors, and the endogenous opioid ligands (endorphins, enkephalins, and dynorphins) appear before receptors are present (25). In addition, in the rat pup descending inhibitory pathways are present at birth but are not functional before day 10 of life (23). Thus, extrapolating from the animal model, the human preterm or even full-term neonate may have a...

Production Media and Culture Conditions

Whatever the culture collection or mutant strain used, batch alginate production seem to be almost similar and of the order of 6 with alginate yield factors on dry cell (YP X) and sugar consumed (YP S) of about 0.4 g.g-1 and 0.15-0.38 g.g-1, respectively. Continuous alginate production resulted in a yield as great as one g of alginate per g of sugar consumed (24). D. Sucrose 20 g K2HPO4 0.64 g E KH2PO4 0.16 g E. Sucrose 20 g Sodium acetate 2 g NCIB 9068 K2HPO4 1.2 g (mutant) KH2PO4 0.3 g F. Sucrose 20 g NCIB 9068 K2HPO4 32 mg (mutant

Test Methodology 46 Urine Glucose

Glucose may be measured as a reducing substance using the Benedict's copper reduction reaction. Glucose reduces cupric ions to cuprous ions to produce a yellow or red cuprous compound. All reducing sugars will produce a positive result. Other reducing substances, such as ascorbic acid, may also produce positive reactions.

Alteration Of Cellulose Formation In Plants

Alteration of cellulose production in economically important plants using a transgenic approach has been the subject of only a few studies (Hu et al. 1999 Tang and Sturm 1999 Levy et al. 2002 Li et al. 2003a). An antisense approach used to reduce lignin content was reported to increase the proportion of cellulose in aspen stem wood (Hu et al. 1999 Li et al. 2003a). Aside from the fact that some of the apparent increase in cellulose level could be explained by the compensatory effects of nonlignin wall constituents, the estimate for cellulose concentration was derived from sugar composition and not from direct measurements of its crystalline form, making it difficult to discern the compounding effect of glucose present in other forms, such as free sugars. An interaction between cellulose and lignin deposition has also been recognized in Arabidopsis and rice. The eli1-1 and eli1-2 mutants, defective in the AtCesA3 gene, had reduced levels of cellulose and showed aberrant deposition of...

Separation Based Analysis of Heparin and Heparan Sulfate

While initial methods of analysis relied on chemical processes for specifically degrading these sugars, the isolation and characterization of the heparin lyase enzymes (5,6) expanded the analytical tool kit with which these molecules can be studied. These enzymes, based on their substrate specificity, enable breakdown of HP and HS into smaller units that can be analyzed by known separation techniques like high performance liquid chromatography (HPLC). Powerful separation technology is a key element for the analysis of these sugars as they are complex mixtures with many structurally variable components. The ability to detect these components effectively after separation is also another important issue that affects the ability to analyze them. Different separation approaches based upon HPLC, gel electrophoresis and capillary electrophoresis have been developed for the analysis of HP and HS. Various different modes of detection including ultraviolet (UV) absorbance and fluorescence have...

Carbonfixing reaction

1 molecule of glucose and other sugars Glucose can be stored in plants in several ways. In some plants, the glucose molecules are joined to one another to form starch molecules. Potato plants, for example, store starch in tubers (underground stems). In some plants, glucose converts to fructose (fruit sugar), and the energy is stored in this form. In still other plants, fructose combines with glucose to form sucrose, commonly known as table sugar. The energy is stored in carbohydrates in this form. Plant cells obtain energy for their activities from these molecules. Animals use the same forms of glucose by consuming plants and delivering the molecules to their cells.

Desacyl Ghrelin A New Peptide

The increased hydrophobicity of the acyl side-chain may explain why acyl ghrelin circulates bound to larger plasma proteins, particularly HDL species, whereas des-acylated ghrelin circulates as free peptide. This could be important in the transport of ghrelin to centres of appetite control 10 .

Isolation of the N1 N2H3H4 Fraction

5 Sucrose buffer 5 (w v) sucrose, 23 mM KCl, 17 mM NaCl, 0.1 mM EDTA, 2 mM MgCl2, 20 mM HEPES, 1 mM DTT, 10 mg mL phenylmethylsulfonyl fluoride, 2 mg mL leupeptin, 2 mg mL pepstatin, pH 7.5. 3. 20 Sucrose buffer 20 (w v) sucrose, 23 mM KCl, 17 mM NaCl, 0.1 mM EDTA, 2 mM MgCl2, 20 mM HEPES, 1 mM DTT, 10 mg mL phenylmethylsulfonyl fluoride, 2 mg mL leupeptin, 2 mg mL pepstatin, pH 7.5.

Analysis of membrane compartments in the endoplasmic reticulumGolgiplasma membrane pathway

Sucrose-D2 O gradients Before iodinated density gradient media became widely used for analysis of membrane trafficking, one particular strategy to reduce the concentration of sucrose solutions necessary to band subcompartments of the ER and Golgi membrane systems was to dissolve the sucrose in D2O (Protocol 5.7). This allowed gradients of lower osmolality and viscosity to be used 19 . The method was used primarily to study the transfer of proteins from the rough endoplasmic reticulum to the ds-Golgi. The gradient showed that this intercompartmental transport involved a low-density vesicle quite distinct from its source and destination membranes. Part of the resolution of such gradients is achieved by collection of the gradients in small fraction volumes (12 ml gradients in 32 fractions). The gradients are also capable of a high degree of resolution of ER-Golgi-trans Golgi network (TGN) events 19,20 and subfractionation of the Golgi membranes in the analysis of N-linked oligosaccharide...

Separation of membrane vesicles from cytosolic proteins

The strategy for separating membranes and cytosol, which involves flotation of the membranes through an iodixanol barrier from a dense sample, has been developed for both eukaryotes and bacteria (Protocol 5.13). This strategy allows maximum resolution of the two components, since the proteins, which are much denser than the membranes, tend to sediment rather than float. It removes any ambiguity in the results from top-loaded sucrose gradients, in which the membranes sediment and the proteins move more slowly in the same direction due to sedimentation and diffusion. The difference in density between the proteins and the membrane vesicles is also much greater in iodixanol than in sucrose, primarily due to the much lower osmolality of the iodixanol gradients. Similar methods are used for the separation of liposomes from denser proteoliposomes. Sucrose gradients can be used to separate the endocytic compartments on the basis of density 32 but unless the ligand is attached...

Serotonin and Nitric Oxide

Nitric oxide (NO) has also been recently proposed as a neuromodulator of the central pathways of appetite control. Central blockade of NO production inhibits food intake 62 . This condition is reversed by administration of NO donors (l-arginine) 7 . NO seems also to be involved in many central feedback systems, such as those of leptin and 5-HT 63 .

Michaelis Menten Kinetics

Working in the early part of the twentieth century, Michaelis and Menten set about to explain several key experimental facts regarding the conversion of substrate to product catalyzed by simple enzymes. Figure 4.12 illustrates their results for the enzyme invertase, which converts sucrose to glucose and fructose. These graphs give the time course of accumulation of product (measured as the change in optical rotation of the solution) for a fixed total enzyme concentration E T. In the three curves the initial concentration of sucrose, S T, is increased from 5.2 mM to 10.4 mM to 20.8 mM. The initial rate of increase of product, given by the slope of the three curves (V(0)), is plotted for these and similiar experiments in Figure 4.12. As the concentration of sucrose increases, the initial rate saturates at the value Vmax. This function is hyperbolic and can be fit with the expression The concentration of substrate at which V(0) Vmax 2 is called the Michaelis constant or Km for the...

Raphael Sandaltzopoulos and Peter B Becker 1 Introduction

Chromatin reconstitution in crude extracts from Xenopus oocytes or eggs or Drosophila embryos provides a powerful means to study structure function relationships in chromatin organization (see Chapters 12, 13, and 15) (1,2). For many of those analyses (e.g., the evaluation of the transcriptional potential of a chro-matin template), the chromatin must be purified from the complex reconstitution reaction. The most common method for chromatin purification is its centrifuga-tion through a sucrose gradient. Although efficient, this method is time-consuming, does not allow parallel processing of many samples, and many loosely associated chromatin components that may be of pivotal importance (e.g., for chromatin dynamics), may be lost during the long centrifugation.

Isolation of Core Histones

Lysis buffer I 50 mM Tris-HCl, pH 8.0, 25 mM KCl, 25 mM NaCl, 4 mM MgCl2, 10 mM Na-butyrate, 5 mM 2-mercaptoethanol, 0.2 mM PMSF, 0.5 M sucrose, 0.5 Nonidet P40 (v v) (Sigma, Deisenhofen, Germany), 10 ng mL of Trichostatin A. 5. Lysis buffer II 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 4 mM MgCl2, 10 mM Na-butyrate, 5 mM 2-mercaptoethanol, 0.2 mM PMSF, 0.34 M sucrose, 0.5 Nonidet P40.

N M OBrien and T P OConnor

Early work demonstrated that the incorporation of dairy products into the diet greatly decreased the development of dental caries in rats. Later work indicated that if enamel is treated with milk in vitro and subsequently washed, the solubility of the enamel is greatly reduced. This effect was attributed to the high levels of calcium and phosphate in milk or to the protective effects of casein. It has also been reported that both casein and whey proteins significantly reduced the extent of caries, with the former exerting the greater effect. Thus, evidence exists that milk proteins, calcium and phosphate all exert an anticariogenic effect. Additionally, a study in humans found that the consumption of Cheddar cheese after sweetened coffee or a sausage roll increased plaque pH, possibly due to increased salivary output. Rats fed additional meals of cheese while on a high-sucrose diet developed fewer smooth surface carious lesions and exhibited increased salivary output (which buffers...

See also Phosphorolysis Glycogen phosphorylase Figure 1318 Kinase Cascade Figure 1316 Figure 1317 Polysaccharides

Glycosidic bonds between monosaccharides give rise to oligosaccharides and polysaccharides. The simplest oligosaccharides, the disaccharides, include compounds such as sucrose and lactose, which are referred to as sugars (like the monosaccharides). Other common disaccharides include trehalose, maltose, gentiobiose, and cellobiose. Thus, oi-D-Glcp(l- 2)-. -D-Fru corresponds to sucrose.

Preparation and UV Treatment of DNA Templates

Purify pBS plasmid from bacteria using a Qiagen plasmid purification kit. Resuspend the collected plasmid in TE pH 8.0 and determine the DNA concentration by measuring the optical density at 260 nm (OD260 of 1 50 g mL). Up to 2 mg of supercoiled plasmid can be obtained from a 500-mL culture. While keeping the bulk of the plasmid preparation on ice, briefly check its quality by running a 200-ng aliquot on a 0.8 agarose minigel containing 0.25 g mL of ethidium bromide. If nicked form (migrates above the supercoiled form) is detected it is necessary to further purify the supercoiled form through 1 or 2 sucrose gradients as described (19), otherwise directly aliquot the plasmid preparation in 50-100- iL aliquots at 50-100 g mL and store at -80 C (see Note 3).

Enzymes Are Protein Catalysts

Life depends on complex networks of chemical reactions. These are mediated by enzymes. Enzymes are catalysts of enormous power and high specificity. Consider a lump of sugar. It is combustible but quite difficult to set alight. A chemical catalyst would speed up its combustion, and we would end up with heat, a little light, carbon dioxide, and water. Swallowed and digested, the sucrose is broken down in many steps to carbon dioxide and

Transcriptional Analyses of SV40 MCs

Mix together on ice 0.8 yL of 25X NTPs, 10 yCi a-32P UTP, KCl and or MgCl2 (as required to obtain the final, optimal concentrations), H2O (such that the final reaction volume will ultimately be 20 yL), and poly d(I-C) d(I-C) . Add either MCs (up to 7 yL) (see Note 29) or viral DNA. When a comparison is desired between transcription from the MCs vs. viral DNA, supplement the transcription reaction containing viral DNA with 22.5 sucrose at the same volume as the volume of MCs that are being transcribed. Add HeLa whole cell extract (up to 7 yL), mix, and incubate the reactions for 1 h at 30 C (see Note 30).

And Robust Extract From Wheat Embryos

Strate, indicating irreversible damage of ribosomes by contaminants from endosperm (Fig. 3B). High protein synthesis activity with washed embryos was also confirmed by sucrose density gradient analysis, where significant formation of polysomes was observed after 1 h of incubation, and at 2 h a shift to heavier polysomes with a concomitant decrease of 80S monomers (24). Further, in order to check the long-term stability and productivity of this new cell-free translation, it was performed using Spirin's CFCF with continuous supply of substrates and removal of small byproducts (30) the reaction proceeded for more than 60 h, yielding 0.8-4 mg of functionally active proteins per mL of reaction mixture (24). The high efficiency of our system, therefore, can be attributed to

Nucleiand nuclear components see Protocols 4146 Nuclei

Detergent-free methods for the purification of nuclei have routinely involved the pelleting of the organelles from a crude nuclear pellet suspension, through a dense (approx 2 M) sucrose barrier. The nuclei lose water to the grossly hyperosmotic medium and attain a density 1.32 g ml. Pelleting the nuclei is thus the only option since this is the limiting density of sucrose solutions. The high viscosity of the solutions also requires a g-force, normally of 100 000g or higher, for at least 1 h. It is possible that DNA-protein interactions become destabilized by the loss of water and the high hydrostatic pressure at the bottom of the tube. On the other hand, in a discontinuous gradient of iodixanol (centrifuged at 10000g for 20 min), which is close to being isoosmotic, the nuclei exhibit a much lower density (approx 1.23 g ml) and can therefore band at an interface. The method, developed for mammalian liver 20-23 has been used for other tissues such as brain 24-26 , a wide variety of...

Quantitative Analysis for Clustering of HMGContaining Nucleosomes

Calculate the clustering value C (for definition see Note 7) of the HMG protein within a nucleosomal array using the formula C R(N-1) (1-R) (1). R equals the HMG protein core histone ratio. It is calculated by dividing the densitometric value of the HMG by the average of the densitometric value of the core histones (i.e., the total volume of the core histones divided by four). N equals the length of the oligonucleosome fraction taken for analysis. When N is not a integer (the oligonucleosome fraction is not homogeneous), it is possible to calculate N from the relative amounts of the various oligonucleosome species in a sucrose gradient fraction.

Tuber Storage Parenchyma Ultrastructure

The dormant parenchyma cells contain plastids (Gerola and Dassu, 1960 Tulett et al., 1969), mitochondria, dictyosomes (Kaeser, 1988), a nucleus, and nucleoli (Williams and Jordon, 1980). The cells have high levels of arginine, glutamine, and asparagines, very low metabolism of DNA and RNA, low amounts of polysomes, and low levels of polyamines (Favali et al, 1984). They are highly vacuolated, causing the nuclei and other organelles to be adjacent to the cell walls. The vacuoles are the storage site for fructans, and vesicles are formed in the cytoplasm, facilitating fructan synthesis from sucrose entering the cell (Kaeser, 1983). There is a close association of the plastids with mitochondria and the nucleus (Figure 4.6A) (Ishikawa and Yoshida, 1985). The nuclei display regions of condensed chromatin and contain several nucleoli (Figure 4.6C) (Jordan and Chapman, 1971). The plastids vary in structure and are found both scattered in the peripheral

Substrate Supply For Cellulose Synthesis And Its Stress Sensitivity In The Cotton Fiber

Research on cotton fiber has figured prominently in the first steps toward understanding the metabolic control of cellulose biogenesis under normal and stressed conditions for at least two reasons. First, fiber secondary walls are composed of almost 100 cellulose that is deposited over a period of at least 20 days. Second, these extraordinary seed epidermal trichomes can be readily isolated in bulk as a pure cell type or cultured in vitro as part of an easily manipulated ovule fiber system. This chapter summarizes changes in the amount and physical characteristics of cellulose throughout cotton fiber development. Mechanisms of cellulose biogenesis are reviewed, including the role of plasma membrane rosette protein complexes containing cellulose synthases and the relationship of cellulose microfibril orientation to microtubules. Biochemical mechanisms participating in P-1,4-glucan chain polymerization are discussed, including a possible lipid-linked primer, cycling of carbon between...

NLinked Oligosaccharides

N-linked oligosaccharide residues of a protein are all derived from a common 14-sugar-lipid complex. Five of the 14 core sugars are conserved. The conserved core N-oligosaccharides are Man3GlcNAc2. The core sugars are extensively modified by the addition and deletion of simple sugars following attachment to the protein (Fig. 2). These modifications are termed the trimming and branching reactions. The final N-linked oligosaccharide can have three distinct configurations complex, high man-nose, and hybrid. The complex N-oligosaccharides contain disaccharides like Galb1,4-GlcNAc. The high-mannose N-oligosaccharides contain only mannoses at the ends of the oligosaccharide branches. Hybrid N-oligosaccharides have one complex branch or antennae, and one oligosaccharide mannose antennae branching off the core Man3GlcNAc2 group. Figure 2 shows the core N-oligosaccharides with the conserved pentasacchrides boxed. Also in Fig. 2, a generalized oligosaccharide is shown with common antennae as...

Crystallization of Amorphous Sugars

Amorphous Lactose Sorption

Water sorption studies of dairy powders have often shown that during storage at above 40 relative humidity (RH) and room temperature, large amounts of water may be sorbed initially, but during further storage the sorbed water content is reduced with storage time. For example, as early as in 1926, Supplee (5) found a gradual decrease in sorbed water content of milk powder when the material was stored at 50 RH at room temperature. Such decrease in sorbed water content has often been suggested to be a result of time-dependent crystallization of amorphous lactose (e.g., 13, 35, 52-59). Similar crystallization behavior from the amorphous state has been found to apply to other sugars, including glucose and sucrose (7, 60), and the development of crystallinity can be followed from the loss of sorbed water (32, 60, 61). Sugar crystallization occurs in both pure amorphous sugar preparations and sugar-containing foods. Development of crystallinity of amorphous lactose and other sugars, as a...

Case Study Variation in Metarhizium spp 851 Introduction


Since the publication of the Rombach et al. (1987) key, our knowledge of diversity within Metarhizium has increased greatly due to new strains being found and the use of biochemical and molecular techniques to provide new taxonomic characters. Morphological studies have shown that the distinction between M. anisopliae with cylindrical phialides and conidia and M. flavoviride with its swollen phialides and ovoid conidia is difficult to apply, and some isolates can display both forms depending on factors such as cultural conditions and age of culture (Glare et al., 1996). In addition, detailed measurements of conidia have failed to show a distinction between these two species (T.R. Glare, Christchurch, 1997, personal communication). Various authors have used RAPDs to establish differences between strains from sugar cane insects (Fegan et al., 1993) from cercopids and the soil in Brazil (Tigano-Milani et al., 1995c Fungaro et al., 1996), from different countries (Leal et al., 1994a),...

Licorice Glycyrrhizin

Glycyrrhizin (licorice root extract) is extracted from the roots of the plant Glycyrrhiza glabra. Evidence of licorice root has been found in Egyptian tombs, and writings through the ages have described its use in suppressing cough, and soothing sore throats and upset stomachs. Because it is 50x sweeter than sucrose, its traditional use was as a sweetener of food and medications. A typical preparation contains between 5 and 25 percent of the product as glycyrrhizin. Reported healing properties include a reduction in inflammation via vasoconstriction, inhibition of inflammatory cell migration within tissues, and perhaps antiviral activities against a variety of viral agents, that may result from the induction of IFN-y.

Buffers and Preparation of Foods

Add 166.7 g sucrose to beaker a, 66.7 g palmitic acid to beaker b, 366 g Mori-Nu Silken Tofu (extra firm) to beaker c, and 114 g extra lean (95 ) ground beef (pre-boiled, dried, and granulated) to beaker d. 8. Because the foods resemble one another and standard sucrose fly food, the vials, bottles, and dishes were carefully labeled with an easily remembered marker stripe system to distinguish each type.

Cryoprotectant Removal

Cosity of the macromolecular matrix or through a mechanism similar to that afforded by removal of the cryoprotectant in the presence of a non-permeating solute such as sucrose. Both conditions may restrict cell swelling, as the cryoprotectant diffuses out of the cells along its concentration gradient.

Nutritional Counseling for Patients with MetS

NCEP ATP III recommendations for diet composition for patients with MetS are consistent with general dietary recommendations (Table 25-4).13 Guidelines for healthy anti-atherogenic diet call for (1) low intake of saturated fats, trans fats, and cholesterol (2) reduced consumption of simple sugars (3) increased intakes of fruits, vegetables, and whole grains.17 Such principles also concern diet recommendations for the treatment and prevention of diabetes mellitus and related disor-ders.59 The so-called Mediterranean diet is in agreement with these basic recommendations and may be expected to improve some of the main metabolic abnormalities present in MetS and thus to reduce the incidence of CVD.

Rein Aasland and A Francis Stewart 1 Introduction

Usually, nuclease digestion is performed with isolated nuclei. This classical protocol has been described in detail elsewhere (1,3). Here we describe a simplified protocol (4) where the DNaseI treatment takes place as the cells are permeabilized with the nonionic detergent Nonidet P-40 (NP40). We describe two variations of the protocol. In the first variation, cells grown on tissue plates are overlaid with a sucrose-containing buffer which also includes NP40 and DNaseI. After a brief incubation (4 min) at room temperature, the combined permeabilization and nuclease treatment is quenched by addition of a SDS-containing solution. In the other variation, cells are first trypsinised and then resuspended in the sucrose-, NP40-, and DNaseI-containing buffer. After permeabilisation and cleavage, the reaction is quenched as above. The latter variant is preferable if the cells in culture do not grow as a flat monolayer attached to the plate. The cell lysate is subsequently treated overnight...

Implication for the medical use of marijuana

The use of cannabis for both recreational and medicinal purposes dates back to thousands of years. In recent times, there has been an increase in calls for marijuana to be legalized for medicinal use in AIDS, cancer, multiple sclerosis and other medical conditions where patients might benefit from the biological effects of cannabis. Synthetic cannabinoids such as dronabinol, marinol and nabilone already have an established use as antiemetics in nausea and vomiting associated with cancer chemotherapy. The reported beneficial effects in cancer and AIDS patients might reflect improved weight gain, owing to the well-documented anti-emetic and appetite stimulating effects of cannabinoids. This might be a major advantage for cancer patients undergoing rigorous chemotherapy, or advanced AIDS patients. Interestingly, although cannabis is widely used as a recreational drug in humans, only a few studies revealed an appetitive potential of cannabinoids in animals. However, evidence for the...

Longevity Studies With Mated Males and Females

Seven dietary conditions used in this chapter. In the top four conditions, the larvae were raised in control (high sucrose) food. The adults were placed in control, palmitic acid, beef, or soy food for the duration of their lives for the longevity experiments. For the microarray, live weight (LW), triglyceride (TG), and protein level measurements, flies were sacrificed at 10 d (arrow). In conditions five to seven, the eggs were laid in the indicated foods, and the larvae were raised in the same foods (palmitic acid PA , beef, or soy food). Notice that the time from egg laying to eclosion time is longer than in the control food (see Fig. 2). Fig. 1. Seven dietary conditions used in this chapter. In the top four conditions, the larvae were raised in control (high sucrose) food. The adults were placed in control, palmitic acid, beef, or soy food for the duration of their lives for the longevity experiments. For the microarray, live weight (LW), triglyceride (TG), and protein...

Food Plants Containing Fructans

Degree of polymerization can have a pronounced impact on the potential use of the inulin and fructooligosaccharides. Short-chain fructooligosaccharides (i.e., GF5) are of interest because of their health benefits, sweetness ( 30 of sucrose), and as a substrate for the synthesis of certain chemicals (e.g., fermentation products). Inulins with higher degrees of polymerization can be used for fat replacement and high-fructose syrups (longer chain lengths decrease the percentage of glucose in the syrup). Likewise, longer chain lengths can be systematically reduced in size by partial hydrolysis using an endo-inulinase, while lengthening is not a commercially viable option.

Anion Exchange Chromatography

Elute sucrose synthase with a linear salt gradient (7.5 L buffer E and 7.5 L buffer F) with a linear flow rate of 0.77 cm min. 5. Determine the activity of sucrose synthase in the fractions (fraction size 100 mL) with the sucrose synthase assay followed by the photometric analysis of the UDPglucose concentration. 6. Adjust the pH of the pool of the fractions containing sucrose synthase immediately to 7.2. 8. Take a sample, and determine the activity of sucrose synthase and the protein concentration.

Regulation of Autophagy by Amino Acids

The specificity of amino acid inhibition of autophagic sequestration has been intensively studied. Based on experiments with the perfused rat liver, Mortimore and colleagues defined a regulatory group of amino acids consisting of leucine, phenylalanine, tyrosine, glutamine, proline, histidine, tryptophan and methionine together with alanine as a synergistic coregulator, this group of amino acids was able to mimic the inhibitory effect on autophagy of a complete mixture of amino acids at near-physiological concentrations.24-26 In isolated rat hepatocytes, high concentrations of leucine together with either glutamine or histidine acted in synergy and were particularly effective in inhibiting autophagic sequestration of electro-injected cytosolic 14C sucrose.27 A combination of near-physiological concentrations of leucine with either alanine, glutamine, proline or asparagine was also extremely effective in inhibiting autophagic proteolysis in hepatocytes,28,29 and the same was true for...

Fractionation of Subcellular Membranes in Studies on Membrane Trafficking and Cell Signalling

Protocol 5.1 Separation of basolateral and bile canalicular plasma membrane domains from mammalian liver in sucrose gradients 160 Caco-2 cells in a sucrose gradient 163 Protocol 5.7 Analysis of Golgi and ER subfractions from cultured cells using discontinuous sucrose-D2O density gradients 172

The Initial Velocity of an Enzyme Reaction

Product Time Enzyme

However, if either the substrate or the product has a property that can be measured while the reaction is proceeding, then the whole assay can be performed in one test tube. Many enzyme assays are done by using changes in the absorption of light when the substrate is converted to product. Other optical properties can be used. In their original work Michaelis and Menten studied an enzyme that breaks the disaccharide sucrose down into glucose and fructose. The mixture of glucose plus fructose rotated polarized light differently from sucrose, and they used this property to follow the course of the reaction. If there is no convenient optical property, then others may be available for example, one can monitor the progress of a reaction in which the polysaccharide glycogen (page 30) is hydrolyzed by digestive enzymes by measuring the resulting fall in viscosity.

Why does LMMC brown excessively on cooking

Browning during cooking results from heat-induced Maillard reactions between the carbonyl groups of reducing sugars and the amino groups of peptides and amino acids 188 . Reducing sugars in low-moisture Mozzarella cheese (LMMC) consist of unfermented lactose and galactose at concentrations that may vary considerably depending on the sugar-fermenting characteristics of the starter culture used and the extent to which the starter bacteria survive the heat treatment during stretching 147 . Short peptides and amino acids in LMMC 146 made from high-quality milk occur primarily through secondary proteolysis by the starter bacteria 23 and may vary considerably in concentration, depending on the proteolytic characteristics of the starter culture used and the extent of starter inactivation during stretching. Browning reactions occur when adequate concentrations of both reducing sugars and products of proteolysis are present in the cheese during heating. Furthermore, browning reactions are...

Substrate Supply For Cotton Fiber Cellulose Biogenesis

Cotton Fiber Synthesis

3.1 A role for sucrose synthase Substantial evidence implicates UDP-Glc as the immediate substrate for cellulose polymerization in vascular plants including cotton fiber (Franz 1969 Carpita and Delmer 1981). UDP-Glc was the most abundant nucleotide in the fiber and its concentration increased in parallel with stage-specific increases in the cellulose synthesis rate 0.2, 1.2, and 2.1 imol boll at 13, 17, and 21 DPA, respectively. The UDP-Glc for cellulose synthesis could be supplied by (1) sucrose synthase (SuSy E.C. Suc + UDP UDP-Glc + Fru) or (2) by UDP-Glc pyrophosphorylase (Glc-1-P + UTP UDP-Glc + PPi) (Figure 9-1). Addressing first the possible participation of UDP-Glc pyrophosphorylase, this enzyme activity increases 1.8x along with the increased rate of cellulose synthesis at the onset of cotton fiber secondary wall deposition (Wafler and Meier 1994). Pulse-chase experiments with 14C-Glc fed to cultured cotton ovules fibers were consistent with carbon flow through...

Overlapping Differential Expression

Found that all three experimental diets significantly decrease longevity, increase the length of time to develop from egg to adult, and alter global gene expression patterns compared with the control high-sucrose diet. Preliminary microarray analyses suggest that a total of 60 genes have significantly (FDR 0.1) altered gene expression diets by all three experimental diets compared with the control diet. The life-shortening effect of palmatic acid confirms previous studies by Driver's laboratory (26). We interpret that time required from egg laying to eclosion as being inversely proportional to the quality of food, at least as it applies to proper Drosophila larval development. If this interpretation is correct, then it suggests that the control (high sucrose) food also has the highest quality. This is not surprising because fruit flies typically lay eggs in rotting fruit, hence their name. Rotting fruits are high in fructose and fermentation products, which like sucrose, are quickly...

Identification and quantification of small quantities in a complex mixture by 2D 1H31Phmqctocsy Nmr

The sea urchin system constitutes one of the best models for NE (nucelur envelope) assembly (Larijani et al., 2000). Its primitive genome simplifies signalling pathways involved in the regulation of NE assembly. Following fertilization, the sea urchin sperm nucleus undergoes the breakdown of its inactive envelope. The disassembly of the lamina and the chromatin decondensation are followed by fusion of fertilized egg membrane vesicles (MVs). It is known that the cytoplasmic membrane vesicles (MV) participate in the formation of the NE but details of how and which molecules participate depend on identifying and quantifying the role of the various fractions and their components. The cytoplasmic membrane can be separated on a sucrose density gradient into fractions MV1, MV2, MV3 and MV4. The MVs are made up of phospholipids and it is a major analytical challenge to identify and quantify them.

Rough and smooth endoplasmic reticulum ER

The microsome fraction (see 'Differential centrifugation' above) will contain, in addition to the membrane vesicles derived from the smooth and rough ER, contaminating organelles that failed to sediment at 15 000g, together with core structures from peroxisomes, which have lysed during the previous manipulations. The smooth vesicle fraction will also contain vesicles derived from other sources such as the Golgi and TGN. Rough ER and smooth ER can be effectively separated in a continuous sucrose gradient 48 as described in Protocol 4.18. To resolve the rough ER from the peroxisomal cores (and other contaminating organelles), the density of the RER vesicles is reduced by stripping off the ribosomes with pyrophosphate and they are purified on a second continuous sucrose density gradient 48 . As both the SER and RER band broadly, the method may offer some scope for subfractionation of both membranes. Protocol 4.19 offers an alternative method for separating these membranes in a...

Scientific Foundations

Fermentation is a chemical change that produces energy through the anaerobic breakdown of simple sugars. An anaerobic process is one that does not need oxygen. Fermentation usually refers to the conversion of sugar to alcohol by yeast. This process is used to make beer and wine.

Cleaner Burning Fuels

Ethanol also can be made from cellulose-based biomass, such as trees and grass. Cellulose (found in plant cell walls) is made up of chains of sugar molecules. Enzymes are added to break up the chains, in order to produce simple sugars. The sugars are then fermented using yeast to produce alcohol.

Buffers for Nuclei Extraction from Rat Liver or Cells in Culture

Lysis buffer Buffer A (1X), 0.34 M sucrose, 2 mM EDTA, 0.5 mM EGTA, 0.65 mM spermidine, 1 mM DTT, 0.1 Triton X100, 0.5 mM PMSF. 3. Buffer B1 Buffer A (1X), 0.34 M sucrose, 2 mM EDTA, 0.5 mM EGTA, 0.65 mM spermidine, 1 mM DTT, 0.5 mM PMSF. 4. Buffer B2 Buffer A (1X), 1.5 M sucrose, 0.65 mM spermidine, 1 mM DTT, 0.5 mM PMSF. 5. Buffer D Buffer A (1X), 0.34 M sucrose, 0.65 mM spermidine, 1 mM DTT, 0.5 mM PMSF.

The Properties Of Wood

Xylogenesis Wood Fiber Cells

Wood has a structure that ideally combines strength with flexibility. This is achieved by the production of complex multilayered cell walls composed of cellulose microfibrils embedded in a matrix of hemicellulose and lignin. In a tree, over 90 of the wood cells are arranged along the axis of the trunk or branches to transport water from the roots to the leaves. All the different cell types in a tree originate from a single layer of multipotent dividing cells termed the vascular cambium. There are two types of cambial cells - the long narrow fusiform initial cells, which develop into the axial cells and the ray initials, which form the radial cells. Some of these cells continue to divide whereas others differentiate either inwards into the xylem cells, or outwards into the phloem cells. The xylem has two main functions. One is to transport water, nutrients, and hormones whereas the other is to provide mechanical support to the plant. The phloem provides paths for the distribution of...

Womens Health Premenstrual Syndrome PMS

Premenstrual syndrome (PMS) is a group of symptoms that generally appear 4 to 10 days before menstruation and end, often abruptly, as menstruation begins. The most common symptoms are irritability, nervous tension, depression, mood swings, craving for sugary foods, breast tenderness, water retention, and weight gain.1 The symptoms of PMS can be mild or severe about one in five women have severe symptoms that interfere with daily activities. In many women, an imbalance of too much estrogen and too little progesterone triggers the symptoms of PMS.

Fructose and Short Chain Fructans

The characteristic inulin biochemistry of Jerusalem artichoke makes it an excellent source of fructose. Fructose is the sweetest of the natural sugars its sweetness is around 16 greater than sucrose (Shallenberger, 1993). Fructose syrups are widely used by the food industry. They have a high solubility in water, fewer calories than sucrose, and are less viscous. With these properties, fructose has gained in importance within the food processing industry as a sweetener. It is an ideal sugar for use in reduced-calorie foods, foods for diabetics, and products to combat obesity. A range of fructose-containing products can be obtained from Jerusalem artichoke, including sugar solutions, pure fructose syrup, and crystalline fructose. Inulin, fructooligosaccharides, fructose, and other useful compounds can all be purified from the juice extracted from Jerusalem artichoke tubers. Fleming and GrootWassink (1979) describe a process for obtaining high-fructose syrup (75 yield) using enzyme...

How may browning of heated cheese be controlled

Much cheese manufactured now is used for ingredient applications 187 and hence cheese may be exposed to heat during processing or cooking. Browning of heated cheese is due to the Maillard pathway, which is a very complex series of reactions involving amino groups of free amino acids, peptides and proteins and a reducing sugar, and which is favoured by intermediate water activities and high temperatures. A certain degree of Maillard browning may be desirable in certain applications of cheese as an ingredient (e.g. slight browning of low-moisture part-skim Mozzarella used as a pizza topping) or it may be undesirable. Control of Maillard browning in heated cheese involves removing one of the necessary reactants and or providing conditions unsuitable for browning to occur. Browning is favoured at high temperatures, pH above 6 and at intermediate aw. However, it is often impractical to vary these conditions to avoid browning and hence control of Maillard browning in cheese usually involves...

Complete Hydrolysis Fructose Syrups

Fructose syrups are widely utilized in the food industry in that they are sweeter than sucrose, thus allowing less sugar to be used to achieve a given level of sweetness (i.e., fructose is 1.2 times sweeter than sucrose on a weight basis (Shallenberger, 1993)). In addition, fructose metabolism in humans is not insulin dependent, and it produces less tooth decay than other sugars (Roch-Norlund et al., 1972). Currently, much of the fructose used by the food industry is produced from corn starch, a glucose polymer, via hydrolysis followed by isomerization. The fructose content is around 42 but can be increased to 95 by chromatographic separation of the residual glucose and further isomerization.

Chromatin Preparation

Nuclear incubation buffer 15 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 60 mM KCl, 0.1 mM EDTA, pH 8.0, 0.1 mM EGTA. Filter-sterilize (0.22 pm) and store at 4 C. Add fresh immediately before use 0.5 mM dithiothreitol (DTT), 1 mM PMSF (Sigma, St. Louis, MO, stock solution 200 mM PMSF in 100 ethanol).

Chemical Modification Of Inulin

An inherent problem with reducing sugars (e.g., glucose, fructose, and fructosylfructose) is their tendency to undergo undesirable discoloration and side reactions under alkaline conditions or at high temperature. Inulin also has sufficient residual reducing power to discolor (Kuzee, 1997). Exposure of inulin to a reducing agent or by electrochemical reduction prior to subsequent modification minimizes potential color alterations.

Buffers and Solutions

Fixation buffer 3.5 Paraformaldehyde, 2 sucrose in PBS (see the chapter by Everts et al., this volume, for details). 5. Freezing buffer PBS, 2.3 M sucrose, 0.05 sodium azide. 7. Triton permeabilization buffer 0.5 Triton X-100 (Pierce, UK), 20 mM acid (HEPES), 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 0.05 sodium azide, pH 7.0.

Multicolor Labeling of Cells by Fluorescence Immunostaining

Rinse the discs in PBS, and permeabilize the cells for 5 min in 1 mL of ice-cold Triton X-100 permeabilization buffer (20 mM HEPES, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 0.5 Triton X-100, 0.05 sodium azide, in PBS at pH 7.0). Alternatively, use 0.1 saponin permeabilization buffer (0.1 saponin, 80 mM disodium PIPES, pH 6.8) for 30 min on ice.

Synthesis of other polysaccharides involves many of the same mechanisms as for glycogen

Dextran - an oi(l- 6) polymer of glucose with ot(l- 2), ot(l- 3), and oi(l- 4) branch points. Dextran is the only polysaccharide listed here that does not use activated nucleotide sugars (or derivatives) in making the polymer. The polymerization, catalyzed by the enzyme dextran sucrase, is a transglycosylation of sucrose n Sucrose - glucosen (dextran) + n fructose

Modern Ief With Carrier Ampholytes

IEF is commonly carried out on polyacrylamide gels. The preparation and polymerization of the gel are carried out in the presence of the ampholine mixture, in a vertical column or slab. The upper (anodic) reservoir is then filled with an acidic solution of, for example, phosphoric acid, and the lower (cathodic) reservoir is filled with a basic solution, for example, aqueous ethanolamine. These solutions are allowed to diffuse toward the center of the gel, to generate a pH gradient from the acid and base reservoir solutions. This step is followed by a prefocusing step before sample introduction, to allow the migration of the carrier ampholytes to their respective pI values. The carrier ampholytes thus focus at their pIs and stabilize the initial pH gradient. The sample is then layered at the anodic end in a sucrose solution, and the electrofocusing step is begun this step may take several hours to reach steady state, with components migrating according to their mobilities until they...

Two Proteins Are Transported by the Cvt Pathway Aminopeptidase I and aMannosidase

Aminopeptidase I, hereafter referred to as Apel, is a metallopeptidase belonging to the Ml8 family of the MH clan. It is a leucine aminopeptidase with preference for neutral or hydrophobic amino acids using zinc as a cofactor, and its peptidase activity is also enhanced by chloride ions.4 Unlike most vacuolar proteins, which pass through the secretory pathway, precursor Apel (prApel) does not contain a signal sequence that would direct the newly synthesized peptide chain to the endoplasmic reticulum, but is instead synthesized free in the cytosol. The first 45 amino acids constitute an inhibitory propeptide, which is cleaved off by proteinase B after the protein enters the vacuole. This cleavage generates an active aminopeptidase and can be monitored as a molecular mass shift from 6l kDa to 50 kDa. This shift can easily be detected by SDS-polyacrylamide gel electrophoresis and western blotting and provides a convenient assay that can be used to monitor the transport...

The Cvt Complex Localizes to the Pre Autophagosomal Structure PAS

Subcellular fractionation using isotonic density gradients separates the autophagic proteins into at least two different populations.28 It is therefore possible that the PAS is not a traditional membrane compartment but rather an assembly point for the autophagic membrane and the autophagic machinery. Apgl, Apg2 and Cvt9 peak with the membrane protein Apg9 at high density, while other proteins including Cvtl9, Apg5 and Apgl2 peak at a somewhat lower density that is similar to the density of the completed Cvt vesicle. Completed autophagosomes, on the other hand, have a much lower density. 8,32,33 Subcellular fractionation using OptiPrep and sucrose density gradients separate PAS marker proteins from plasma membrane proteins (Pmal), vacuolar membrane proteins (Pho8), Golgi proteins (Pepl2 and Kex2) and ER marker proteins (Dpml).28,30,32 Although the PAS is localized close to the vacuole, it does not stain with the dye FM 4-64, which is taken up by endocytosis and labels the...

Materials Used to Generate a Gradient

The most common materials used to generate density gradients are sucrose, Ficoll, and Cs salts. Sucrose solutions can be as concentrated as 65 w w, with a maximum density of 1.32 g cm3 at 4 C however, concentrated solutions of sucrose have high osmotic strength and viscosity. Ficoll is a brand name for a synthetic polysaccharide with an average MW of 400,000 and a maximum density in aqueous solution of 1.23. It is very useful to separate osmotically sensitive particles, like mammalian cells.

The Components of the Cvt Vesicle Forming Machinery

Role for Apgl also in autophagy in S. cerevisiae. The ATP binding site of the kinase domain of Apgl was mutated to allow binding and inhibition by 1-NA-PP1.51 1-NA-PP1 is a bulkier, cell-permeable analogue of the non-specific protein kinase inhibitor PP1 pyrimidine) that is only active at ATP binding sites where a methionine is replaced with a smaller amino acid such as alanine.52 The inhibitor not only selectively abolished the autophosphorylation of the mutated form ofApg1 in vitro it also blocked the uptake of prApe1 in vivo in rich medium.51 However, the block in prApe1 uptake was reversed upon starvation even in the presence of the specific inhibitor, and bulk autophagy was found to be normal. These data suggest that the kinase activity of Apg1 may be more important for the Cvt pathway than for autophagy, and that the role of Apg1 in autophagy may rather be of a structural nature. Concomitant with the shift to starvation, a structural change was seen for Apg1 using sucrose...

Histone Modifications and Chromatin Structure

FIGURE 1 Epigenetic regulation of chromatin. Each nucleosome consists of 146 base pairs of DNA wrapped around a core of histone octamers (QJ). (A) Euchromatin An unmethylated promoter and a normal balance between histone deacetylases (HDAC) and histone acetyltransferases (HAT) allows for acetylation of histone tails, creating a chromatin that is open and transcriptionally active. Aceylated histones associate preferentially with transcriptionally active chromatin. Histone acetylation as well as methylation of histone H3 at lysine 4 may also facilitate binding of transcription factors to promote or disrupt higher order chromosome structure. (0) A histone H3 methylated at lysine 4. (B) Heterochromatin Promoter methylation and hypoacetylation of histones create a chromatin that is condensed and transcriptionally inactive. Methylation of lysine 9 on histone H3 (H3-MeK9) is associated with gene transcription. Abbreviations DNMT, DNA methyltransferases HAT, histone acetyltransferases HDAC,...

Cannabinoid effects on naturally rewarding behaviors

The literature dealing with cannabinoid effects on voluntary consumption of sweet foods and liquids is smaller. And, in the view of this reviewer, it is more germane to the considerations of the present review, in light of the following facts (1) animals genetically selected for high rates of electrical brain-stimulation reward (Lieblich et al., 1978 Gross-Isseroff et al., 1992) show significantly enhanced consummatory responses to sweet stimuli (Ganchrow et al., 1981) and (2) a number of other, non-cannabinoid, addictive drugs share the common feature of augmenting consumption and increasing the reward value of sweet stimuli (for review, see Milano et al., 1988). With respect to cannabinoids, a number of studies have found them to augment choice and consumption of sweet foods and solutions (e.g. sucrose) (Sofia and Knoblock, 1976 Brown et al., 1977 Milano et al., 1988) and to increase their reward value as assessed by increased progressive-ratio break-points (Gallate et al., 1999)....

Inulin and Diabetes Mellitus

Of sucrose or glucose prompts blood sugar and insulin changes, but no corresponding effects are noted when equivalent amounts of inulin or fructooligosaccharides are ingested (Roch-Norlund et al., 1972). Therefore, consuming inulin-rich foods helps to restore normal levels of blood sugar, whereas foods containing starch and sucrose further raise blood sugar levels. Experiments during the 1980s and 1990s confirmed the beneficial role of inulin-rich foods in diabetic diets. Daily intake of fructooligosaccharides has been shown to reduce blood sugar levels in both diabetic and healthy subjects (Luo et al., 1996 Yamashita et al., 1984), for instance, while inulin reduced insulin peaks compared to diets containing other carbohydrates (Rumessen et al., 1990). An intake of around 16 to 23 g of inulin per meal has been recommended in diabetic diets (Van Loo et al., 1995). There is a long history of inulin-containing plants being used to treat diabetes. The Greek physician Theophrastus used...

Physicochemical Diet Stevia Soft Drink

A In soft drinks in the European Union. b 5 equivalent sucrose 1. a In soft drinks in the European Union. b 5 equivalent sucrose 1. This ingredient has a fast-onset sweet taste with a marked bitter aftertaste that is more noticeable at high concentrations. Combinations of acesulfame K and aspartame are synergistic with respect to sweetness intensity and sweetness quality when compared with acesulfame K alone. Synergy has also been reported with cyclamate, glucose, fructose, sucrose and sucralose (Nutrinova, 2003 Von Rymon Lipinsky & Huddard, 1983). Negative synergy (i.e. suppression) occurs with acesulfame K saccharin combinations. The taste profile of aspartame is similar to sucrose sweetness (Ripper et al., 1985). It is approximately 200 times as sweet as sucrose. It is synergistic with saccharin, cyclamate, stevioside, acesulfame K and many sugars, in particular fructose, but has little sweetness intensity synergy with sucralose. water. Alitame is approximately 2000 times as sweet...

Seperation Of Cho From Soft Drinks

Wide range of parameters - ethanol, pH, volatile acidity, total acidity, levels of tartaric, malic, gluconic, lactic and acetic acids, reducing sugar content, glucose to fructose ratio and density - in wines and musts from one simple measurement (http, which is perhaps an indication of things to come. Anon (1998a) Determination of sucrose enzymatic. No. 56, in International Federation of Fruit Juice Producers (IFU) Handbook of Analytical Methods, Swiss Fruit Juice Union, Zug, Switzerland. Bergmeyer, H. U. and Brent, E. (1974a) Enzymatic analysis of sucrose, in Methods of Enzymatic Analysis (ed. H.U. Bergmeyer), Academic Press, London, pp. 1176-9. Martin, G.G., Jamin, E. Gonzalez, J., Remaud, G., Hanote, V., Stober, P. and Naulet, N. (1997) 'Improvement of the detection level of added sugar with combined isotopic and chemical analysis. Fruit Processing 9, 344-49.

Methods for Nutrigenomics and Longevity Studies in Drosophila

Effects of Diets High in Sucrose, Palmitic Acid, Soy, or Beef Nutrigenomics is the study of gene-nutrient interactions and how they affect the health and metabolism of an organism. Combining nutrigenomics with longevity studies is a natural extension and promises to help identify mechanisms whereby nutrients affect the aging process, life span, and, with the incorporation of age-dependent functional measures, health span. The topics we discuss in this chapter are genetic techniques, dietary manipulations, metabolic studies, and microarray analysis methods to investigate how nutrition affects gene expression, life span, triglyceride levels, total protein levels, and live weight in Drosophila. To better illustrate nutrigenomic techniques, we analyzed Drosophila larvae or adults fed control diets (high sucrose) and compared these with larvae or adults fed diets high in the saturated fat palmitic acid, soy, or 95 lean ground beef. The main results of these studies are, surprisingly, that...

Identification of PrfAControlled Target Genes

Ponents of an ABC transport system predicted to import disaccharides, oligosaccharides, and polyols. The second transcription unit, lmo0278, is monocistronic and codes for the ATPase component of a sugar transporter. It is possible that the products of the two group II transcription units act together to mediate sugar import. lmo0278 has a potential PrfA box located only about four nucleotides upstream of its -10 region. The proximity of these binding sites to the -10 might inhibit transcription by preventing binding of RNAP. In contrast to the situation with lmo0278, lmo0178 lacks an upstream PrfA box near predicted -10 or -35 regions of the promoter, and the mechanism by which PrfA downregulates expression of this gene is unclear. It is also not obvious why it would be advantageous for L. monocytogenes to impair expression of this transport system through PrfA. One possible explanation might be that the group II genes encode a transporter of sugars that are readily available in the...

Microbiology of soft drinks and fruit juices

Simple soft drinks such as orangeade and lemonade are too acidic for the growth of most organisms, so that spoilage is generally by carbonation-resistant species such as Dekkera anomala (Stratford and James, 2003). Yeasts usually require a carbon source such as a hexose sugar, a nitrogen source such as amino acids or ammonium salts, simple salts (phosphate, sulphate, potassium and magnesium ions), trace minerals and vitamins. Some yeasts have particular sugar requirements for example, Z.bailii and Z.rouxii cannot utilise sucrose (Pitt & Hocking, 1997 Stratford et al., 2000).

Mass Action And Metabolic Control

Sucrose synthase (SuSy) provides another potential pathway for controlling cellulose production. SuSy cleaves sucrose in the presence of uridine diphosphate UDP - glucose + Fructose Sucrose + UDP Considering the free energy of hydrolysis of sucrose as -6.6 Kcal'mol-1 and the in favor of sucrose synthesis (Cardini et al. 1955). The apparent equilibrium con Figure 5-1 shows the sensitivity analysis for changes in sucrose concentration and the ratio of reverse and forward reaction velocities. The parameters for Figure 5-1 were calculated using the ranges of substrate and product concentrations found in the literature. UDP-glucose was reported to vary between 1.4 to 3.2 mM (Dancer et al. 1990 Krause and Stitt 1992 Barreiro 1999). Reported fructose levels range from 0.1 to 1.5 mM (Winter et al. 1994 Krapp and Stitt 1995), however, the concentration of cytoplasmic fructose assumed in the calculations could be higher than the concentration found in vivo because of the existence of high...

Backward Conditioning

Keith-Lucas and Guttman (1975) designed an experiment to test this adaptive-value hypothesis. Rats were placed in an experimental chamber and fed a sugar pellet in a particular location. While eating the pellet, the rats were given a one-trial presentation of electric shock (US). After the shock, the chamber was made completely dark for 1, 5, 10, or 40 s. When the light in the chamber came back on, a toy hedgehog (CS) was presented to the rat. To make this experiment clear, eating sugar pellets was viewed as the laboratory equivalent of grazing, the shock represented an attack, and the appearance of the toy hedgehog substituted for the predator. Two control groups were run under identical conditions, except that one group saw the hedgehog but did not get shocked, and the other group got the shock but did not see a hedgehog.

Procedure 2 Immunofluorescence analysis of vimentinGolgi interaction

Glass Coverslips

Figure 6.26 Binding of vimentin to Golgi membranes in vitro. (a) Recombinant vimentin alone, or recombinant vimentin incubated with purified Golgi membranes were loaded at the bottom of a sucrose density gradient and subjected to equilibrium centrifugation. Fractions were collected from the top of the gradient. An equivalent amount of each fraction was processed by SDS-PAGE, transferred to NC and immunoblot-ted with anti-vimentin and anti-GM130 antibodies. Vimentin remains in the load when centri-fuged alone, but is recovered in a lower density fraction containing the Golgi marker GM130 when incubated with Golgi elements. (b) Fractions 3 and 4 from panel A were combined, loaded at the bottom of a sucrose density gradient and subjected to another round of equilibrium cen-trifugation. Fractions were collected and analysed as in (a). Vimentin remains associated with Golgi membranes. Reproduced from The Journal of Cell Biology, 2001, 152(5), 877-893, by copyright permission of the...

Selective Oxidation of the Primary Hydroxyl Group

Platinum (catalyst) and molecular oxygen (oxidant) have been used to selectively oxidize carbohydrates (Abbadi and van Bekkum, 1996 Gallezot and Besson, 1995) such as sucrose (Fritsche-Lang et al., 1985 Kunz and Recker, 1995). Inulin is readily oxidized using O2 and platinum in the presence of sodium hydroxide (Verraest et al., 1998). Oxidation occurs selectively at the C-6 position with a relatively high yield (79 ). Sucrose is also selectively oxidized at the C-6 and C-6' positions under similar conditions (Edye et al., 1991, 1994). The molecular weight of the inulin influences the reaction rate and degree of oxidation. Nystose (GF3) had a degree of oxidation of 40 . As the chain length increases, the degree of oxidation declines. Inulin with an average degree of polymerization of 10 had a degree of oxidation of 28 , while at an average degree of polymerization of 30, it decreased to only 20 . In addition, the amount of by-products increases with increasing chain length and degree...

Dynamical Transitions in Amorphous Solids

Zation within the cooling time. (Some materials, however, form glasses only under extreme conditions formation of metallic glasses, for example, requires cooling rates in excess of 106 K s_1.) Many biomolecules readily form amorphous solids these include simple sugars, oligosaccharides and carbohydrates, and proteins. Synthetic polymers also readily form amorphous solids even a simple polymer, such as polyethylene, which readily forms crystalline phases, has an appreciable fraction of amorphous-state polymer (low-density and high-density polyethylene differ in the content of amorphous and crystalline polymer).

Diagnostic Tool For Cookie Doughs

In order to develop a diagnostic DSC assay and facilitate the interpretation of complex DSC profiles that exhibit multiple thermal events, model systems of increasing complexity and variable water content were explored, as illustrated by the DSC heating profiles shown in Figure 1A (2). For a model batch-mix cookie dough containing 100 54 27 24 parts by weight flour sucrose fat water, the schematic DSC profile (top curve) in Figure 1A shows three broad endother-mic transitions (labeled endo-1, endo-2, and endo-3, with peak temperatures centered around 55 , 90 , and 135 C, respectively), followed by an exothermic transition with a peak around 160 C (labeled exo). An actual DSC profile (bottom curve) for a model continuous-mix dough of the same composition is also shown in Figure 1A. To identify the thermal events underlying the four transitions manifested in the DSC profiles in Figure 1A, various two- or three-component model mixtures were examined. For example, DSC analysis of the...

Stress Development Models

The viscoelastic stress model accounts for the time required for deformation and stress relaxation to occur. Several researchers have modeled heat and mass transfer with Luikov's model and viscoelastic stresses in foods. Cummings (57) and Litchfield (61) modeled pasta drying. Itaya et al. (62) modeled three-dimensional heat and mass transfer and stress development in starch sucrose bars, and Aki-yama et al. (63) studied crack formation and propagation in viscoelastic foods. Kim (60) modeled checking in crackers during cooling.

Allergy diets and pollutants

Yeast allergy is one of the most universally common - a fungus parasite of the gut normally kept in check by the immune system, but it can become systemic, or cause a skin infection. It causes leaky gut mucosa, letting toxic substances into the body and causes immunosupression and increased production of corticosterone. It causes many symptoms like ME and allergy - food cravings, feeling bad all over, hypoglycaemia, chemical sensitivities.

The Normal Adult Brain

In long-lasting metabolic processes, glucose and other sugars are able to glycolyse proteins irreversibly via non-enzymatic reactions which finally generate advanced glycation end products (AGEs). These posttranslational modifications were found to be prominent during aging. AGEs show a high affinity to its receptor (RAGE). Receptor binding is followed by an expression of both growth factors and transcription factors as well as by changes of membrane permeability (Vlassara et al 1994). RAGE have been demonstrated to be present in CNS. Interestingly, PA4 has been shown to bind to RAGE. With aging, AGEs have been found to accumulate in the perikarion of large neurons in the hippocampus and the dentate gyrus, and in pyramidal cells of the cortical layer III, V and VI. These cell layers have been demonstrated to be particularly prone to degeneration in sporadic Alzheimer disease.

Energy Distribution Among Nutrients

Sucrose 400 However, in addition to the importance of controlling the total amount of dietary energy, food choices should be directed towards a balanced distribution of energy among nutrient sources. Epidemiological and experimental studies have led to the establishment of correct energy distribution among carbohydrates, fats, and protein, in order to prevent the onset of chronic diseases and to assure the maintenance of a good nutritional and health status. In this view, in the USA, more than 20 years ago, a Senate Select Committee stated that the energy distribution compatible with good health and that should be reached by the American population (dietary goals) should consist of 58 of energy from carbohydrates, 30 from fats, and 12 from proteins. Among carbohydrates, 15 of calories should derive from sugars and 40-50 from complex carbohydrates, while, among fats, 10 of calories should come from saturated fats and 20 from unsaturated fats 13 . Therefore, the actual recommendation is...

Density gradient centrifugation

It is now widely recognized, particularly for some of the larger organelles (nuclei, mitochondria, lysosomes, peroxisomes), which can be pelleted at relatively low g-forces (1000-15 000g), that the use of viscous, hyperosmotic sucrose gradients at g-forces of more than 100000g in long sedimentation path length swinging-bucket rotors, is both inconvenient and potentially deleterious to the organelles. Consequently, there is a pronounced trend to the use of low-viscosity gradient media such as Nycodenz or iodixanol 17 , whose gradients have a much lower osmolality than those of sucrose (or A disadvantage of Percoll gradients is that it is normally a requirement to remove the colloidal silica particles prior to any analysis the particles are light scattering and can interfere with any spectrophotometric measurements they also cause irregular banding of proteins in SDS-PAGE. Moreover, removal of the silica colloid by sedimentation can also lead to the loss of organelles into the gel...

CD36 and Insulin Responsiveness in the Mouse

It cleared glucose faster, reflecting a several-fold enhancement in glucose utilization by muscle. In vitro, insulin responsiveness of glycogenesis by the null soleus was enhanced. However, when the chow diet was switched to one high in fructose, the CD36- - but not the wild-type mouse developed marked glucose intolerance and hyperinsulinemia, reflecting decreased muscle glucose uptake. Both mice groups had glucose intolerance with feeding a high-fat diet but muscle insulin responsiveness was partially protected in the CD36-null mouse. In conclusion, CD36 deficiency in the mouse enhances insulin responsiveness on a high-starch low-fat diet. It predisposes to the insulin resistance induced by high fructose and, partially protects from that induced by high fat. If these data could be extrapolated to the human case, CD36 deficiency would constitute an important risk factor for the insulin resistance linked to high consumption of simple sugars. It is also possible, based on...

Enzymatic Hydrolysis

The products of the partial hydrolysis of maize starch, called hydrolysates, are classified by their dextrose equivalent (DE). By industry definition, the DE of a maize syrup is the total amount of reducing sugars expressed as dextrose on a dry basis. Since starch contains only one reducing group per molecule, it is essentially zero DE. Complete hydrolysis, on the other hand, yields a product that is 100 DE. Partial hydrolysis produces a range of products that provide the functional properties illustrated in Figure 6. The arrow indicates the direction in which the property increases (Austin and Pierpoint 1998).

The Calvin Benson Cycle

The immediate product of CO2 fixation, 3-phosphoglycerate, must undergo a series of transformations before the net synthesis of carbohydrate is realized. Among carbohydrates, hexoses (particularly glucose) occupy center stage. Glucose is the building block for both cellulose and starch synthesis. These plant polymers constitute the most abundant organic material in the living world, and thus, the central focus on glucose as the ultimate end product of CO2 fixation is amply justified. Also, sucrose (a-d-glucopyranosyl-(1 n 2)- -d-fructofuranoside) is the major carbon form translocated out of leaves to other plant tissues. In nonphotosynthetic tissues, sucrose is metabolized via glycoly-sis and the TCA cycle to produce ATP.

Plasma membrane domains

Bile canalicular and basolateral domains of rat liver plasma membrane are prepared from the plasma membrane sheets, isolated in Protocol 4.26, after vesiculation, which is usually produced either by sonication 2,3 or liquid shear 4-6 . The two domains are then separated in a continuous or discontinuous sucrose gradient (Protocol 5.1). Sometimes Nycodenz gradients are used to provide additional purification 7,8 . The sinusoidal domain is rather less easy to purify by density gradients and it is more usually obtained by density perturbation with protein A-sepharose beads (Protocol 5.2). The beads are bound to an antibody to the polymeric IgA receptor, often called the secretory component (SC), which is a specific marker for the sinusoidal plasma membrane domain 9 . Once bound to the beads, the sinusoidal membrane vesicles can be harvested in a microcentrifuge. The methodology has been widely reviewed 10,11 . Protocols 5.3 and 5.4 provide methods for the fractionation of apical and...

Synthesis Using Microorganisms

An alternative to transgenic plants is expression of either p-fructofuranosidases (EC or p-fructosyltransferases (EC in a microorganism such as Escherichia coli for the synthesis of inulin or short-chain fructooligosaccharides from sucrose. In this case, sucrose acts as both the fructose donor and initial acceptor (Fishbein et al., 1988). p-Fructofuranosidases with sufficient transfructosylation activity can produce fructooligosaccharides for example, the enzyme from Aspergillus niger ATCC 20611 has been used successfully for industrial production (Hidaka et al., 1988 Hirayama et al., 1989). The product is separated into two commercial classes Neosugar G and Neosugar P (Hidaka et al., 2001). Neosugar G contains 35 glucose and fructose (w w), 10 sucrose, and 55 fructooligosaccharides. To increase the fructooligosaccharide concentration to 95 , the monosaccharides are removed from Neosugar G, yielding Neosugar P. The sweetness of each fraction is about 60 (G) and 30...

Prodrugs Under Development

Is readily processed to ACV by xanthine oxidase (365). However, desciclovir is more toxic than ACV. Modification of desciclovir by adding reducing sugars to the 2 position of adenine provided good water solubility, but poor adsorption after oral dosing in rats. They were also inefficiently processed to ACV (366).

Biological Roles for Surface Carbohydrates and Potential Uses

Because polysaccharides represent the predominant structures on the bacterial cell surface, they are important in the interaction between the pathogen, host, and environment. To summarize the reports described in Subheadings 2.-4., C. jejuni carbohydrates are involved in many cellular functions, including assembly of the flagellar filament (73), which we now realize is a type III-like secretory apparatus necessary for the release of invasion colonization proteins (14,15), motility (73), assembly of the TFSS that affects DNA uptake and natural transformation (79), adherence and invasion in vitro (41,54,76), colonization, and disease in vivo (41,64,68,76-78), molecular mimicry of gangliosides (82), autoimmunity leading to GBS (48), maintenance of cell surface charge (41), serum resistance (41,53), antigenic (53,60,83) and phase variation (41,54,57,83,84). Considering the importance of glycoconjugates in C. jejuni biology and based on the hypothesis that glycoconjugates have evolved in...

Carbohydrates serve as fuel and building material

Carbohydrates include both sugars and the polymers of sugars. The simplest carbohydrates are the monosaccharides, or single sugars, also known as simple sugars. Disaccharides are double sugars, consisting ol two monosaccharides joined by a condensation reaction. The carbohydrates that are macromolecules are polysaccharides, polymers composed of many sugar building blocks. Monosaccharides (from the Greek monos, single, and sacchar, stigpx) generally have vaokcttur formulas that are some multiple of the unit CH2( (figure 5.3), Glucose (C6Hi206), the most common monosaccharide, is of central importance in the chemistry of life. In the structure of glucose, we can see the trademarks of a sugar The molecule has a carbonyl group ( C 0) and multiple hydroxyl groups ( OH). Depending on the location of the carbonyl group, a sugar is either an aldose (aldehyde sugar) or a ketose (ketone sugar). Glucose, for example, is an aldose fructose, a structural isomer of glucose, is a ketose. (Most names...

Density Gradient Centrifugation and Protein Concentration

Fig. 1. (A) Analysis of the membrane lipid raft-associated and actin cytoskeleton-associated forms of the TCR -chain in SLE T cells. (A) SLE T cells were treated with the lipid raft-disrupting agent methyl-p-cyclodextrin for 30 min. The cells were lysed, and the detergent-soluble and detergent-insoluble fractions were collected by cen-trifugation, separated by SDS-PAGE, transferred, and blotted with the TCR -chain C-terminal monoclonal antibody (MAb). On treatment with methyl-p-cyclodextrin, the increased level of the p16 and p21 23-kDa forms of the TCR -chain in the detergent-soluble fraction represent lipid raft-associated TCR -chain. Densitometric analysis showed that, in SLE T cells, the lipid raft-associated TCR -chain is increased compared to normal donors. (B) Sucrose density gradient analysis showing distribution of LAT in raft and nonraft fractions 100 x 106 cells were lysed with a lysis buffer containing 1 Brij58 and fractionated by...

Receptor Stability

GABAA receptor internalization is blocked by hypertonic sucrose in both heterologous recombinant systems and in cultured hippocampal neurons (Kittler et al. 2000), conditions which have been shown to impair clathrin function (Heuser and Anderson 1989). In addition, blocking dynamin function by using a peptide that disrupts the association between dynamin and amphiphysin, or by overexpression of the catalytically inactive dynamin K44A mutant, also results in a reduction of GABAA receptor internalization (Kittler et al. 2000 Herring et al. 2003 van Rijnsoever et al. 2005). Together, these suggest that GABAA receptors are internalized via clathrin-mediated endocytosis (Fig. 3). Moreover, both p and y subunits have been shown to bind the clathrin adaptor protein complex AP2, an integral member of the clathrin endocytosis machinery (Kittler et al. 2000). In addition, a form of clathrin-independent GABAA receptor endocytosis has been reported in HEK293 cells although there is not yet any...

Analytical Methods

Diagram Xanthan Production

To check the state of the fermentation, some analytical techniques must be used. The main components to be analyzed are biomass, xanthan, carbon source (sucrose), and nitrogen source (ammonium) concentrations. Dissolved oxygen is also a very important component to consider, but it is usually monitored on line by means of an electrode. Sucrose Analysis The best method to determine the concentration of sucrose in samples of xanthan gum production is an enzymatic kit (see Note 5).

Smith BCA Method

The Smith assay takes 1 h, and requires a minimum volume of 500 p.L with 0.2-50-mg protein (0.4-100 mg mL). As with the Lowry assay, calibration curves are nonlinear, with negative deviation at high protein concentration. Interferences include copper reducing agents (such as reducing sugars) and complexing agents as well as acidifying agents in the unknown sample. Common membrane lipids and phospholipids have also been shown to interfere, and an apparent protein concentration of 17 mg of protein (as BSA) was found for 100 mg of 1,3-dilinoleoygly-cerol.4 Since BCA is a stable reagent at alkaline pH, it can be added to the alkaline copper reagent, so that only one reagent addition is required. The Smith assay has been adapted for 96-well microtiter plates,5 and a linear dependence of absorption (570 nm) on protein concentration was observed over the 1-10-p.g mL concentration range, with 10-mL sample volumes.

The Sugar Solution

The Sugar Solution

Curb Sugar Cravings Once And For All With These Powerful Techniques. Sugar sensitive people might be low in specific neurochemicals that help us feel calm, centered, confident, and optimistic. Sugar is a drug that temporarily makes the sugar sensitive feel better, but with damaging consequences.

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