Natural Remedies for Food Cravings

Sugar Belly Secret

Joe Bovino is not only the creator of this book of strategies but also the author of other four amazon number one bestsellers. Having done extensive research and consulted professionals, he has formulated a strategy on how to get rid of that extra fat and lose weight. That is after almost a decade and a half year. He has also experienced other products prior to researching the natural ways of having a good strategy for weight loss. He can, therefore, be trusted. It entails a fun and simple strategy of having weight loss that melts away the extra pound without exercise or dieting. At times, it is quite hard to stay motivated to work out on a daily basis, especially when you are busy with work and getting older, it is hard to find the time and maintain your workouts! With this book of strategies, you learn how to continue with your usual work and enjoy life with your friends and family while at the same time lose that extra weight and belly, without any shed of sweat. It will help you; Rejuvenating and refreshing your skin, Supercharge your energy levels and become activated most of the time, You will still continue eating your preferred food and drinks without restrictions., Melt away extra pounds and keep them off for a long time. Read more...

Sugar Belly Secret Summary

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Reducing Sugar Method

Reducing sugar assays measure the increase of new reducing ends on fragments produced by the hydrolytic action of -amylase on soluble starch. In the 3,5-dini-trosalicyclic acid method, the concentration of the nitroaminosalicyclic acid formed at the reducing hemi-acetal is measured colorimetrically (83). The measurement of -amylase activity by this method is accurate only if the sample is not contaminated with other amy-lolytic enzymes. A modified procedure for small-scale analysis of cereal -amylases is described 250 L of enzyme in buffer (200 mM sodium acetate, pH 5.5 10 mM CaCl2) was combined with 250 L of 1 soluble starch and incubated at 37 C for 10 min. To the reaction mixture 500 L of dinitrosalicyclic acid reagent (1 nitrosalicyclic acid, 30 potassium sodium tartrate, 40 I N NaOH) was added, heated for 5 min in a 100 C water bath, cooled to room temperature in cold water, and then clarified by centrifu-gation. The absorbance at 547 nm was measured, and the activity was...

Chromogenic Reactions

Many different enzymes produce the same molecules (e.g., NADH, NADPH, orthophosphate, ammonia, hydrogen peroxide, etc.) or different molecules with essentially the same chemical properties (e.g., aldehydes, ketones, reducing sugars, thiols, etc.). This means that very similar or identical chromogenic reactions can be used to detect different enzymes. Thus, classification of chromogenic reactions based on properties of products that are detected seems to be the more useful and practical approach. Such classification is advantageous because it allows one to choose an adequate chromogenic reaction to visualize activity bands even of those enzymes that have not yet been detected on electrophoretic gels.

Discovery of Enzymes

Invertase was discovered in 1860 by Berthelot in yeast (37). The enzyme catalyzes the hydrolysis of sucrose to glucose and fructose Sucrose + H2O glucose + fructose During this period of enzymology, polarimeters were developed that could manually or continuously follow the changes in optical rotation during the conversion of sucrose to glucose and fructose by the change in optical rotation from 56.5 to -23.7 based on the difference in optical rotation of the sucrose (the substrate) to the products (glucose and fructose). This ability eventually led to discovery and quantitation of the effect of pH, temperature, and substrate concentration on the kinetics of enzyme reactions.

The Period From 1890 TO 1940 A Growing Interest

A few years after Fischer's investigations, Eduard Buchner published a series of papers (23, 24) which signaled a breakthrough in fermentation and enzymol-ogy. The experiments began in 1893 (24c). In his first paper on alcoholic fermentation without yeast cells (23) he stated, in a remarkably short and precise manner, that a separation of the (alcoholic) fermentation from the living yeast cells was not successful up to now'' in the following year (24) he described a process that solved that task. He gave the experimental details for the preparation of a cell-free press juice from yeast cells with disruption, filtration under high pressure, and further filtration. He then described the formation of carbon dioxide from carbohydrates (sucrose, glucose, fructose, and maltose). No fermentation was observed with lactose or mannitol. No microscopic organisms were present. Chloroform, an antiseptic inhibiting microbial growth, did not inhibit the fermentation'' (the enzymatic reaction). At...

Cultivation and Media for Metabolite Profiling

In general, agar media for optimal secondary metabolite and mycotoxin production have been based on media containing yeast extract. Yeast extract sucrose (YES) broth was introduced as a semisynthetic broth medium for aflatoxin production by Davis et al. (1966). It was later shown to be a very effective general secondary metabolite production medium when used with a crude yeast extract (DIFCO or SIGMA) and formulated as an agar medium (YES agar) by Frisvad (1981) Frisvad and Filtenborg (1983), and has been used for Penicillium, Aspergillus, Fusarium, Alternaria, and many other fungal genera (Andersen et al. 2002 Thrane 2001). Other media including Czapek yeast autolysate (CYA) agar, Potato dextrose (PD) agar can be used to supplement YES agar, depending on the genus being considered, as seen in Table 1. Some of these semisynthetic agar media can occasionally give problems as certain brands of yeast extract, malt extract, potato extract, agar, peptone, or tryptone, etc. may differ...

Overview of Dietary Assessment in Behavioral Medicine

Nutritional status is one of the most important predictors of health risk. Research consistently demonstrates that diets rich in fruit, vegetables, whole grains, and lean meats from poultry and fish are inversely associated with risk of age-related chronic diseases such as cardiovascular disease, cancer, and diabetes (Kushi et al, 2006 Lampe, 1999 Neuhouser, 2004 Pool-Zobel et al, 1997 Prentice et al, 2004 World Cancer Research Fund AICR, 2007). Conversely, diets high in refined grains and added sugars, but low in diverse plant foods, increase risk for obesity and obesity-related disorders including cardiovascular disease, cancer, and diabetes (Boynton et al, 2007 Kristal et al, 2000 National Research Council Committee on Diet and Health, 1989 Patterson et al, 2004). Despite the strong and consistent diet-disease associations and recommendations to the public to make healthy food choices and limit or avoid added fats, sodium, and empty calorie-type foods, consumers still, for the most...

Purification and characterisation of proteins

Once in the lab, the tissue needs to be disrupted. This is a critical step Cells should be broken open, but cell organelles should remain intact. Usually the tissue is minced first by hand, then cut into a fine pulp by rotating knifes (for example in a Warring blender like those used to make milk shakes in the kitchen) and finally homogenised by the application shearing forces in specialised equipment (Potter-Elyehjem- or DoUNCE-homogeniser, French press). All these steps are performed on ice, buffer solutions are used to keep the pH at the required value, they usually also contain protease inhibitors, antioxidants and sucrose or mannitol to keep the osmotic pressure in the solutions at the same level as in the cell ( 300-350 mosm). Ions like Na+,

Monosaccharides Disaccharides and Polysaccharides

Carbohydrates include simple sugars, or monosaccharides, and longer molecules that contain a number of monosaccharides joined together. The suffix -ose denotes a sugar molecule the term hexose, for example, refers to a six-carbon monosaccharide with the formula C6Hi2O6. This formula is adequate for some purposes, but it does not distinguish between related hexose sugars, which are structural isomers of each other. The structural

The Starch Biosynthetic Pathway

A current view of the starch biosynthetic pathway in maize endosperm is diagrammed in Fig. 1. The immediate precursor of endosperm starch is sucrose. Sucrose is synthesized by photosynthesis in the leaf, and transported through the phloem. By the process of photoassimilate partitioning, sucrose is taken into the sink or storage tissue, which in this case is the endosperm of the kernel. Sucrose is converted into starch by a series of enzymatic steps (Fig. 1). In the cytosol, sucrose synthase and UDP-glucose pyropho-sphorylase convert sucrose into Glc-1-P.*

Proteins RAPs and Natural Compounds in Corn That Inhibit Aspergillus flavus Growth and Aflatoxin Contamination

In another investigation, an examination of kernel protein profiles of 13 corn genotypes revealed that a 14 kDa trypsin inhibitor protein (TI) is present at relatively high concentrations in seven resistant corn lines, but at low concentrations or is absent in six susceptible lines (Chen et al. 1998). The mode of action of TI against fungal growth may be partially due to its inhibition of fungal-amylase, limiting A. flavus access to simple sugars (Chen et al. 1999b) required not only for fungal growth, but also for toxin production (Woloshuk et al. 1997). The TI also demonstrated antifungal activity against other mycotoxigenic species (Chen et al. 1999a). The identification of these proteins may provide markers for plant breeders, and may facilitate the cloning and introduction of

C1anion Approaches

The chemistry associated with the anomeric center is cationic by nature. One interesting approach to C-glycoside synthesis is to reverse the electronic character of this center from electrophilic to nucleophilic. Hence anions at the anomeric center (gly-cosyl anions) have now become fairly common in the synthesis of C-glycosides. Of course, one limitation of this method was the presence of heteroatoms at C2, since any discrete anion at C1 would surely cause an elimination reaction to occur and in turn produce the corresponding glycal (55 58, Scheme 13). This is in fact a very common way of preparing sugar glycals from sugars. As a consequence of this limitation, the chemistry of 2-deoxy C1 anions (56 59) has been extensively explored. An interesting development in this area came with the observation that if the hydroxyl group at O2 is unprotected, deprotonation followed by anion formation at C1 can occur to give species 57, which can go on to react with a suitable elec-trophile to...

Starch Debranching Enzyme

In maize endosperm, the identification and characterization of SDBE, an isoamylase, was achieved using a su1 mutant which carries mutations on the SDBE gene (61). In maize kernels, these mutations lead to increased sucrose levels, reduced amylopectin, and accumulation of the highly branched glucopolysac-charide phytoglycogen (62). The correlation between the su mutation and the deficiency of SDBE was first reported by Pan and Nelson (58). This study showed that SDBE activities from wild-type maize endosperm could be separated into three peaks on a hydroxyapa-tite chromatography column. Their key observation was that the su1 endosperm lacked one of the three activities while the other two were greatly reduced. They also observed that SDBE activity was proportional to the number of copies of the Su allele, suggesting that the Su allele was the structural gene for SDBE. This publication classified SDBE as an R-enzyme or pullulanase, and assumed that the biochemical lesion in the su1...

Detailed Preparation of 500 ml

Dissolve 3.03 g Tris (Boehringer Mannheim) and 85.575 sucrose (BBL) in 250 ml H20. Adjust pH to 8.4 with 5 N HC1. Bring to 300 ml. Autoclave for 15 min in a 500-ml bottle. 4. To the cooled 300-ml Tris sucrose solution, add 25 ml of the following sterile stocks 10 NaCl (filter), 10 yeast extract (autoclave), 10 Bacto-Tryptone (autoclave), and 10 glucose (filter). Then add 1 ml of these filter-sterilized stocks 0.5 M CaCl2 (3.68 g of CaClr2H20 in 50 ml H20) , 1.0 M MgS04 (12.32 g of MgS04-7H20 in 50 ml H2OJ, and 0.5 M MnS04 (1.69 g of MnS04 H20 in 50 ml H20).

[1 Arrays of Transfected Mammalian Cells for High Content Screening Microscopy

Add 7.5 ul EC buffer (EC Buffer is part of the Effectene Transfection kit, Qiagen) containing 0.2 M sucrose and mix thoroughly by pipetting three times up and down. It is important to mix the transfection components just prior to the spotting, to achieve a high reproducibility in transfection efficiency. The optimal incubation times and amounts of transfection reagent are determined empirically for different transfection reagents. However, for optimizing the transfection mix for transfection reagents different from Effectene, the protocol described previously is a good starting point for optimization by simply replacing Effectene with equal amounts of the alternative transfection reagent. The EC buffer from the Effectene kit can be replaced by water without significant loss of transfection efficiencies, when tranfection reagents different from Effectene are used. The presence of sucrose in the EC buffer reduces the loss in transfection efficiencies when the dried arrays are stored...

Conformational Studies

A detailed appreciation of the biological function of a molecule requires that its conformation be understood. Therefore, in recent years a great deal of effort has been directed toward elucidating the solution conformation of oligosaccharides 92 . The work, however, has been heavily focused on oligopyranosides. At the same time, a wealth of information detailing the conformation of furanose rings in nucleotides and nucleic acids has accumulated 93 . However, except for sucrose 94,95 , only recently have conformational investigations of oligosaccharides containing furanose residues been reported 96,97 . Outlined in this section are some recent investigations that have explored the solution conformation of methyl a-d-arabinofuranoside and oligosaccharides containing a-d-arabinofuranosyl residues.

Mass Spectra Interpretation

A systematic nomenclature for labeling fragment ions observed in MS MS spectra has been introduced by Domon and Costello in 1988 (12). As shown in Scheme 1, Ai, B , and C are used to designate fragments containing a terminal (nonreducing end) sugar unit, whereas Xj, Yj, and Zj represent ions still containing the aglycone or the reducing sugar unit. Subscripts indicate the position

Two Basic Strategies

The chemical synthesis of oligosaccharides has been a major focus since the 1980s 8 as a result of our increased understanding of the importance of these sugars in many life processes 3 . Although with low efficiency and sometimes with tremendous difficulties, almost all the sugar linkages known in nature have been synthesized.

Generation of Adenoviruses by Manipulation of Viral DNA

Recombinant adenoviruses were originally created by isolating fragments of the viral chromosome, followed by ligation or recombination to regenerate a complete viral backbone. Generally, DNA isolated from virions is digested with a restriction enzyme that removes the left end of the chromosome, including the viral packaging signals. This renders the large DNA fragment noninfectious. The unique Clal site located at 2.6 m.u. is commonly used for this purpose (Fig. 3A). Undigested viral DNA and the smaller DNA fragments are often removed by sucrose gradient ultracentrifugation. The large fragment is ligated in vitro to a DNA fragment containing the left end of the chromosome and containing the desired modifications (Stow, 1981). If the viral DNA contained in this fragment has a deletion of the Ela region, the resulting recombinant Ad will be a replication-defective El mutant. Inclusion of unique restriction sites in the left end-containing plasmid (generally in the place of the Ela gene)...

Biocontrolspecific Gene Expression In Trichoderma

In the laboratory, high-level induction of extracellular cellwall lytic enzymes is usually obtained by growing Trichoderma on purified chitin, fungal cell walls, or mycelia as sole carbon sources. No, or much less, induction is normally obtained when related compounds such as chitosan, cellulose, unpurified chitin, or laminarin are used. In addition, formation of most chitinolytic enzymes does not occur or is even inhibited by glucose, sucrose, and chitinolytic end-products (Carsolio et al. 1994 Garcia et al. 1994 Lorito et al. 1996a Margolles-Clark et al. 1996 Peterbauer et al. 1996), suggesting that direct induction and or catabolic repression are major regulatory parameters for chitinase formation. Some researchers also found trace quantities of some chitinases (e.g. the 102-kDa N-acetyl- -D-glucosaminidase, the 42-kDa endochitinase and the 33kDa endochitinase) are produced constitutively (Carsolio et al. 1999 Garcia et al. 1994 Haran et al. 1995 Inbar and Chet 1995 Margolles-Clark...

Enzyme Assay Methods

Analysis by colorimetric or chromatographic (TLC, HPLC, CPG) techniques of aglycone or sugar moieties (monosaccharides or disaccharides) released from glycosides (2, 5, 48, 51, 103). In the case of agly-cone liberated by enzymatic hydrolysis from natural glycosides, the volatiles are recovered from the enzyme assay before GC analysis, by either liquid-liquid extraction with a solvent or selective retention on hydrophobic adsorbents (2, 6, 12). A colorimetric assay of monoterpenes (aglycones) liberated from sugars has also been proposed (104).

Direct Enzyme Analysis

Systems employing a sequence of enzyme reactions can be used to increase the ease with which certain enzymatic products can be assayed. Peroxidase is frequently employed to detect the peroxide product of oxidase reactions at low applied potentials (45), thus avoiding electrochemical interferences that may be present in a sample. Difficulties with these systems include the possibilities that the optima for performance of the enzymes in the sequence may not be the same and that multiple enzymes decrease specificity. Such systems are therefore only used for important analytes such as sucrose (36) and aspartame (46) or to overcome major problems, such as electrochemical interference (45). Sucrose is hydrolyzed with the enzyme invertase, where the resultant glucose can be detected with glucose oxidase. Mutarotase is commonly added to such systems to speed up the conversion of a-D-glucose to y8-D-glucose.

Design Enzyme Stability

Until the advent of genetic modification, immobilization was routinely used for improving the stability of enzymes in industrial processing. Immobilization anchors the enzyme onto a suitable carrier such as agarose, alginate, gelatin, or polyacrylamide either by covalent coupling, adsorption, matrix entrapment, or encapsulation. The primary advantage of immobilization is to increase the stability of the enzyme, as well as convenience in separation and recovery, and reuse of the enzyme from the product mixture. In the manufacture of high-fructose corn syrup, glucose isomerase is immobilized by direct crosslinking of lysed cells with glutaraldehyde and extruded with structural support substances, such as gelatin and polyamine (19). Although not directly relevant to food applications, immobilization of enzymes has been shown to have higher stability in reactions carried out in water-miscible organic solvents (20). Apart from the conventional approaches, Stempfer et al. (21) engineered a

Thinking Beyond Communications

Appropriately priced, made available in convenient and attractive outlets, and backed by relevant and engaging communications. This mix of variables (the so-called marketing mix) is manipulated to maximize satisfaction. Again the rubric can be applied to social and health behaviors. Case Study C demonstrates how the marketing mix was used to think through a strategy for encouraging doctors to prescribe sugar-free medicines.

Alternative Substrates

While isozymes are apt to have different energies of activation, even under the same assay conditions, an enzyme acting on different substrates can in some circumstances exhibit the same energy of activa-tion10,11. Yeast sucrase, for example, has an energy of activation of 46 kJ-mol 1 (or 11.0 kcal-mol1) for both sucrose and raffinose11-13. The rate-determining step in the enzyme-catalyzed reaction may differ with alternative substrates, and this may be reflected in the observed energy of activation. Likewise, if the rate-determining step changes with protein modification, assay conditions, or through site-directed mutagenesis, Arrhenius plots should reflect those changes. An example is the myosin ATPase which exhibits biphasicity in the Arrhenius plot with ITP as a substrate, but a typical linear Arrhenius plot with ATP as the substrate. Levy, Sharon, and Kosh-land14 suggested that this may be the result of the 6-amino group on ATP interacting with some functional moiety...

Thinking Beyond the Individual

Social marketing, then, recognizes the collective determinants of human behavior and one of the first decisions to be made in any intervention, as we have already noted, is whose behavior has to change. Sometimes this will be individuals. For example, attempts to improve dental health might consider targeting parents to encourage tooth brushing and reduced sugar consumption. However, as Case Study B shows, water fluo-ridation is likely to be much more effective -and the individual's control over this is very limited fluoridation of water supplies easily trumps the potential health promotion power of other, more individual-based, health promotion interventions.

Paying Careful Attention to the Competition

This final principle recognizes that people -whether parents or water providers - have a choice. They can - and often do - continue with their current behavior. It is therefore very important to look closely at this competition in order to understand what benefits it is perceived to bring and how our alternative behavior can be made more attractive. In Case Study C, for example, the doctors were not prescribing sugar-free alternatives because it was easier to go with the option automatically suggested by their software. As in so many other aspects of life, convenience matters. The solution was to get rid of the competition and change the software so that it favored sugar-free.

The Vital Role of Strategic Planning

In nudging this or that behavior a few degrees in the right direction we do not just want more people to visit cessation services, or a few GPs to prescribe sugar-free medicines - we want to help everyone to become smoke-free and all doctors to offer healthier medicines. More broadly our interest is improving the public health of whole societies not just behavior change, but social change.

The role of dairy products in preventing dental caries

Sucrose, are recognized as being a major contributor to dental caries' etiology. Other social factors such as alcohol and tobacco use, drug abuse, poor hygiene, Lactose is less fermented by indigenous oral microflora than sucrose only lowering the biofilm microenvironmental pH to around 6.0 compared to pH 5.0 with sucrose (Rugg-Gunn et al., 1985). Most anticaries properties are Weiss and Bibby (1966) utilized an in vitro test with extracted teeth and with similar test variables (milk and flavored-milk) to show tooth enamel demineralization was reduced when teeth were exposed to cariogenic substances in an acetate buffer (pH 4.0) system. They concluded casein proteins were rapidly absorbed onto the tooth enamel surface and provide resistance to acid demineralization. Concurrently, Jenkins and Ferguson (1966) studied the effects of milk on acid production in human plaque. Subjects refrained from tooth brushing for three days to allow plaque accumulation on their teeth. Subsequently,...

Chromatin Remodeling Enzymes

The ability to remodel nucleosomes is vital for accurate and regulated transcription and efficient DNA replication. Nucleosome remodeling is defined as an enzymatic activity capable of altering the position or stability of the nucleosome. The flagship of the ATP-dependent, enzyme complexes that remodel chromatin is the SWI SNF complex. The SWI SNF class of chromatin remodeling complexes is named for the genetic screens used to identify sucrose non-fermenting (Snf) or mating-type switching defective (Swi) mutants. The SWI SNF chromatin remodeling complexes contain an enzymatic ATPase protein subunit, which on its own is capable of remodeling chromatin in vitro (Cote et al., 1994). In yeast this protein is called Snf2 Swi2 and in humans there are two homologous proteins Brgl and Brm. Each of these proteins contains a structural motif called a bromodomain, which binds to acetylated lysines. Acetylation of histones by HATs promotes recruitment of SWI SNF remodeling complexes through...

Quality deterioration of fresh produce respiration ethylene senescence and breaking of dormancy

Senescence is the natural ageing of the of ethylene and anything else that speedsuprespiration ratesasdescribed above. Senescence ultimately affects all aspectsofquality,endinginthedeathof the product. Some senescence changes can specifically affect certain types of fresh produce processing, for example, changes to the chemical and physical structure of the cell wall (Jimenez et al., 1997). Although in fresh produce, texture is highly dependent on cell turgor (see section 7.4 below), the integrity of the cell wall is important to the texture of some processed products (Femenia et al., 1998). In some fruits and vegetables (e.g. apples and tomatoes), the breakdown of intercellular adhesion between cells leads to a condition known as mealiness which is generally perceived as a loss in textural quality (Van der Valk and Donkers, 1994). In potatoes, so-called senescence sweetening is where, over time, storage starch is gradually converted to sugars. Concentrations of reducing sugars of...

Other Cytokine Receptor Superfamily Members

The notion of preformed dimers on the cell surface is not solely associated with the cytokine receptor super-family. The epidermal growth factor (EGF) plays important roles throughout development including cell proliferation, differentiation, and survival of multicellular organisms 29 . The EGF receptor (EGFR) is a member of the growth factor receptor tyrosine kinase family. Using chemical cross-linking experiments and sucrose density-gradient centrifugation, experiments have demonstrated that EGFR forms a dimer in the absence of bound ligand 30 . A flexible rotation model was proposed for EGFR activation in which the binding of EGF induces rotation of the juxtamembrane domain and, accordingly, the transmembrane domain. Consequently, the dimeric intracellular domains dissociate, allowing for the catalytic kinase domains to become accessible to their substrate tyrosine residues.

Removal of glucose

Glucose is a reducing sugar that can react with amino groups in, for instance, proteins, to form colored Maillard components that are often undesirable in food products. Additions of GOX to the food system in the presence of air or an oxygen donor like hydrogen peroxide will result in the conversion of glucose to the non-amine-reactive gluconic acid and thus prevent the formation of these Maillard components. The best known application is the prevention of nonenzymatic browning in egg white powder. A detailed description of this application is given by Scott (10). Treatment can be done up to 50 C and in the pH range from 4 to 7. Egg white is neutral at the time of laying, but the pH rises quickly as carbon dioxide is lost, such that it is generally close to 9 when the egg white is processed. The pH is brought below 7 by adding citric acid addition of hydrogen peroxide as oxygen donor is advantageous. Owing to the low affinity of GOX for glucose, high quantities of enzyme and or long...

DNAs Plasmids Cloning and Transfection

30 Acrylamide to-acrylamide solution (29 1 Sigma), N,N,N,N'-tetramethyl-ethylenediamine (TEMED Biorad, Hercules, CA,), and 66 (w v) sucrose. Store at 4 C. 5. 5X Laemmli (5) SDS-sample buffer 0.5 M of Tris-HCl (pH 6.8), 7.5 (w v) SDS, 25 mM of EDTA, 5 M of sucrose, and 0.05 (w v) of bromophenol blue. Store at -20 C.

Western Immunoblotting of Intact C2GnTl and the EGFPConjugated Protein

Prepare 3-mm-thick SDS gel by mixing 2.6 mL of 30 acrylamide Ms-acrylamide, 1.4 mL of separating buffer, 1.2 mL of 66 sucrose, and 5.1 mL of water. Add 104 L of 10 SDS, 60 L of 10 ammonium persulfate, and 6 L of TEMED. Pour the gel (leaving space for a stacking gel) and overlay with water. The gel should polymerize in 20-30 min. 3. Prepare stacking gel by mixing 700 L of 30 acrylamide to-acrylamide, 700 L of stacking buffer, 600 L of 66 sucrose, and 3.2 mL of water. Add 52 L of 10 SDS, 30 L of ammonium persulfate, and 3 L of TEMED. The gel should polymerize within 30 min.

In Vivo Expression Technology Approaches

Nine of the 72 ivi genes appeared to encode sugar-related functions, including genes involved in ribose, cellobiose, sucrose, and sorbitol transport. Another nine genes encode functions involved in acquisition and synthesis of amino acids, nucleotides, cofactors, and vitamins, indicating their limited availability in the GI tract. Four genes involved in stress-related functions were identified, reflecting the harsh conditions that L. plantarum encounters in the GI tract. Another four genes encoding extracellular proteins were identified that could mediate interactions with host GI-tract epithelial cells. Remarkably, the protein encoded by one of the hypothetical proteins identified in this study in L. plantarum is a homologue (32 identity) of the only conserved hypothetical protein that was identified with IVET in L. reuteri (43). Moreover, a large number of the functions and pathways identified in L. plantarum have previously been identified in pathogens as...

Importance Of Starch Phosphorylase To Cold Storage Of Potato And Plant Starch Deposition

Interconversions between starch and sucrose are of great interest, particularly with respect to a phenomenon referred to as cold-induced sweetening in potato. Cold-induced sweetening generally occurs when storage temperatures are reduced from 10 C to 2 C over the course of several days. Since starch phosphorylase acts upon oligoglucans to form hexose phosphates, which are presumed precursors to sucrose, starch phosphorylase activity has been proposed as a logical catalyst for the initial process of cold-induced sweetening of potato during storage (2). However, despite numerous efforts, a direct role for starch phosphorylase in cold-induced sweetening has yet to be demonstrated. Temperature shift experiments, either from 10 C to 2 C or vice versa, were accompanied by negligible changes in starch phosphorylase activity (3). A follow-up study (2) on the dual role of glycolytic intermediates and amyloplast envelope integrity showed that visible deterioration to the double envelope was not...

Detection of the Amylosucrase Activity

The simplest way to detect amylosucrase activity is in a small in vitro reaction assay. An aliquot of the enzyme sample is mixed with 100 L reaction buffer (5 (w v) sucrose, 50 mM Na citrate pH 6.5) and incubated for some minutes up to several days at 37 C. Very high activity samples show a white precipitate, and samples with a lower activity are mixed with Lugols solutions (0.5 mM I2KI). This test can be used for relatively rough quantitative determination by comparing the color intensity with a calibration row. A more accurate activity test can be done by measuring the real-time fructose release in an optically coupled enzymatic assay. The reaction cuvette contains 20 sucrose, Na citrate buffer (100 mM, pH 7), glycogen (0.05 ), NAD (0.4 mM), ATP (1 mM), and the helper enzymes hexokinase (1.5 units mL), phospho-glucose-isomerase (2 g mL), and glucose-6-phosphate dehydrogenase (5 g mL), which are added directly to the reaction mixture (200 L). The assay is started by addition of the...

Substrate Specificity

Amylosucrase has a high substrate specificity. Several substrates like 6-desoxy-6-fluorosucrose, 6-desoxysu-crose, 4,6-dideoxysucrose, 3-desoxysucrose, and a-D-allopyranosyl-ft-D-fructofuranoside were used as substrates. None of the tested substrates were incorporated into a polymer, but 6-desoxy-6-fluorosucrose, 6-desoxysucrose, and 4,6-dideoxysucrose could inhibit the enzymatic reaction (9). Under optimal conditions the amylosucrase achieves nearly 100 conversion of its substrate sucrose to polymers (or oligomers) and fructose. Invertase activity (the splitting of sucrose into fructose and glucose without any glycosylation step) is negligible for the amylosucrase reaction under all reaction conditions tested. Remaud-Simeon et al. (5) showed that under special reaction conditions a transglycosylation reaction of oligomaltose may be possible. When the substrate sucrose was lacking, the amylosucrase could disproportionate oligoglucans. The glycosidic residues were mainly transferred...

Introduction A Chemical Reactions Catalyzed

In 1861, Louis Pasteur discovered the microbial origin of the gelification of cane sugar syrups (1). In 1874, the corresponding product was named dextran because of its positive rotatory power. The microorganism causing that gelification was isolated in 1878 by Van Tieghem and named Leuconostoc mesenteroides (2). Hehre demonstrated in 1941 that dextran could be synthesized from sucrose by a cell-free filtrate (3). The corresponding extracellular enzyme was named dextransucrase by Hestrin et al. (4). n sucrose dextran + n fructose (glucose)n Sucrose is the only natural D-glucosyl unit donor used as substrate by the enzyme. The energy necessary for D-glucose polymerization simply comes from the cleavage of the high-energy glycosidic bond. No activated intermediate or cofactor is needed. The most widely used dextran is produced by the dextransucrase of the strain L. mesenteroides NRRL B-512F, which synthesizes a very highly linear polysaccharide containing 95 a-1,6 bonds. Besides...

Synthesis of Nondigestible Oligosaccharides

The dextransucrase from the soil bacterium Leuconostoc mesenteroides NRRL B-1299 is known to catalyze the synthesis of dextran polymers containing a-1,2-linked branched chains (Table 1). When this specific glucosyltransferase is used in the presence of maltose as acceptor and of sucrose as D-glucosyl donor, a-gluco-oligosaccharides are obtained, which contain a-1,2 glucosidic bonds (24, 25) at their nonre-ducing end and a maltose residue at the reducing end.

Isomaltooligosaccharides

Using glucose as an acceptor, a European patent (31) describes the formation of isomalto-oligosacchar-ides from sucrose with 10-20 anhydroglucose units the average molar weight may be in the range of 20005000. The molar ratio of sucrose to glucose is in the range of 2-5. The sucrose is added to the reaction solution of 0.2-0.5 M glucose and 1000 units dextran-sucrase quasi-continuously. The sucrose concentration should not exceed 25 of the total dry substance the reaction is conducted to high conversion. The product mixture contains fructose in a molar range corresponding to that of the sucrose added and may be used as a sweetener. Some further data are presented by Pereira et al. (32) with, however, limited yields of oligosaccharides of up to 45 . Systematic investigations concerning reaction engineering were undertaken by K. Demuth (23).

Properties As Enzyme A Mechanism

In a secondary reaction, the so-called acceptor reaction, the glucosyl moiety is transferred from sucrose to the acceptor molecule instead of the growing dextran chain to give the acceptor molecule elongated by one single glucosyl unit as the primary product. A specific acceptor binding site at the active center of the enzyme

Kinetic Characteristics

Sucrose is the only simple carbohydrate used by dex-transucrase as a D-glucosyl donor. Initial reaction rates follow Michaelis-Menten kinetics up to 200 mM sucrose concentration, but the enzyme is inhibited by higher substrate concentrations (55). The inhibitor constant for sucrose is 730 mM (56). This inhibition can be removed by acceptor or dextran addition (6, 57, 58).

Mathematical Modeling and Reaction Engineering Considerations

The enzyme is assumed to have three binding sites one covalent glucosyl-, one dextran, and one acceptor (53, 54). Reactions 1,2, and 3 represent the growth cycle of dextran formation. A sucrose molecule S is bound at the enzyme E, fructose F is released, and the glucosyl G is transferred to the dextran chain Gi, which is always linked to the enzyme. Fructose can be linked to the acceptor site, and react with G to form leucrose FG (reactions 4 and 9). The same reactions are possible with other acceptors A (reactions 6 and 13), and acceptor products (except leucrose) may again react as acceptors (reactions 6 + j and 13 + 2xj). Sucrose is able to bind at the acceptor site (reaction 5) but does not react to an acceptor product, thus leading to inhibition at high concentrations. For approximate modeling, reactions 10-12 and 14 may be neglected. Booker et al. (54) presented approximate kinetic correlations as well as a rational model based on the reaction mechanism for different...

HighMW Dextran Synthesis

Standard reaction conditions are 20 mM acetate buffer, pH 5.4, containing 100 g L sucrose, 0.05 g L calcium chloride (CaCl2 2H2O), 1 g L sodium azide at 30 C. Dextransucrase activity must be in the range of 0.3-1.0 U mL (1 unit corresponds to the amount of enzyme which catalyzes the production of 1 mol of fructose per min in the above reaction medium). Dextransucrase activity is measured by determining the initial rate of production of reducing sugars followed by the 3,5-dinitrosalicylic acid (DNS) method (70). Reaction samples (0.1 mL) are withdrawn at various time intervals. Dextransucrase reaction is stopped by addition of 0.1 mL DNS solution (10 g L DNS, 300 g L sodium potassium tartarate, 16 g L sodium hydroxide). After heating for 5 min at 95 C followed by ice cooling, 1 mL water is added and absorbance is read at 540 nm. A standard curve is prepared with standard fructose solutions ranging from 0 to 2 g L in fructose concentration. Any catalytic activity producing reducing...

LowMW Oligosaccharide Synthesis

Standard reaction conditions are 20 mM acetate buffer, pH 5.4, containing 100 g L sucrose, 20-100 g L acceptor (maltose, isomaltose, ), 0.05 g L calcium chloride (CaCl2 H2O), 1 g L sodium azide at 30 C. Dextransucrase activity must be in the range of 0.31.0 U mL. The key factor determining the molecular-weight distribution of the products is the sucrose donor acceptor molecular ratio. Reaction samples (0.1 mL) are heated for 5 min at 95 C to stop the reaction. They are diluted with ultrapure water to reach a total carbohydrate concentration < 5 g L and analyzed by HPLC using a C18 column and ultrapure water as eluent. Fructose, maltose, leucrose, sucrose, and gluco-oligosaccharides with a polymeriza For immobilized dextransucrase it is obvious that systematic errors in the activity measurement can be avoided only when dextran formation is suppressed as far as possible. This can be achieved by adding an excess of good acceptor like maltose (71). A test in which maltose was applied in...

Iisources And Function Of Levansucrase

There are a number of microorganisms reported as levan producers beyond the genus (7). Levansucrases from some bacteria are inducible and exocellular, whereas some are constitutive and endocellular. The synthesis of levan by levansucrase in a sucrose-containing medium gives the microorganisms a mucoid morphology. Researchers have demonstrated that this mucoid structure of the microorganisms plays a role in the symbiosis (Bacillus polymyxa) or phytopatho-genesis (Erwinia amylovora) of plant interactive bacteria. In addition, the enzyme also participates in the defense mechanism against environmental stress (Bacillus subtilis) (8). The function of levansucrase located intracellularly in some bacteria is not understood. The diversity of the enzymatic properties of levansucrase may be caused by the different biological function of each enzyme in each microorganism. Recently, the enzyme was overexpressed in E. coli for the mass production of levan with high yield and purity (9). An...

Utilization Of Levansucrase

Levan, produced by levansucrase from sucrose, consists entirely of fructose and glucose at one terminal in each molecule (Fig. 1). Many potential applications of levan in food and pharmaceutical industries exist, but the use of this polymer is not yet practical due to the lack of information about its polymeric properties needed in industrial fields. Novel applications of levan include such uses as emulsifier, formulation aid, stabilizer, thickener, surface-finishing agent, encapsulating agent, and carrier for colors or flavors and fragrances in the food industry (7). It has been reported that levan has certain biological activities, such as the promotion of infection and necrosis, tumor inhibition, and an increase in cell permeability to a cytotoxic agent (11). Calazans et al. (12) postulated that levan showed antitumor activity against sarcoma 180 and Ehrlich carci

Biochemical Properties Of Levansucrase

The substrate specificity of levansucrase is extremely low. Sucrose and raffinose serve as fructosyl donors. The glucose moiety of sucrose can be replaced by various sugars such as D-xylose, L-arabinose, lactose, etc. Substrate specificity for the acceptors is, however, relatively broad. Several kinds of alcohol mono-, di- or oligosaccharides sugar alcohols and levan are known acceptors. The specificity toward acceptor molecules differs depending on the catalytic properties of each enzyme. In B. subtilis levansucrase, a turnover number of 17,000 moles of sucrose per minute per mole of enzyme at 37 C and pH 6.0 was obtained (21). Sucrose is directly converted to levan by the enzyme without the need of any primer, nucleotide sugar intermediate, or sugar phosphate. The Km values of levansucrase from B. subtilis are 20 mM for sucrose and 50 mM for the analog glu-cosido-sorboside at pH 6.0 and 37 C. The affinity constant for the short-chain levan (DP 40) acting as an acceptor-activator is...

Levansucrase Activity

Several analytical methods can be employed for the measurement of levansucrase activity. Isotopic procedure utilizes 14C sucrose as a substrate and measures the incorporation of isotopes into the products. This procedure is powerful when a wild-type strain with very little activity is employed as an enzyme source. The measurement of the weight of dried levan that is formed by the enzyme reaction is conducted after ethanol precipitation of the reaction mixture. Another method for the determination of levan content, which gives a linear curve at low concentrations, is to measure the optical density at visible range (450550 nm), corresponding to the turbidity of the reaction mixture generated by the formation of levan. By paper and TLC chromatographic analyses, one can distinguish the polymeric products (nonmobile), oligomeric products, substrate, and glucose (byproducts), qualitatively or quantitatively, after visualization. Sucrose hydrolysis is commonly related to determination of...

Mechanisms of Interferon Action

Interferon is known to activate the proteasome, which is a ubiquitously expressed multi-subunit complex that degrades proteins. To examine the possibility that IFN treatment induced the proteasome that specifically degrades GFP translated from the IRES construct, inhibition of GFP expression from IRES clones by interferon was assessed in the presence of two proteasome inhibitors lactacystin and epoxomicin (Fenteany & Schreiber, 1998 Meng et al., 1999). Inhibition of GFP expression from the IRES clones by IFN treatment was not affected when Huh-7 cells were pretreated with the proteasome inhibitors, indicating that interferon action on the IRES inhibition is not due to activation of proteasome pathways. We also examined the possibility that interferon treatment impaired the loading of polyribosome with IRES containing mRNA in the transfected Huh-7 cells. This was accomplished by examining the distribution of IRES containing GFP mRNA in polysome fractions by northern blot analysis....

Separation Of Dna In The Ultracentrifuge

Velocity or equilibrium ultracentrifugation of DNA represents the only serious alternative to DNA electrophoresis. For certain applications it is a powerful tool. However, for most applications, the resolution of ultracentrifugation just isn't high enough to compete with electrophoresis. DNA can be separated by size in the ultracentrifuge by zonal sedimentation. Commonly density gradients of small molecules like sucrose are employed to prevent convection caused by gravitational instabilities. Sucrose gradient sedimentation is tedious because ultracentrifuges typically allow only half a dozen samples to be analyzed simultaneously.

Medium Chain Fatty Acids

Polyhydric alcohol fatty acid esters have great potential for use as emulsifiers in food formulations (Razani-Rohani and Griffiths 1994). They also possess antifungal properties and, therefore, may exert a preservative effect in foods. Kato and Shibasaki (1975) demonstrated strong fungistatic activity of glycerol monocaprate and glycerol monolaurate toward Aspergillus niger, Penicillum citrinum, Candida utilis, and Saccharomyces cervisiae. Sucrose monocaparte and sucrose monolaurate were found to be slightly inhibitory to a spoilage film-forming yeast inoculated into a soy sauce substrate (Kato 1981). Six sucrose esters substituted to different degrees with a mixture of palmitic and stearic acids were examined by Marshall and Bullerman (1986) for antifungal properties. Growth of Aspergillus, Penicillium, Cladosporium, and Alternaria spp. were inhibited in media containing 1 of the sucrose esters.

Diffusion vs Convection

The effect of different sizes of molecules and their distribution within brain tissue was studied by several investigators (11,12). Bobo et al. measured the volume of distribution of small (14C-sucrose, Mr 359) and large molecules (111In-Transferrin, Mr 80,000) (10). After initiating interstitial infusion, the flow rate was gradually increased from 0.5 L min to 4 L min and maintained at the higher level for 24 h. The VD within brain tissue increased linearly with the volume of infusion (VI) of both molecules, although 14C-sucrose distributed faster and occupied a larger volume compared with 111In-Transferrin. Importantly, there was no evidence of any increase of intracranial pressure during the infusion period (10). Kroll et al. confirmed findings that showed a linear increase of VD proportional to increase in time, volume, and dose with a constant infusion rate and concentration (13). They studied the pharmacokinetics of iron oxide nanocompound (MION) and CED characteristics in a rat...

Purification of Enzyme Product

Using a flash evaporator and a vacuum pump, enzyme product is exchanged three times with 99.99 deuterium oxide and analyzed by 1H NMR spectroscopy. This will indicate the linkage of the newly-added sugar. 3. For further linkage analysis, radioactive enzyme product is analyzed for its susceptibility to cleavage by galactosidases. The specificity of galactosidases helps to identify the linkage point and anomeric configuration of the newly-added sugar in the enzyme product. Incubate an aliquot (2000 cpm) of enzyme product at 37 C for 60 min with 0.01-1 U ( imol min) of a- or P-galactosidase in 100 L containing citric acid sodium phosphate buffer and 0.01 BSA at pH 4.3. After the incubation, add 800 L of water and pass the mixture through a C18 Sep-Pak column. Undigested product should elute with methanol as described in Subheading 3.3. Digested product elutes as free 3H galactose when eluted with water, and as non-radioactive substrate when eluted with methanol. A comparison of controls...

Acyl Transfer Reactions

Between N-acetyl-L-(amino acid)-2-chloroethyl esters and amides of L or D amino acids (78). This reaction can be utilized in the production of optically active amides by taking advantage of the regio- and stereo-specificity of the enzyme (79). Using hydroxyacid esters, bifunctional molecules containing both a methyl ester and an OH group as substrates, lipases can catalyze intermolecular or intramolecular condensation to give corresponding oligomers or lactones as the product (80, 81). The preference for oligomerization or lactonization depends on the chain length and degree of substitution of the substrate. This reaction has been exploited for the synthesis of optically active y-substituted lactones from symmetrical hydroxy-carboxylic acid esters (82). Lipases have been used in the regioselective acylation of sugars, the process of which can rarely be effectively performed in organic synthesis and often requires multisteps of protection and deprotection of the various hydroxyl...

RA BIoSynthesis In cELL cuLTuRES

In terms of regulation of RA in cell cultures it is constitutively expressed in Coleus blumei without any medium manipulation (65). Plant cell cultures are known to accumulate 8-10 of their dry weight as RA, a content much higher than parent plants (53,66), contradicting the callus and shoot culture comparisons of the Ocimum study (63). The pathway of RA biosynthesis is through the aromatic amino acids, phe-nylalanine, and tyrosine (65). Cell suspension cultures of C. blumei (66), Rosmarinus officinalis, Salvia officinalis (67), and Anchusa officinalis (68,69) have been used to produce RA in cell suspension cultures. The influence of various macronutrients and growth regulators on growth of A. officinalis has been investigated (68,69). Concentrations of 3 sucrose, 15 mM nitrate, 3 mM phosphate, and 0.25 mM calcium were best for increasing both biomass and RA contents with yields in the range of 10-15 (68). Similar nitrogen, potassium, and phosphate optimizations proved useful for RA...

Behavioral analysis of DISC1 mouse models Table

How to differentiate between tests relevant to negative symptoms and tests relevant to depression. Since DISC1 is a candidate gene not only for SZ, but also for other mental disorders including depression, such phenotypes are of interest even if not clearly attributed to a specific disease. Social withdrawal can be modeled by a three-chamber sociability test. It has been applied to the Q31L and L100P point mutants (Clapcote et al., 2007) and to the inducible CaMK-Ac Tg (Li et al., 2007) and showed abnormalities in all of them, except for L100P. The constitutive (Hikida et al., 2007) and inducible CaMK-AC Tg (Pletnikov et al., 2008) were tested in other social tests, in which only the inducible CaMK-AC Tg males differed from the wild-type littermates. Anhedonia can be manifested in mice by decreased reinforcing properties of rewards (Nestler et al., 2002). The Q31L, but not the L100P mutants consume less sucrose in the sucrose consumption test. The forced swim test is widely used to...

Human Flora Associated Animals

HFA animals are created by inoculating germ-free animals with a human fecal homogenate (94). The resulting microbial profile of HFA animals is partly dependent on the differing ability of the various microorganisms in the human fecal sample to colonize the animal GIT. Previous studies have shown that certain microorganisms of human fecal origin were unable to colonize the rodent GIT (95). There may be several reasons for this, such as diets or host factors like transit times and physiological conditions. It has been demonstrated that mice, fed with a commercially available animal feed, may have a reduced, or even undetectable level of bifidobacteria in feces. However, after feeding these mice an alternative diet for several weeks, bifidobacteria could be detected in the mice that were fed sucrose or amylose, with particularly dense populations of bififodobacteria

Factors Affecting AflatoxinST Biosynthesis

The best-known nutritional factors affecting aflatoxin biosynthesis are carbon and nitrogen sources (Adye and Mateles 1964 Bennettetal. 1979 Luchese and Harrigan 1993). It is clear that simple sugars such as glucose, sucrose, fructose, and maltose support aflatoxin formation, while peptone and lactose are not (Buchanan and Stahl 1984 Payne and Brown 1998). Woloshuk et al. (1997) reported the connection between alpha amylase activity and aflatoxin production in A. flavus. Yu et al. (2000c) identified a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus next to the aflatoxin pathway gene cluster. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes in aflatoxin-conducive medium. This is the first evidence that primary metabolism (sugar metabolism) and secondary metabolism (aflatoxin biosynthesis) are genetically linked on the chromosome. A...

Products Available

Cranberry is available in a variety of forms such as fresh or frozen cranberries, cranberry juice cocktail, other cranberry drinks, cranberry sauce, and powder in hard or soft gelatin capsules (2,10). Cranberries are approx 88 water and contain flavonoids, anthrocyanins (odain), cetechin, triterpinoids, y-hydroxybutyric acid, citric acid, malic acid, glucuronic acid, quinic acid, benzoic acid, ellagic acid, and vitamin C (2). Fresh or frozen cranberries are a good source of cranberry because they contain pure fruit however, because of their high acidity and extremely sour taste, they are less readily used in clinical practice (1). Pure cranberry juice is tart like lemon juice because of the high citric and quinic acid content (2). Cranberry juice cocktail is more palatable, but is only 25-33 juice and contains corn syrup as a sweetener (2,10), whereas other cranberry juice drinks contain as little as 10 juice (2). These sweetened beverages are relatively high in calories (approx 140...

Isabell A Schmitt Milana Dolezal Michael F Press

The initial in vivo human studies demonstrating a correlation between estrogen uptake by the tumor tissue and responsiveness to hormonal ablation therapy were followed by in vitro observations in primary breast cancer slices or ho-mogenates. Procedures for cell fractionation and sucrose density gradient analysis were developed, which permitted the identification and quantification of estrogen binding in breast cancer samples. Using these techniques, the characteristics of an estrogen receptor (ER) were defined as a protein which bound its estrogen ligand in a highly specific fashion, with high affinity (Kd of 10 10 M) and with saturation by excess ligand. This interaction with the receptor protein was considered to mediate a variety of biological responses. Use of in vitro assays of tumor tissue samples improved the predictions of patient response to hormonal therapy. Approximately 50 -60 of women with ER-rich breast cancers responded to such therapy, while fewer than 10 with ER-poor...

Biomass As A Sugar Source

Cellulose is a long linear polymer ranging from 1000 to 1000 000 D-glucose units. Glucose monomers are linked together with b-1,4-glycosidic bonds to form highly stable chains, and these chains further aggregates together via hydrogen bonds to form a rigid crystalline structure that is water-impermeable, water-insoluble, and resistant to enzymatic hydrolysis (Linko 1987). The boundary and sequence structure of the molecules determine the chemical properties of cellulose (Alen and Sjostrom 1985). On the other hand, hemicellulose, which is alkali-soluble, is composed of short, highly branched copolymer of both six-carbon and five-carbon sugars. The branched structure allows hemicellulose to exist in an amorphous form that is more susceptible to hydrolysis. Compared to cellulose, which is similar across all biomass sources, hemicellulose is quite diverse in structure and composition, depending on the source. The hydrolysis product of hemicellulose is typically a mixture of xylose,...

Utilityutilization Of Enzymes In Foods

In the food industry, the main use of fiF is for producing chocolate-coated candies with a soft and creamy center. A hard and firm center is prepared with sucrose and molded into the desired shape. After coating with chocolate the fiF is inoculated into the center to change its consistency to a permanent and noncrystallizable cream. Purified fiF is preferred to yeast since variations in both the added amount and in the strains of yeast utilized does not assure uniform quality of softer centers. Other fiF uses are related to the production of artificial honey and to the enzymatic determination of glucose and sucrose in dietetic foods (29). The productivity value, the operational stability and mechanical stability of F, the low cost of the immobilization on a variety of carrier, and the absence of undesirable reaction byproducts make the scale production of inverted syrups from sucrose and nonrefined sugars economically very attractive. In the confectionery industry fructose is...

Functional genomics of macromolecular and nutrient metabolism

Based on comparative genomics and putative gene expression patterns of LAB, it is clear that these organisms are equipped to utilize a variety of mono-, di- and oligosaccharides. The coordinated regulation of expression of genes involved in sugar transport depended on sugars present in the local environment (Barrangou, 2006). Phosphoenolpyruvate (PEP) sugar transferase systems were important for the uptake of glucose, fructose, sucrose, and trehalose, whereas ATP-binding cassette (ABC) transporters were important for the uptake of raffinose and fructo-oligosaccharides (FOS) in L. acidophilus (Barrangou, 2006). The PEP sugar phosphotransferase and ABC transporter systems appear to be important features of LAB that are particularly adept at sugar uptake and utilization. For example, L. johnsonii encodes 16 phosphotransferase-type sugar transporter systems, representing a relatively large number of sugar transporter systems when compared to other bacteria with similarly sized genomes...

Km Vmax Substrates Nonsubstrates and Activation Energy

Tables 4 and 5 report the Michaelis constant for sucrose, Kmuc, and raffinose, KRaf, of Fs from several plants and microorganisms, other identified substrates and nonsubstrates, the optimal pH, and maximal velocity. In plants, Kmuc for alkaline F is higher than that of the acid form. The activation energy, Ea of plant F has been reported to be 57.3 kJ mol for Ricinus communis (25) and 147 kJ mol for nodules of Cicer arieti-num L. (51). The Ea value is 37.63 kJ mol in whole yeast cell for acid F (56) and 29.30 kJ mol for a soluble preparation of acid F from yeast (57).

Specific Mechanism of Action

The mechanism of yeast F action involves a nucleo-phile and a proton donor in a two-step reaction. The Glu204 residue acts as a proton donor to the glycosidic oxygen of sucrose. The C-2 of the fructose moiety is attacked by the nucleophile Asp23. A covalent fructo-syl-enzyme complex is formed and glucose is released. In the second step a proton from the water molecule is abstracted by Glu204. The hydroxyl group displaces fructose, and the original active center residues are restored. Glu204 is essential for sucrose hydrolysis while Cys205 possibly plays a supportive role in catalysis by maintaining a suitable microenvironment in the active site, or perhaps in aiding substrate binding. A carboxylate group, along with histidine, methionine,

Mechanisms Of The Bifidogenic Effect

Interestingly, while many bifidobacteria grow well when cultured with prebiotic oligosaccharides as their sole carbon and energy source, they often do not grow when supplied only with the monosaccharides from which these oligosaccharides are composed (74,171,172). This physiology may be another consequence of their evolution in an environment with a limited availability of simple sugars. It suggests that bifidobacteria lack transport mechanisms for many monosaccharides and import prebiotic oligosaccharides before hydrolyzing and metabolizing them. This presumably minimizes the availability of released simple sugars for cross-feeding by other intestinal bacteria and may be another factor contributing to the specific bifidogenic effect of NDOs.

Solubilizing Activity of Carbohydrases

When enzymes are tested on insoluble substrate (e.g., cell wall polysaccharides), a relatively simple assay to monitor the solubilization of pectic material is by using (automated) colorimetric assays like the m-hydroxybi-phenyl assay (32, 33) for uronic acids and the phenol-sulfuric acid or orcinol-sulfuric acid assay for neutral sugars (34, 35). Obviously, the latter two methods are not very discriminative for the various neutral sugars, but can be used quite satisfactorily to monitor enzyme activity. More information on the enzyme is obtained when the determination of concentration of the solu-bilized carbohydrates is combined with information on their size (e.g., reducing sugar assay or size exclusion chromatography).

Preclinical Vaccine Development

In 1991, Zhou and Frazer reported that coexpression of HPV16 L1 and L2 in monkey CV-1 cells via a vaccinia virus vector resulted in the generation of viruslike particles that could be concentrated by sucrose gradient centrifugation 31 . These irregular particles had a mean diameter of 35-40 nm, compared to the 50-55 nm symmetrical particles reported for authentic virions, and were described as incorrectly assembled arrays of HPV capsomeres. Particles were not detected when L1 was expressed separately. The vaccine potential of these L1 L2 capsomere arrays could not be critically evaluated because no HPV16 neutralizing assay was available at this time. Whether this study was the foundation for the subsequent development of the HPV prophylactic vaccines or taught against their development became the subject of much discussion, particularly among patent lawyers.

Iipolysaccharides And Product Development

Figure 1 gives a simplified product development scheme. The raw material may come from seeds (e.g., guar, locust bean gum, corn, wheat starch), tubers and roots (e.g., potato and tapioca starch, konjac), fruits (pectin), seaweeds (e.g., agar, alginate, car-rageenan, furcellaran), plant exudates (e.g., ghatti, gum arabic, karaya, tragacanth), and various plant sources (cellulose), or microorganisms (dextran, gellan, pullulan, xanthan). These substances are collected through appropriate means, cleaned, purified, and often modified. The modification may be chemical (e.g., to form substituted celluloses or modified starches), or microbial (e.g., to convert sucrose to dextran).

Feeding ecology and diet

Bombycillids feed predominantly on small fruits, which are the mainstay of the diet for the north temperate species for seven months of the year. They also eat insects, plucking them off of vegetation and tree bark or swooping down from high perches and taking them in flight. The cedar waxwing can store ingested fruits in a portion of the esophagus, presumably to maximize the amount of food ingested while foraging. Unlike fruit-eating thrushes, they have the enzymes to digest sucrose. In recent years, waxwings have come to rely

Experimental Procedures For The Purification And Characterization Of The Arabinanases And Galactanases

An assay in which both arabinofuranosidase activity and endoarabinanase activity can be determined is measuring the appearance of reducing sugars from ara-binans with different degrees of branching (e.g., linear arabinan, heavily branched sugar beet arabinan, and moderately branched arabinans isolated from apple

LTryptophan dehydrogenase and 23dioxygenase

Another study found that rat brain MAOA is heat stable, whereas B is labile. A and B are partially separated by sucrose density gradient centrifugation A1227, A1309, Al841 more MAOB is found in high-density mitochondria A1909 . Both A and B are fairly evenly distributed throughout brain A2386 . Brain enzyme is inhibited reversibly by b-propiolactone and b-nitropropionate A1800 , by apomorphine A2069 and atropine (also heart enzyme) A1898 . The specificity of A and B are similar to human enzyme (above) A1324 . Brain mito-chondrial enzyme has an optimum pH of 7.5 for dopamine and tyramine, 8.2 8.5 for serotonin and tryptamine, and 9.1 for kynuramine. Similar results were obtained with beef brain mitochon-drial enzyme A1285 . Brain MAOA oxidizes dopamine better than MAOB A2484 m- and p -tyramine are substrates for both isozymes, whereas o-tyramine is substrate only for MAOB A3667 . The ratio of B to A in synaptosomal mitochondria is about 1 2 that for extrasynaptosomal mitochondria from...

Qualitative and Quantitative Determination of Activity

The most common way to follow EX activity is to determine the reducing sugars formed from selected type of xylan. Two basic procedures for determination of reducing sugars have been widely used the Somogyi-Nelson procedure (109-111), and the 2,4-dinitrosalicylic acid (DNS) test (112-114). The methods are sufficient to prove the presence of the enzyme, to assay its activity, and to evaluate the extent of poly-saccharide hydrolysis, which may be the first criteria for xylanase differentiation. The two methods for the determination of reducing sugars have been compared in an interlaboratory evaluation organized by Finnish scientists (115). Although the Somogyi-Nelson procedure is almost 10 times more sensitive than the DNS method, the latter always gives higher values of xylanase activity. When standardized with respect to substrate and procedure, the assay employing the DNS method afforded reproducible results in various laboratories with a standard deviation of 17 (115). Recently...

Transcription Sites and Nuclear Territories Functional Organization ofInterphase Nuclei

Using chromatin precipitation, sucrose gradient sedimentation, and array comparative genomic hybridization (aCGH) techniques, Gilbert et al. (86) showed that gene-rich domains are enriched in open chromatin fibers and suggested that domains of open chromatin may create an environment that facilitates transcriptional activation and could provide an evolutionary constraint to maintain clusters of genes together along chromosomes.

Introduction the development of edible coatings

Several attempts have been made to develop other materials that could be used to coat produce and modify internal gas composition for short-term storage. Zhang and Quantick (1997) suggested that chitin and chitosan (deacetylated chitin) from marine invertebrates could be used to make a transparent film for application as an edible coating on fruits and vegetables. In 1982, Lowings and Cutts (1982) reported an edible coating material that is non-phytotoxic, tasteless, odorless and effective in preserving fruits. This coating material is a mixture of sucrose fatty acid esters (SFAE), sodium carboxymethyl cellulose and mono-and diglycerides. SFAE was originally developed as an emulsifier. However, it has been established that the ripening of fruits can be retarded by a coating of SFAE. SFAE mixtures have been commercially available for coating fruits and vegetables since the 1980s, under the trade names 'TAL Pro-long' and 'Semperfresh' (Banks, 1984 Chu, 1986 Santerre et al., 1989). Park...

Boon Huan Tan Jian Lin Fu and Richard J Sugrue

The full-length and truncated forms of recombinant envelope (E) glycoprotein from Dengue virus type 1, Singapore strain S275 90 were expressed in the yeast, Pichia pastoris, using a secretory vector. A truncated form of the E protein in which the transmembrane domain was deleted was secreted successfully into the culture medium. The E protein was also co-expressed with C and prM proteins using a non-secretory yeast vector. The co-expression of C, prM and E proteins resulted in the spontaneous formation of virus-like particles (VLPs), which were confirmed by sucrose gradient analysis and transmission electron microscopy. Furthermore, the VLPs were used to immunise rabbits, and shown to be immunogenic by immunofluorescence staining of dengue virus-infected Vero cells. The yeast-expressed E protein was treated with PNGase F, which showed that although the protein was modified by the addition of W-linked glycans, the recombinant expressed E protein was not hyperglycosylated. Key Words...

Digestion and Absorption of Carbohydrates

Most carbohydrates are ingested as starch, which is a long poly-saccharide of glucose in the form of straight chains with occasional branchings. The most commonly ingested sugars are sucrose (table sugar, a disaccharide consisting of glucose and fructose) and lactose (milk sugar, a disaccharide consisting of glucose and galactose). The digestion of starch begins in the mouth with the action of salivary amylase. This enzyme cleaves some of the bonds between adjacent glucose molecules, but most people don't chew their food long enough for sufficient digestion to occur in the mouth. The digestive action of salivary amylase stops some time after the swallowed bolus enters the stomach because this enzyme is inactivated at the low pH of gastric juice. Maltose, maltriose, and oligosaccharides are hydrolyzed to their monosaccharides by brush border enzymes located on the microvilli of the epithelial cells in the small intestine. The brush border enzymes also hydrolyze the disaccharides...

Formation of Virus Like Particles

Sucrose Gradient Centrifugation 3. Prepare a gradient in the centrifuge tube from 5 to 50 of sucrose solutions made up in PBS (see Note 4). 1. Place 10 L of each sucrose fraction onto the EM grid. Drain dry. To determine whether the co-expression of C, prM, and E could form VLPs, the yeast transformmant containing the CprME construct was induced, the cells lysed, and the clarified lysate applied to a 5 to 50 sucrose gradient, which was centrifuged for 16 h at 100,000g (Fig. 5A). Our results indicate that fraction 3, collected from the top of the gradient, indicates the presence of E protein when analysed by Western blotting with the antiserum raised to bacterial-expressed E protein (lane 3). When the peak fraction 3, was further analyzed by TEM, VLPs were observed at a high magnification of X60,000 (Fig. 5B). Fig. 5. Biophysical analysis of recombinant E proteins. Protein expression in the yeast transformant containing the CprME construct was induced, the cells lysed and the...

Major and satellite signals

We should perhaps add that all the signalling molecules we have considered are essentially plant produced and plant orientated, but our consideration is not exhaustive. For example, polyamines are common to both eukaryotes and prokaryotes and as low molecular weight compounds they can regulate and influence metabolic events (Kumar et al., 1997). Sugars (sucrose or glucose) levels in a cell operate a complex signalling network with ethylene and abscisic acid to inhibit or promote specific growth responses (Leon and Sheen, 2003). Such substances are universal signal molecules. Perhaps as every cell is a target cell, so, given the appropriate circumstances, every molecule can become, at least temporarily, a signal molecule.

Nonwestern Medical Systems Traditional Chinese Medicine

Traditional Chinese Medicine (TCM) has existed for thousands of years, long before Western medicine. Rather than following the disease model of Western medicine, TCM focuses on a symptom approach such that a person with PD who has mostly tremor would be evaluated and treated differently than another person whose symptoms were mostly gait and balance difficulty with no tremor. The specific symptoms of the individual signal a deficiency in the body fluids blood that is unable to properly nourish the energy flow or chi or Qi of the entire organism. There are three main symptom approaches under TCM (5). The first is Qi and blood deficiency, which is believed to arise from anger, emotional stress, frustration, and resentment. The second is phlegm-fire-agitating wind (yang), which is the result of poor diet, in particular eating greasy, fried, sweet, sugary foods and alcohol. The third is kidney and liver (yin) deficiency, which results from a lack of rest and overwork as well as part of...

Overview Of Metabolic Substrate Engineering

The complex glycoconjugates expressed on eukaryotic cell surfaces (Figs. 3-8) comprise primarily 10 monosaccharides Glc, Gal, Man, Fuc, GlcN, GlcNAc, GalNAc, Xyl, GlcUA, and sialic acid (Neu5Ac). These sugars can be biosynthesized de novo within a cell, often from Glc or, in many cases, key metabolic intermediates can be supplied exogenously to a cell. The pathways for these interconversions are summarized in Figure 9 33-36 . If the enzymes in these biosynthetic pathways could tolerate unnatural substrates, subtle modifications, such as replacement of a hydroxyl group with a hydrogen atom or halogen, could be introduced into cell surface gly-cans. This possibility attracted the attention of many groups interested in understanding how specific carbohydrate structures dictate molecular and cellular interactions.

Chemical Reaction Catalyzed

Xylose isomerase catalyzes the reversible isomerization of monomeric keto sugars to their enol isomers. The natural substrate of this enzyme is D-xylose that is isomerized to D-xylulose. In the food industry, only the isomerization of D-glucose to D-fructose is of importance since this is the final step in the production of high-fructose corn syrups (HFCS for the industrial application of glucose isomerase see Sec. II.B) (4-6). These HFCSs are used as sweeteners in soft drinks and other food products. Therefore, the enzyme is usually referred to as glucose isomerase in both the industry and the scientific community. Other sugars that are substrates for this enzyme are L-arabinose, L-rham-nose, D-ribose, and others (1-6).

Production of High Fructose Corn Syrups

High-fructose corn syrup is a mixture of glucose and fructose produced from cornstarch (4-6). HFCSs find widespread use in the food industry. Their most important application is as a sweetener in soft drinks where they replace beet and or cane sugar. They have the advantage that at an equal sweetener level (see Table 3), they are some 10-20 cheaper than sucrose and are less caloric because of the lower resorption of fructose. A technical advantage of HFCS is the better solubility of glucose and fructose compared to sucrose and therefore the lesser tendency to crystallize in a wide range of food products. This has led to their application in confectionery, jam and jellies, ice cream, canned products, baking, pickles, sauces, meat products, etc. (5). High-fructose corn syrups are produced in a process that comprises three consecutive enzymatic steps (Fig. 2). The raw material used in this process is starch, mainly derived from corn. Starch consists of two polymers of D-glucose the...

Origins of celltocell signalling

Traces of sucrose have been shown to increase the production of ethylene and stimulate xylogenesis in lettuce pith explants (Warren-Wilson et al., 1994), and so it is interesting to speculate that the transportation of inductive concentrations of sucrose along the conductive tissues of the phloem in higher plants and along the elongated cell pathways carrying metabolites in primitive plants could have been one of the causal signals to the evolutionary development of lignin-like elements in the plant body.

Developments Since 1940

The largest immobilized enzyme product still today in volume is immobilized glucose isomerase. The first commercial enzymatic production of high-fructose corn syrups (HFCS) was in Japan in 1969 (Takasaki and coworkers), utilizing heat treated Streptomyces cells containing glucose isomerase in a batch reactor. In the United States, Clinton Division of Standard Brands (now ADM) was the first company using Takasaki immobilized glucose isomerase'' to make industrial quantities of HFCS around 1971 (33). The skyrocking sucrose prices, during 1973-75, where the price of sucrose increased from 5-70 lb to 300 lb speeded the interest for HFCS. Thereby, immobilized glucose isomerase increased dramatically in price. Companies like Novozymes, and later Gist-brocades developed more stable enzyme products, which were easier and cheaper to use. Resources were spent on optimizing fermentation for glucose isomerase production, immobilization processes, and the application processes for the immobilized...

Lifestyle Modification of the Metabolic Syndrome Type 2 Diabetes and CVD

The first of these RCTs was conducted in China beginning in 1986 (Pan et al, 1997). Over 100,000 men and women from health clinics in the city of Daqing were screened for impaired glucose tolerance and type 2 diabetes. Using WHO criteria, 557 subjects were determined to have impaired glucose tolerance and subsequently were randomized into one of the four conditions control, diet only, exercise only, or diet-plus-exercise. The goal of the diet intervention was to promote vegetable intake and lower alcohol and sugar consumption. Overweight subjects were encouraged to lose weight by reducing total caloric intake. The goal of the exercise intervention was to increase leisure time physical activity. Follow-up examinations were conducted every 2 years over a 6-year period to determine the incidence of type 2 diabetes. Over the 6-year follow-up period, the cumulative incidence of diabetes was 67.7 for the control group compared to 43.8 in the diet group, 41.1 in the exercise group, and 46.0...

Dairy components and food intakesatiety

Of the milk proteins, whey protein has been studied the most, probably because it is readily available as a by-product of cheese making. It suppressed food intake more than sucrose and egg albumen at a pizza meal one hour later in young men (Anderson et al., 2004). Similarly, intake of a buffet meal at 3 hours (Bowen et al., 2006a Bowen et al., 2006b) after a 50 g preload of whey was lower than after a glucose preload. However, no differences were found between whey and casein (Bowen et al., 2006a) or among whey, gluten and soy protein preloads (Bowen et al., 2006b). In other reports, however, casein and whey had different effects on food intake. In a study by Hall et al. (2003), a preload containing 48 g of whey resulted in lower ad libitum intake of a buffet meal 90 min later than a preload containing the same amount of casein. In contrast, no differences were found in the intake of a pizza meal 90 min after 50 g pure preloads of casein and whey, but casein suppressed energy intake...

Cyclopropane ring fission

Both enzyme quantity and total enzyme activity. This is caused by an increase in the half-life of the enzyme from 2.3 to 3.9 h. A2059 . In contrast, treatment with sucrose or ethanol for two days resulted in no change in the amount of 'holoen-zyme', but a 30 per cent decrease in 'total enzyme'. The effect of cortisol in conjunction with sucrose appears to result entirely from decreased 'holoenzyme', but with no effect on 'total enzyme'. On its own, cortisol markedly increases both enzyme forms and eliminates the effects of ethanol. In this study it appears that the two forms were distinguished by measuring activity with and without added haematin A2062 . Rat cerebral activity is increased by administration of l-tryptophan A1289 .

Tcc Receptors And Signal Transduction

Lipid raft isolation protocol Lipid rafts are insoluble in nonionic detergents such as Triton X-100, and application of a sucrose gradient (45 to 5 sucrose) allows them to be separated from the other membrane domains. For this purpose, cell lysates in Triton X-100 1 are overlaid with sucrose and ultra-centrifuged for 16 h at 100,000 rpm at 4 C. Fractions from the top are collected and analyzed for lipid raft markers, among the best of which are gangliosides (GM1 and GM3). However, recent data indicate heterogeneity in lipid raft composition in each cell type. In this connection, flotillin can be used, even for functionally different rafts. Once the lipid raft fractions are identified, several experiments including protein localization phosphorylation (Western-blotting), kinase activity, and immunoprecipitation can be performed to assess composition. Lipid raft fluidity can be assessed using diphenylhexatriene as a probe for fluorescence anisotropy.

The Start Of A Collaboration

Therefore we began trying to purify and concentrate the virus to escape the toxicity and to obtain antigen of sufficient potency to develop antisera in rabbits and guinea pigs. The lipids in white matter presented a real challenge. How could we free the virus from that mass of myelin lipid and cell membranes It became a matter of trial and error and using any clues available from the work of other investigators. Many techniques were tried, including genetron extraction and centrifuging to a pellet through 5-20 sucrose, but we eventually settled on one that started with homogenizing the tissue in a mortar or a blender. It was then sonicated and treated with sodium deoxycholate and trypsin and subjected to differential centrifugation. Supernatant fluid from a final low-speed centrifugation was diluted to the equivalent of a 10 tissue extract in buffered saline. Extracts of diseased tissue and normal human brain tissue prepared in this way were used in...

Immobilized Enzyme Processes That Have Been Commercialized In The Food Industry

Production of High-Fructose Corn Syrup Using Immobilized Glucose Isomerase This industry has grown from the initial commercial production in 1970 by Clinton Corn Pocessing to become the largest industrial use of an immobilized enzyme process in the world (16, 17). Most recent data indicate that the global annual production of high fructose corn syrup is 10 million metric tons dry substance(dsb) which is produced with 1500 metric tons of immobilized enzyme (J. Shetty, personal communication, 1999). Currently, the major producers of immobilized enzyme are Novo Industry A S and Genencor. The Genencor immobilized enzyme is produced by adsorption of substantially purified enzyme on ion exchange resins, crosslinked with polyethylenei-mine and glutaraldehyde, and granulated by extrusion. This immobilized enzyme typically has a productivity of 12,000-15,000 kg dsb kg and a half-life of 80-150 days (1920-3600 h) (J. Shetty, personal communication, 1999). The immobilized enzyme produced by...

Molarity and Molality

Glucose is a monosaccharide with a molecular weight of 180 (the sum of its atomic weights). Sucrose is a disaccharide of glucose and fructose, which have molecular weights of 180 each. When glucose and fructose join together by dehydration synthesis to form sucrose, a molecule of water (molecular weight 18) is split off. Therefore, sucrose has a molecular weight of 342 (180 + 180 - 18). Since the molecular weights of sucrose and glucose are in a ratio of 342 180, it follows that 342 grams of sucrose must contain the same number of molecules as 180 grams of glucose.

Phenolic Phytochemical Ingredients And Benefits

Flavonoids are subdivided into several families such as flavonols, flavones, flava-nols, isoflavones, and antocyanidins, which are formed as a result of hydroxylation, meth-ylation, isoprenylation, dimerization, and glycosylation of the substituents in the aromatic rings (2,11). Phenolic phytochemicals are often esterified with sugars and other chemicals such as quinic acid to increase their solubility and to prevent their enzymatic and chemical degradation. Esterification also helps to target the phenolics to specific parts of the plant (11). Phenolic phytochemicals esterified via their hydroxyl groups to sugars are called glycosides. The sugar most commonly involved in esterification is glucose. However, the glycosides of phenolics with galactose, sucrose, and rhamnose are also found in some plant species (11).

Culture Conditions

Both rich and synthetic minimal media are used to culture Saccharomyces. Rich medium, called YEP or YP, is made from commercially available yeast extract and peptone (a complex protein digestion product). It contains all essential nutrients including ammonia (a rich nitrogen source), phosphate, sulfate, sodium, magnesium, calcium, copper, iron, etc. and certain other compounds that all Saccharomyces strains are unable to synthesize. In addition, rich medium provides many macromolecular precursors such as amino acids and nucleotides that wildtype Saccharomyces strains are able to synthesize if necessary. A sugar or other carbon energy source must be added, such as glucose (dextrose), sucrose, lactic acid, or others depending on the genotype of the strain and its ability to utilize various carbon sources. Glucose is the richest and most readily available carbon source and a rich medium containing glucose is referred to as YEPD or YPD. Because of the abundant nutrient supply, cells...

Diet

Hydrothermal vent and cold seep worms live in total darkness. They rely on the billions of bacteria living in their bodies to make food. The worms provide the bacteria with carbon dioxide from their own bodies and hydrogen sulfide collected from the hydrothermal vents. As the bacteria convert these chemicals into energy for themselves, they produce simple sugars and other compounds that the worms absorb as food. The conversion of chemical reactions into food is called chemosyn-thesis (KEY-moh-SIN-thuh-sihs).

Nonparental ditype

It is important to note that the phenotype of the double mutant may be unique. This occurs most often when the two genes encode functions involved in the same process. If mutant strains containing alterations in unrelated gene functions, such as ade2 and suc2, are crossed, then the double mutant is expected to exhibit both phenotypes, adenine requiring and unable to utilize sucrose. If mutant strains containing alterations in two related gene functions are constructed, such as MCM2 and MCM7 encoding different components of the origin recognition complex (ORC), then the double mutant could exhibit an unexpected phenotype. For example, the double mutant combination could be lethal even though each single mutant strain is viable. Often mutant genes are crossed for the purpose of determining the double mutant phenotype. As will be discussed in detail in the chapters on epistasis, suppression, and enhancement, a great deal of insight into the function and relationship between gene products...

Cupin Superfamily

Cupin comes from cupa, the Latin term for a small barrel. The characteristic cupin domain comprises two conserved motifs, G(x)5HxH(x)3,4E(x)6G and G(x)5PxG(x)3N (31,32). Based on structural features, cupin proteins are classified as either a monocupin having one cupin motif per one monomer, and a bicupin having two cupin motifs per one monomer. The 7S and 11S globulins are bicupins. Cupin proteins are also known to have diverse functions. For example, in addition to seed storage proteins, an oxalate oxidase and a sucrose binding protein belong to this group (33,34).

Cell Fractionation

Equilibrium density gradient centrifugation separates subcellular components based only on their density. For this method, one must first prepare a density gradient in a centrifuge tube. A nonionic molecule like sucrose, glycerol, or Ludox is used to vary the density of the buffer solution. The concentration of the molecule is varied, and therefore the density of the solution, and the concentration, is greatest at the bottom of the centrifuge tube and decreases slowly towards the top of the tube. Special devices are available for making these gradients. A step gradient can also be prepared. Here a series of solutions of different concentration (30 , 25 , 20 , etc.) are layered on top of one another with the step with the highest concentration at the bottom.

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