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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 here...

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Highly Recommended

I usually find books written on this category hard to understand and full of jargon. But the writer was capable of presenting advanced techniques in an extremely easy to understand language.

In addition to being effective and its great ease of use, this eBook makes worth every penny of its price.

Rate Zonal Centrifugation Using Sucrose

Rate zonal centrifugation is a nonequilibrium density gradient procedure for fractionating cells, in which sedimentation depends on the size, density, and shape of the particles as well as the viscosity and density of the medium. Typically, samples are loaded atop a continuous sucrose gradient during subsequent reasonably short centrifugations (length of run and rate often need to be determined empirically), they separate as distinct bands (zones) enriched in particular organelles. This procedure can be adapted to various-sized gradients available models of gradient-forming devices can be used to generate gradients in tubes ranging from 5 to 38 ml. To prepare gradients in smaller tubes (e.g., for a tabletop ultracentrifuge), investigators often approximate continuous gradients by preparing step gradients having several layers of progressively decreasing density, then letting them stand a few hours before use. Diffusion between the layers makes the approximation quite good. The example...

Velocity Sedimentation of Connexons in Sucrose Gradients

20 (w w) sucrose solution Dissolve 20 g of sucrose in incubation buffer + TX-100 and bring the final volume to 100 mL. Aliquot and store at -20 C. 4. 5 (w w) sucrose solution Dilute 20 sucrose solution with incubation buffer + TX-100 to a final concentration of 5 sucrose.

Prepare and run the continuous sucrose gradients

Place sucrose cushions consisting of 0.5 ml of 2.5 M sucrose in the bottom of 13-ml ultracentrifuge tubes. 16. In each tube, generate a 12-ml continuous (60 to 30 ) sucrose gradient above the cushion using the gradient solutions as follows place 6 ml of 30 sucrose solution in the back chamber of the gradient maker, and fill the junction tube to the mixing chamber. Place 6 ml of 60 sucrose solution in the mixing chamber. Open the

Prepare sucrose density gradients

In the bottom of each centrifuge tube, place a cushion of 2.7 M sucrose. 7. Using the gradient-forming device and working at room temperature, generate a linear sucrose gradient above the cushion extending from 1.9 M sucrose (density 1.25 g ml) to 0.8 M sucrose (density 1.10 g ml) as follows place the 0.8 M sucrose gradient

Dietary Guidance For Carbohydrates

Occurring sugars and refined sugars, despite the recognition that metabolically and vegetables (6). About half of the total digestible carbohydrate intake is made up of monosaccharides and disaccharides. These are found naturally in fruits and milk and also enter the diet as sugars in soft drinks, candies, jams, and sweet desserts mainly composed of sucrose and high-fructose corn syrup. Complex carbohydrates, which constitute the other half of digestible carbohydrate intake, are starches found predominantly in cereal grains, potatoes, legumes, and a few other vegetables. In 1985 sugars and starches provided an average of 45.3 of the energy in the diet of adult men in the United States. This is less than the levels recommended for overall carbohydrate consumption and disproportionately high in simple sugars.

Immunofluorescence and Immunoperoxidase Assays

A freshly prepared paraformaldehyde solution containing 3 paraformaldehyde (3 g), 2 sucrose (2 g) in 100 mL of PBS. The paraformaldehyde solution must be neutralized with 2 N NaOH to a 4. Permeabilization buffer (0.5 Triton X-100 and 300 mM sucrose diluted in PBS). Make up 100 mL of this solution and store at 4 C.

Prenutrition Labeling And Education Act Of 1990

Even before the issuance of the first Dietary Guidelines for Americans in 1980, the FDA and the food industry had realized the problems created by there being no indication on the food label of the complex carbohydrate content of a food. At the same time dietary guidance almost universally advised consumers to increase their consumption of complex carbohydrates. For example, in 1978, the Kellogg Company had submitted a petition to the FDA to voluntarily permit the declaration of (1) grams per serving of starch and related carbohydrates, (2) grams per serving of sucrose and other sugars, and (3) grams per serving of total carbohydrates the sum of (1) and (2) . The Agency did not grant the Kellogg petition, but in the years from 1980 to 1993 many cereal companies listed either complex carbohydrates or starches and related carbohydrates on the labels of their products and the FDA took no regulatory actions.

The Hazards of Tobacco

Step 5 When you quit smoking, you probably will feel like eating more often and may gain a few pounds. Don't stop yourself from eating when you feel tense during the first few weeks it will be hard enough to stay away from cigarettes. Stock up on fresh fruits and vegetables, sugar-free candy and soda, and fat-free pretzels or crackers. Drink plenty of water. Your most intense cravings for nicotine will subside after about 8 weeks, when you can resume your usual eating pattern.

Bicinchinonic Acid Bca Assay For Quantitation Of Total Protein

The BCA assay for total protein is somewhat variable it has differing sensitivities in response to incubation time, incubation temperature, standard protein used for calibration, and other factors (Smith et al., 1985). Certain classes of compounds such as reducing sugars and ammonium ions interfere with the assay, sometimes severely. However, if interfering compounds are eliminated (e.g., by dialysis see Support Protocol 2), the BCA assay has a good combination of sensitivity and simplicity, and it has some advantages over the Lowry technique (see Background Information).

Thawing and Detergent Treatment

As soon as the replicas, complete or in pieces, are detached from the bulk of the biological material, they are transferred to 2 SDS-Tris-sucrose buffer, taking care to immerse the replica completely in the detergent solution. Intermittent agitation, with a plastic pipet, is produced. Change the SDS solution 3-4 times. The total SDS treatment is prolonged for 1-2 h maximum (see Note 4).

Basic Principles Of Centrifugation

This equation states that the sedimentation velocity, dx dt, per unit centrifugal field w2x (which is set by the instrumentation) increases with the square of the particle radius and the difference in density between the particle and the medium and decreases with increasing viscosity. In practical terms, when sedimentation is performed by differential velocity in a medium such as 0.25 M sucrose, which is less dense than all particles and has a low viscosity, the radius of the particle is the dominant factor that determines the sedimentation rate. In contrast, when isopycnic density gradient centrifugation is used, the density of the medium changes from top to bottom in the centrifugation tube, and the range of densities typically includes the buoyant densities of most particles (vesicles). Thus individual types of vesicles will sediment until pp pm, and the vesicles will then stop at their isopycnic densities. Alternatively, in flotation procedures pp < pm initially and dx dt is...

Gradient Fractionation Using Iodinated Nonelectrolyte Solutes

Separation by density gradient with iodinated nonelectrolytes substituted for sucrose to achieve high density. Substituting nonionic solutes for sucrose makes it possible to prepare media that span the range of densities of all membrane-bounded organelles, yet have quite low viscosities and only moderate osmolarities. For organelles with a high buoyant density, this significantly reduces efflux of water, which can lead to irreversible changes in some organelles.

Differential Centrifugation By Velocity

This is the classical standard method of cell fractionation using centrifugation, often used as the starting point for a more complex purification procedure. A cell homogenate is subjected to serial centrifugations of increasingly higher force and longer duration, generally in isoosmotic (0.25 to 0.3 M) sucrose medium (see Background Information for discussion of sucrose concentration). Resulting supernatant fractions or pellets (often prepared as a band atop a cushion of high-density medium rather than a bona fide pellet at the bottom of the tube, in an effort to minimize aggregation) are suitable as starting material for further purification or other procedures. This method gives relatively low resolution in that it does not yield fractions of high purity. However, the postnuclear supernatant (PNS) from step 6 and the supernatant from step 8 serve as useful starting materials for procedures that yield high-purity fractions mainly by exploiting differences in organelle density.

Differential Centrifugation By Equilibrium

This procedure can be used alone (as presented here) but most often is used as a follow-up to differential centrifugation by velocity (Basic Protocol 1). Typically, the samples are applied above (or beneath) a sucrose or other nonelectrolyte-based medium having a density that varies either discontinuously (in steps) or continuously from top to bottom of the tube. The sample must of course be even lower in density than the lowest density of the gradient (if applied to the top) or higher than the highest density of the gradient (if applied at the bottom). During centrifugation, organelles sediment (or float) until reaching their isopycnic positions, where the buoyant density of the particle equals the density of the medium (unit 4.1). In a continuous gradient, organelles separate as bands distributed throughout the gradient in a discontinuous gradient the organelles concentrate at the interfaces between the particular steps whose densities are lower and higher than that of each...

Process individual fractions

Dilute each band at least 2- to 3-fold with buffer or a low-concentration sucrose medium. Pellet by velocity sedimentation for > 100,000 x g (e.g., 40,000 rpm in Beckman SW41 or SW50.1), 4 C, then resuspend in the same medium for further use. Process Golgi fraction by adding dilution solution to 0.25 M sucrose final (use a refrac-tometer, if available, to measure the sucrose concentration), then pelleting 1 1 2 hr at 100,000 x g, 4 C. As an alternative to the continuous gradient procedure, it is also possible to generate Golgi fractions by flotation on a discontinuous sucrose gradient. In this case, a homogenate prepared in 0.25 M sucrose 5 mM TrisCl (pH 7.4) 25 mM KCl 5mM MgCh 4.5 mM CaCl2 (STKCM) is adjusted to 1.15 M sucrose in the same medium. Above this sample in the centrifuge tube are placed layers of 0.95 and 0.4 M sucrose in STKCM. After centrifugation at 200,000 x gfor 90 min, the Golgi fraction is collected at the 0.95 0.4 M interface. By using a discontinuous...

Health Benefits of Complex Carbohydrates

The modern fiber era was launched with a paper by Cleave (1) that attributed many of the diseases of developed countries to consumption of refined sugar and refined flour. Burkitt, Trowell and Walker, all of whom worked in Africa, noticed the low incidence of large bowel cancer, ischemic heart disease, diabetes, and

Collect the gradients at 4C

Assay the gradient fractions for the activity of interest. For some assays it may be necessary to reduce the sucrose content and or concentrate the sample. If so, dilute fractions 2- to 3-fold with 0.3 M sucrose 5 mM MES 1 mM EDTA 0.2 xg ml DPPD and pellet 1 hr (or shorter if the volume is small) at 100,000 x g, 4 C.

Gradient Fractionation Using Percoll

Percoll (polyvinylpyrrolidone-coated colloidal silica) is a useful alternative to high-concentration sucrose gradients, particularly where high densities combined with low viscosity and osmotic activity are desired. Cells are homogenized, mixed with a Per-coll solution, and centrifuged, generally using a fixed-angle rotor. The Percoll forms its own density gradient during centrifugation (a self-forming gradient). As it has very low osmotic activity (pure Percoll as purchased is 10 mOsm), it is generally added to isoos-motic (or slightly hyperosmotic) sucrose for separating organelles from cell homogenates under isoosmotic (or near isoosmotic) conditions. The density range is controlled by the Percoll concentration, and the slope of the density gradient is determined by the time of centrifugation and the centrifugal field (see Fig. 4.2.2). Generally, the duration of separation procedures is selected so that organelles sediment to near equilibrium density

Gradient Fractionation Using Nonionic Iodinated Solutes

Another strategy that is being used to achieve higher-density fractionation media with reduced osmolarity and viscosity involves supplementing or replacing sucrose with iodinated solutes. By far the most successful of these solutes are the nonionic derivatives of triiodobenzoic acid metrizamide Nycodenz and iodixanol, or Optiprep (all available from Nycomed Pharma). Metrizamide Nycodenz (N,N and iodixanol bis N,N are soluble in all aqueous media, stable over the pH range 2 to 12.5 and, unlike related ionic solutes, are not affected by low pH or the presence of divalent cations. Iodixanol is marketed as, Optiprep, a 60 (w v) solution

Preparative Electrophoretic Separation

Homogenization medium 0.25 M sucrose (ultrapure e.g., ICN Biochemicals) 50 mM 4-morpholinoethanesulfonic acid (MES), pH 6.5 6.25 sucrose 6.25 Ficoll 70 50 mM MES, pH 6.5 50 mM MES, pH 6.5 Running buffer 50 mM MES (pH 6.5) with added sucrose as desired 0.15 to 0.2 Isogel agarose (FMC), or equivalent, in running buffer

Prepare sample for immunoadsorption

On the same day the immunoadsorption experiment is to be done, sacrifice a rat that has been starved overnight (to deplete hepatic glycogen) and remove liver. Weigh liver, mince, homogenize, and filter as for velocity differential centrifugation (see Basic Protocol 1), except homogenize at 20 (w v) in 0.5 M sucrose 0.1 M potassium phosphate 5 mM MgCl2 homogenization medium. 6. Place a4-ml cushion of 1.3 M sucrose 0.1 M potassium phosphate 5 mMMgCl2 into a 10-ml centrifuge tube, and layer 6 ml PNS on top. Centrifuge 60 min at 105,000 x g (in Type 50 rotor), 4 C. 7. Collect the band at the surface of the 1.3 M sucrose layer and adjust to 1.1 M sucrose using homogenization medium. 8. Prepare a second set of discontinuous gradients containing 6 ml each of 1.4 M, 1.3 M, and 1.25 M sucrose in 0.5 M potassium phosphate 5 mM MgCl2. Layer 6 ml of the adjusted sample (step 7) over each gradient, overlay with homogenization medium to fill the tubes, and centrifuge 90 min at 80,000 x g (in SW28...

Reagents And Solutions

Prepare three separate stock solutions 0.8 M sucrose 3 mM imidazole, pH 7.4 (density 1.10 g ml) 1.9 M sucrose 3 mM imidazole, pH 7.4 (density 1.25 g ml) 2.7 M sucrose (density 1.34 g ml) The 2.7 M sucrose is used for the cushion and contains no buffer. Sucrose should be ultrapure (e.g., ICN Biochemicals). Sucrose stocks can be filtered using 1.2- m Millipore filter (or equivalent) and stored at least 2 weeks at 4 C or indefinitely at -20 C. 1 M imidazole (pH adjusted with concentrated HCl) can be filtered using 0.22- m Millipore filter (or equivalent) and stored indefinitely at 4 C.

Percoll gradient stock solutions

No-Percoll solution (low sucrose) 0.3 M sucrose No-Percoll solution (high sucrose) 2.15 M sucrose 35 mM MOPS or MES 7 mM EDTA Prepare solution from the following stock solutions 2.5 M sucrose (ultrapure e.g., ICN Biochemicals) 1 M 2-N-morpholinoethanesulfonic acid (MES), pH 6.5 1 M 3-N-morpholinopropanesulfonic acid (MOPS), pH 6.8 0.2 M EDTA, pH 7.0 and 0.4 mM DPPD (diphenyl-p-phenylenediamine Kodak) in ethanol. 2.5 M sucrose can be stored indefinitely at -20 C 1 M MES or MOPS and 0.2 M EDTA should be filtered using 0.22- m Millipore filter (or equivalent) and can then be stored indefinitely at 4 C and DPPD stock should be made fresh before use. 60 Percoll sucrose Mix 60 ml Percoll, 14 ml No-Percoll solution (high sucrose), and 26 ml water. Adjust pH as described above and add 0.2 g ml DPPD (from 0.4 mg ml DPPD stock in ethanol). 86 Percoll sucrose Mix 86 ml Percoll and 14 ml No-Percoll solution (high sucrose). Adjust pH as described above and add 0.2 g ml DPPD (from 0.4 mg ml DPPD...

Rate zonal centrifugation gradient stock solutions

0.5 M sucrose 1.8 M sucrose Prepare gradient solutions from the following stock solutions 2.5 M sucrose (ultrapure e.g., ICN Biochemicals) 50 Ficoll400 1 M 2-N-morpholinoethanesulfonic acid (MES), pH 6.5 0.2 M EDTA, pH 7.0 and 0.4 mg ml DPPD (diphenyl-p-phenylenediamine Kodak) in ethanol. Sucrose and Ficoll stocks are stored long-term at -20 C gradient solutions are used within two weeks of preparation and the DPPD stock is made fresh and used without storage.

Detergent Extraction of Liver Gap Junctions

2 L of 67 sucrose (1780.4 g of sucrose) in Tris-HCl buffer. 5. 1 L of 41 sucrose in Tris-HCl buffer (add 610 mL of 67 sucrose to 390 mL of Tris-HCl buffer). 6. 400 mL of 25 sucrose in Tris-HCl buffer (add 150 mL of 67 sucrose to 250 mL of Tris-HCl buffer). 7. 300 mL of 30 sucrose in Tris-HCl buffer (add 135 mL of 67 sucrose to 165 mL of Tris-HCl buffer).

Alkali Extraction of Liver Gap Junctions

2 L of 67 sucrose (1780.4 g of sucrose) in 1 mM NaHCO3 buffer. 5. 200 mL of 55 sucrose in 1 mM NaHCO3 buffer (made from 67 sucrose solution). 6. 2 L of 37 sucrose in 1 mM NaHCO3 buffer (made from 67 sucrose solution). 7. 5 mL of 30 sucrose in 1 mMNaHCO3 buffer (made from 67 sucrose solution). 8. 2 mL of 41 sucrose in 1 mMNaHCO3 buffer (made from 67 sucrose solution).

Flotationgradient Fractionation Of Tissue Culture Homogenates

Early and late endosomal fractions can easily be prepared from postnuclear supernatants (PNS) by flotation in a step gradient of 40.6 to 8.6 (w w) sucrose D2O. Early endosomes containing the transferrin receptor, the small GTPase Rab5 protein, and annexin II can be separated from late endosomes containing the Rab7 protein, lysosomal glycoproteins (lgps), and the cation-independent mannose-6-phosphate receptor (Table 4.3.1). Fractions from this gradient can be used to determine the general protein composition of the fractions in high-resolution two-dimensional gels (unit 10.4) and to reconstitute the different endosomal membrane fusion events (Fig. 4.3.2).

Endothelial And Microvascular Changes 231 Breakdown of the Blood Brain Barrier

It is clear that the initiation of the breakdown of the BBB occurs within minutes of insult as demonstrated by the use of either 3H sucrose,27 infusion with hyperosmolar L(+)arabinose28 or horseradish peroxidase (HRP) tracer studies.29 However, the opening of the BBB continues over several hours after an ischaemic insult and at least two additional phases of BBB opening may occur.27 There is also evidence for a differential localisation of such openings between different parts of the brain. Thus, after bilateral carotid artery occlusion for 10 to 25 min, followed by recirculation, there is acute opening of the BBB in neocortical regions, possibly due to reactive hyperaemia,30 with recovery suggested to occur by 24 h.27 In the striatum and

Storage As Freezedried Solids

Even if appropriate processing conditions can be developed with a given lyophilizer, it is critical to use the correct additives to prevent protein denaturation during both freezing and drying (reviewed in Carpenter and Chang, 1996 Carpenter et al., 1997). The disaccharides sucrose and trehalose are very effective at protecting proteins during these stresses and during storage in the dried solid. These sugars are nonreducing. Reducing sugars such as glucose and maltose should not be used since they degrade proteins via the Maillard reaction. Thus, for most labs freeze-drying should be considered only when all other methods for enhancing storage stability have been found to be inadequate. Assuming that suboptimal processing will be necessary (i.e., there is no way to control sample temperature in the freeze-drier) and that critical physical parameters cannot be measured, is there any chance of obtaining a stable dried formulation The answer is maybe. The practical approach is to...

Karl Fischer Titration

Unlike gravimetric measurements (unit A1.1), which are indirect methods that assume that all volatiles removed are water, the Karl Fischer titration is a direct method that is almost specific for water. The method is especially useful for low moisture levels (< 1 ), and levels < 0.01 are achievable. The Karl Fischer titration method is especially useful for samples that may be high in sugar, or high in both reducing sugar and protein, which may decompose in the gravimetric methods. The method is a titration of water with an anhydrous methanol solution containing iodine, sulfur dioxide and excess pyridine. The titration is based on the reaction between iodine and sulfur dioxide that will occur only if water is present

Endogenous Advanced Glycoxidation End Products Formation

It is now appreciated that normal living is associated with spontaneous chemical transformation of amine-containing molecules by reducing sugars in a process described since 1912 as the Maillard reaction. This process occurs constantly within the body and at an accelerated rate in diabetes (5,6). Reducing sugars react in a nonenzymatic way with free amino groups of proteins, lipids, and guanyl nucleotides in DNA and form Schiff base adducts. These further rearrange to form Amadori products, which undergo rearrangement, dehydration, and condensation reactions leading to the formation of irreversible moieties called AGEs. Among all naturally occurring sugars, glucose exhibits the slowest glycation rate, although intracellular sugars such as fructose, threose, glucose-6-phosphate, and glyceraldehyde-3-phosphate form AGEs at a much faster rate (5,6,14).

Prepare continuous metrizamide gradient and load sample

The reason for loading the gradient at the bottom is that mitochondria exhibit a higher equilibrium density when initially equilibrated in high-density metrizamide than when equilibrated in sucrose homogenization medium. This strategy is critical to achieving separation of mitochondria and lysosomes in the gradient.

Rapid and automated methods

Samples placed in the instrument are chemically treated to lyse somatic cells and dissolve casein micelles. Bacteria are then separated by continuous centrifuga-tion in a dextran sucrose gradient. Microorganisms recovered from the gradient are incubated with a protease to remove residual protein, then stained with acrid-line orange and applied as a thin film to a disc rotating under a microscope. The fluorescent light from the microscope image is converted into electrical impulses and recorded. The Bactoscan has been used widely for raw milk testing in continental Europe, and correlations with conventional methods have reportedly been good (Kaereby and Asmussen, 1989). The technique does, however, have a poor sensitivity (approximately 5 x 104 cells ml) and this negates its use on samples with lower bacterial counts.

Maintenance Of Ocim1 Cells And Isolation Of Plasma Membranes

This protocol describes the maintenance of OCIM1 cells and the preparation of fractionated plasma membranes from these cultures. OCIM1 cells are grown in quantities sufficient to provide enough plasma membranes for many experiments. The cells are lysed and the membranes are purified through a series of centrifugation steps until they are finally isolated as a band in a sucrose gradient. The membranes are then aliquoted, tested, and stored for future use. The isolated membranes maintain functional receptors (c-kit) that bind human and rodent SCF. sodium pyruvate (GIBCO BRL), ice-cold Sucrose buffer 0.25 (w v) sucrose in TE buffer, ice-cold (sterilize using 0.45- m filter store < 1 yr, 4 C) 36 (w v) sucrose in H2O, ice cold (store < 1 yr at 4 C) TE buffer, pH 7.0 (appendix 2), ice-cold

Dealing with Inclusion Bodies

The formation of inclusion bodies can sometimes be prevented by changing the promoter, host strain, and combinations thereof controlling the growth conditions (especially the pH of the culture) adding nonmetabolizable sugars such as sucrose and sorbitol to the fermentation medium and changing the temperature of induction, usually by lowering it (for reviews, see Schein, 1989 Wetzel, 1992 Baneyx, 1999).

Influence of Suspending Medium Composition on Survival of Live Cells to Freeze Drying

Attempts to freeze-dry cells in water or a simple salt solution typically result in poor survival. A wide range of protective media has been developed for preserving freeze-dried vaccines, including augmented growth media or sugar solutions. Carbohydrates are widely used as freeze-drying protectants either individually or in combination with other solutes. They should be chosen on the basis of experimentally determining their freeze-drying characteristics rather than on a pragmatic basis. Monosaccharides, such as glucose, provide good bioprotection during freezing and freeze-drying but exhibit low glass transition (Tg') or collapse temperatures (Tc) and dry with collapse when orthodox freeze-drying cycles are used. Disaccharides are effective freeze-drying protectants, and because they display higher collapse than monosaccharides, freeze-dry successfully when conventional drying cycles are used. Reducing sugars may induce damaging Maillard reactions, thereby compromising stability,...

Coat the magnetic beads with primary antibody

Coating the beads with antibody (steps I to 4) is usually done the day before the rest of the experiment. The beads are treated with protein to block nonspecific adsorption. Most procedures call for using bovine serum albumin (5 mg ml in PBS or in buffered 0.25 M sucrose) one protocol recommends use of fetal bovine serum (3 in PBS) as the best way to reduce nonspecific binding (Saucan and Palade, I994).

Assembly Assayed by Hydrodynamic Analysis

Connexon assembly was assayed by hydrodynamic analysis using linear 5-20 sucrose gradients. This technique is generally used for assaying assembly of protein subunits into oligomeric structures and was used for connexins by others before (39-41). 1. Nonmembrane integrated connexin polypeptides that are synthesized to a certain extent in the cell-free translation reactions as a byproduct on nonmembrane bound, free ribosomes (see Subheadings 3.1.3. and 3.2.2., and Fig. 5) are separated from membrane integrated connexins prior to gradient analysis by pelleting the microsomes through a 0.5 M sucrose cushion made in 1x PBS, using an Airfuge ultracentrifuge (Beckman Instruments, Palo Alto, CA) as described in 2. To remove unincorporated radioactive label, microsomes are washed twice in 0.25 M sucrose, 1x PBS and pelleted as before. 5. Supernatants are loaded on top of 5-mL linear 5-20 (w v) sucrose gradients containing 150 mM NaCl 50 mM Tris, pH 7.6 and the respective detergent used for...

Assembly Assayed by Coimmunoprecipitation

Connexin specific monoclonal and antipeptide antibodies directed against different regions of axCx43, pxCx32, and p2Cx26 that display no detectable cross-reactivity with other connexin isotypes are used for the immunoprecipita-tion of connexin polypeptides from in vitro translation reactions (see Note 15). Oligomerization of connexin polypeptides into homo- and hetero-oligomeric complexes can be analyzed by immunoprecipitation following general methods described by Harlow and Lane (43). Connexins are translated in different combinations together with other connexin isotypes, connexin mutants, or nonconnexin transmembrane proteins. Connexin polypeptides are then immuno-precipitated either from complete translocation reactions or from microsomes that had been pelleted through sucrose cushions as described in Subheading 3.2.1., followed by their resuspension in 1x PBS, 0.25 M sucrose (see Fig. 8).

Validity of the Method and Determination of the Recovery

For all yogurts, the determination showed only a simple sugar, galactose, and a disaccharide, lactose. The retention times of these sugars differed from those of FOS and therefore should not interfere with the determination of FOS in yogurt. Inversely, the determination of sugars in the control cake with no FOS addition showed two simple sugars, glucose and fructose, one disaccharide, sucrose, and two starch residues, Rs1 and Rs2, the retention times of which correspond respectively to those of GF2 and GF3 peaks (Figure 1). small cake (J. Pasquier p tissier ''madeleine superstar'') after FOS addition showed glucose (G), fructose (F), sucrose (GF), GF2, GF3 and Rs1 and Rs2. After the invertase action (Figure 3) the disappearance of oligosaccharides can be observed, correlated to an increase in glucose and fructose concentrations. On the other hand, Rs1 and Rs2 were not hydrolyzed, and appear on the chro-matogram. If the specific areas of GF2 and GF3 were related to Rs1 and Rs2,...

Handling Storage and Preparation of Human Tissues

In contrast to blood cells, human tissue for use in flow cytometry must first be prepared as an adequate single-cell suspension. Selection of the appropriate methods for specimen collection, transport, storage, and tissue dissociation depend on the cell parameters being measured (e.g., protein, DNA, or RNA) and the localization of the markers (e.g., cell surface, cytoplasm, or nucleus). This unit includes a general method for collecting and transporting tissue samples to the flow cytometry laboratory (see Basic Protocol 1) and preparing a tissue imprint to assess the sample (see Support Protocol 1). Single-cell suspensions of whole cells can be prepared from fresh tissue using mechanical disaggre-gation (see Basic Protocol 2) or enzymatic disaggregation (see Alternate Protocol 1). Single-cell suspensions of whole cells can also be prepared from fine-needle aspitates (see Basic Protocol 3) or pleural effusions, abdominal fluids, or other fluids (see Alternate Protocol 2). Once a...

Planning An Experiment

The long-time success of sucrose gradient analysis. In many cases, these methods offer great advantages because the sample is analyzed after centrifugation by taking fractions (i.e., radial slices) from the centrifuge tube. Because of this, any detection scheme may be used to determine the concentration distribution. This means that unparalleled sensitivity and selectivity are afforded by schemes that use the preparative ultracentrifuge. Moreover, each fraction may be further fractionated e.g., by high-performance liquid chromatography (HPLC) or gel electrophoresis yielding good analytical sedimentation data for complicated mixtures. Detailed protocols for sedimentation velocity and sedimentation equilibrium in a preparative ultracentrifuge are available, and a superb microfractionator is available (from Brandel) specifically for conducting sedimentation analyses (Attri and Minton, 1986).

Removing Debris From Suspensions Of Nuclei

Sometimes filtered nuclear suspensions prepared from fresh, frozen, or paraffin-embedded tissue may contain considerable debris or increased amounts of fibrous connective tissue. In these cases, subcellular debris can be removed by centrifuging the suspension on a sucrose step gradient. 1.75 M sucrose in PBS (appendix 2a) 1.5 M sucrose in PBS (appendix 2a) 1. Add 800 l of 1.75 M sucrose to a 12 x 75-mm centrifuge tube. 2. Carefully layer 800 l of 1.5 M sucrose on top of the 1.75 M sucrose. 3. Carefully add 1.5 ml filtered nuclear suspension on top of the sucrose cushions.

Estimation Of Purity Of Sorted Fractions Using Prins

LB01 lysis buffer (see recipe) 10 (w v) sucrose or PRINS buffer (see recipe) 10 (w v) sucrose PRINS reaction mix (see recipe) containing fluorescein-labeled nucleotides Stop buffer (see recipe) Wash buffer (see recipe) 1. Pipet 15 l LB01 lysis buffer 10 sucrose (for cereal chromosomes) or PRINS buffer 10 sucrose (for legume chromosomes) onto a clean microscope slide.

Triton X100 Solubilization of Connexons

The first step in the biochemical detection of connexons by either chemical crosslinking or sucrose gradient velocity sedimentation is to solubilize these species from membranes in a manner that faithfully preserves their oligomeric state. In principle, this could be achieved by either dissociating fully assembled gap junctional plaques (see Fig. 1, no. 5) into individual connexons or by capturing nascent connexons (see Fig. 1, no. 2 and no. 3) prior to incorporation into junctional plaques. In the cell types we have examined, the first approach is of limited value. This is because most gap junctional plaques are exceptionally resistant to solubilization in common nondenaturing detergents, including Triton X-100 (TX-100), Triton X-114 (TX-114), octylpolyoxyethylene (POE), and octylglucoside (OG) when used in physiological salt solutions at 4 C (6). Harsher detergent treatments cause extensive breakdown of gap junctional plaques to individual connexin subunits rather than into connexon...

Sdspage Analysis of Connexons

After chemical crosslinking or sucrose gradient velocity sedimentation, monomeric and assembled connexin species must be immunoprecipitated and or analyzed by Western blotting using connexin-specific antibodies (11). Immunoprecipitants of connexins metabolically labeled with 35S methionine are detected after SDS-polyacrylamide gel electrophoresis by autoradiography or with a PhosphorImager. Unlabeled connexins are analyzed by immunoblotting either with or without prior immunoprecipitation (see Note 9). In the latter case, the presence of substantial amounts of TX-100 in the samples and their large volumes hinder analysis on minigels. We therefore precipitate the proteins in such samples using the method of Wessel and Flugge (12) prior to SDS-PAGE. This simple procedure yields near quantitative recovery of proteins and removes salts and detergents.

Discharge And Home Healthcare Guidelines

Explain how to calculate the American Diabetic Association exchange list to develop a satisfactory diet within the prescribed calories. Emphasize the importance of adjusting diet during illness, growth periods, stress, and pregnancy. Encourage patients to avoid alcohol and refined sugars and to distribute nutrients to maintain a balanced blood sugar throughout the 24-hour period.

Calibration standards

Table 8.3.8 provides a compilation of commercially available molecular size calibration standards and their suppliers. Some of these are available as kits (e.g., from Amersham Pharmacia Biotech). Protein standards should be prepared in the GF buffer that will be used in the molecular size determination. To prolong the life of the column, samples should be filtered through a protein-compatible filter prior to injection. The exact concentration to be prepared depends on the additives present (e.g., sucrose as stabilizer), detector sensitivity, and the zone broadening of the column. Concentrations of proteins in the range from 1 to 5 mg ml are frequently used for calibration. It is possible to mix several standards to calibrate the separation range of interest in a few runs (it is seldom necessary to calibrate the entire separation range of the column).

Models for Studying Permeability and Transport Across the Blood Brain Barrier

Hydrophilic tracer molecule with negligible cell uptake or binding. Suitable permeability markers of a size similar to small drug molecules (typically 100-400 Da mol.wt.) are sucrose and mannitol (used as radiolabeled probes) or fluorescent indicators such as fluorescein and Lucifer yellow.76 As electrical resistance is the reciprocal of conductance, related to the permeability of small ions, TEER is inversely proportional to the permeability or conductance of Na+ and Cl_ through the tight junctions, these being the major charge carriers in extracellular fluids.92 A plot of TEER versus small solute permeability through an epithelial or endothelial monolayer will give an asymptotic curve reflecting this relationship.95 Net solute flux increases as the sum of fluxes across individual junctional clefts, while TEER is most affected by areas with the lowest resistance, which shunt current flow. Hence a small flaw in a monolayer can cause a large drop in TEER with little drop in sucrose or...

Lectin Affinity Chromatography

The names, sugar specificity, elution conditions, and general types of sugar chains that bind to 25 different lectins are given in Table 9.1.1. This table is not comprehensive (there are at least twice as many lectins commercially available), but those listed are the most commonly used. The list is intended as a guide to possible alternatives, and an attempt has been made to group lectins of similar specificities together. However, specificities are not always hard and fast, and the comments in the table are not always sacrosanct. Some lectin-ligand binding requirements are well characterized, but many of the rules for lectin sugar specificity are based on screening simple sugars to inhibit lectin binding. These competitors can mimic only limited features of the binding of a lectin to its natural ligand. For instance, both pea and lentil lectins bind only sugar chains with a-fucose residues in the core region of N-linked chains, and yet mannose, not fucose, is used to elute bound...

Gradient Fractionation Using Percoll see Alternate Protocol

Percoll's negligible osmolarity but high density and modest viscosity allow its use as an additive to sucrose for achieving a very wide range of densities under isoosmotic conditions. 3. Equilibrium density separations require relatively short centrifugations, especially compared to those in high-density sucrose.

Impact of Freezing on Proteins

Finally, it must not be assumed that, although slower, degradative reactions will be entirely absent in a reduced water environment, and they should be considered and assessed during short or longer term storage in the lyophilized states (5). Saccharides are often chosen as stabilizers for lyophilization processes, but covalent modification of proteins in the presence of reducing sugars is well known, especially at elevated temperatures (6) and the need to control this should be considered when choosing excipients and storage conditions. All of these issues must be addressed by the selection of suitable formulation and processing conditions if stable lyophilized protein preparations, which retain full biological activity, are to be achieved.

High Throughput Assay for aAmylase Activity

Hydrolysis of starch by the a-amylase in the assay mix results in the formation of reducing sugars, which can be quantified photometrically using p-hydroxy benzoic acid hydrazide (PAHBAH) reagent. This reliable method is highly sensitive, easy to use, well suited for high-throughput screening, and does not employ toxic substances or unnatural substrates (8,9).

Formulation Stabilizer Choice for Proteins

Hydrogen bond-forming sugars, such as trehalose or sucrose, are well documented as excellent stabilizers for freeze-drying of proteins. Other stabilizers may be more specific in their effects (such as metal ions, e.g., zinc 7 ), whereas others, such as mannitol or glycine, are valuable as matrix formers without offering the stabilizing benefits of sucrose trehalose.

Intercomparison Study

Before starting the main intercomparison analysis, the participants were asked to analyze a mixture of four polysaccharide materials arabinogalactan (larch gum), fibrous cellulose, sugar free citrus pectin, and galactomannan (alcohol washed guar gum), and to obtain results within the known value 2 SD (based on the performance in the intercomparison study). Participants who were using gas chro-matography were asked, in addition, to carry out a similar exercise on a mixture of sugars. These two preliminary tests proved to be very useful, as they highlighted problems in some laboratories with the filtration step of the AOAC procedures, miscalculations, misunderstandings of the role of blanks, problems with response factors in GC, a problem with the makeup of a sugar standard and of the unknown sugar mixture, and uncovered minor errors in the protocol.

Background Information

Organelle purification procedures capitalize on the differences in size, density, and (occasionally) surface charge density of individual types of organelles. Most fractionation procedures that are based on centrifugation involve some combination of procedures that distinguish both size and density. Initially, a homo-genate is prepared in isoosmotic (or slightly hyperosmotic) sucrose or some other predominantly nonelectrolyte medium. Sucrose solution deviates from ideality while 0.3 M sucrose and 150 mM NaCl have equivalent particle concentrations, 0.25 M sucrose and 150 mM NaCl have the same osmotic activities. Investigators generally homogenize cells and tissues in solutions that are somewhere in the range of 0.25 to 0.3 M sucrose. The homogenate is filtered through cheese cloth or nylon screen to remove residual large connective tissue debris. Then differential centrifugation by velocity is used at low speed to pellet large particulates nuclei, erythrocytes, and larger particles...

Diagnosis And Assays For Human Herpesvirus 8 Infection

An alternative, less-subjective serology test for HHV-8 is the ELISA. A test using sucrose-purified virion has been developed (71). This assay has a sensitivity of 9095 in detecting antibodies in patients with KS, but its ability to detect antibodies in the general population and in epidemic areas like Africa has not been studied extensively. Additional ELISA assays have been developed using recombinant viral antigens, such as capsid protein ORF65.2 and lytic protein K8.1 (72). These assays are comparable to the latent IFA assays in detecting antibodies in KS patients but tend to be more variable in the general blood donor population (72,73). Western blot analyses using either infected whole-cell lysates or recombinant viral proteins have also been used (44). The latency-associated nuclear antigen is the primary antigen detected using the whole-cell lysate (11). These assays in general are much more difficult to perform. They are only about 80-90 sensitive in detecting antibodies in...

Classification Cephalosporin

Contraindications Hypersensitiv-ity to cephalosporins. Special Concerns Although cefti-buten has been approved for pharyngitis or tonsillitis, only penicillin has been shown to be effective in preventing rheumatic fever. Not approved to treat urinary infections. Hypersen-sitivity to penicillins. Use with caution in patients with renal impairement, infants < 6 months, and in patients with pseudomembraneous colitis. Oral suspension contains 1 g sucrose per 5 mL.

Determination Of Sugars And Oligosaccharides

A typical isocratic separation of simple sugars is shown in Figure 9. This method was adopted in 1994 as the official method for the determination of sugars present in sugar cane molasses by the International Commission for Uniform Methods of Sugar Analysis (ICUMSA). Among the advantages cited for the new method were 1) lack of co-elution with non-sugar impurities, 2) greatly reduced possibility of overestimation of sugars due to co-eluting impurities, 3) no column heater is required. Figure 10 illustrates the use of HPAE-PAD with gradient elution to separate maltose oligomers up to DP10. Figure 9 Determination of sugars in sugar cane molasses by HPAE-PAD. Column CarboPac PA1 (10 iM, 250 X 4 mm i.d.) with guard. Eluent 150 mM NaOH. Flowrate 1.0 mL min. Detection pulsed amperometry, Au electrode. Peaks. 1 glucose, 2 fructose, 3 lactose (internal standard), 4 sucrose.

Purification Of Vaccinia Virus

Vaccinia is usually purified by zonal sucrose gradient centrifugation. Purified virus is useful for preparation of vaccinia DNA (see Support Protocol 2), in studies in which contaminating infected-cell proteins are undesirable, and as a very high titer stock. For large-scale purification (as in this protocol, which is for 1-liter cultures or multiples thereof) it is preferable to use HeLa cell suspensions for infection rather than monolayer cultures. If monolayer cells are used, follow the alternative procedure for monolayers (see unit5.12, Basic Protocol 3) then continue with step 9 of this protocol. For many purposes, virus that is partially purified (through step 14 of this protocol) may suffice. For purification of MVA, virus is not trypsinized and either CEF or BHK-21 cells are used instead of HeLa.

Applications Of Nirs To Foods

And snack foods (24, 25) beta-glucan in barley (26) starch in grains (27), pulses (28), and snack foods (25) starch damage in flour (29) amylose in rice (30) sucrose in chocolate (31) and wine (32) soluble solids in vegetables (33) and alcohol in beer (34) and wine (32).

In Vitro Screening Of Enzyme Activity

Both a source of enzyme and an assay are required in order to screen and prioritize newly synthesized inhibitors of TACE. We chose the monocytic leukemia cell line, Mono Mac 6 as a source of TACE, since the cells produced significantly more TNF-a than other monocytic cell lines. Membrane-bound TACE (mTACE) is prepared by lysis of cells followed by subcellular organelle fractionation by a modification of the method of Fleischer and Kervina (19). The cellular homogenate is centrifuged at two low-speed spins to pellet the mitochondrial, lysosomal, and peroxisomal membranes. The supernatant is further centrifuged to generate a pellet thought to consist of endoplasmic reticulum, Golgi apparatus, and plasma membrane. The pellet is resuspended and placed on a sucrose gradient followed by centrifugation overnight in a swinging bucket rotor. Membrane bands at the interfaces of the discontinuous sucrose gradient (Fig. 1) contain functionally purified TACE activity. This activity has a...

Development Of Solidphase Peptidesynthesis Methodology

A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar substitute aspartame to clinically used hormones such as oxytocin, adrenocorticotropic hormone, and calcitonin. Rapid, efficient, and reliable methodology for the chemical synthesis of these molecules is of utmost interest. The stepwise assembly of peptides from amino acid precursors has been described for nearly a century. The concept is a straightforward one, whereby peptide elongation proceeds via a coupling reaction between amino acids, followed by removal of a reversible protecting group. The first peptide synthesis, as well as the creation of the term peptide, was reported by Fischer and Fourneau (1901). Bergmann and Zervas (1932) created the first reversible Na-protecting group for peptide synthesis, the carbobenzoxy (Cbz) group. DuVigneaud successfully applied early classical strategies to construct a peptide with oxytocin-like activity (duVigneaud et al.,...

Basic Dna Content Analysis Of Samples Utilizing Detergents And Protocol 2 Trypsin

Cells are collected via aspiration from tissue samples into a sucrose-citrate buffer that contains DMSO, which allows for long-term storage of samples if needed. After samples are supplemented with an internal DNA standard (a mixture of chicken and trout erythrocytes), cells are lysed, digested with trypsin, and stained with PI. The stained nuclei are then subjected to flow cytometry.

Classical Biochemical Approaches

Figure 4 Sucrose density centrifugation separation of subcellular fractions of housefly larval homogenates showing the distribution of marker enzyme activities between mitochondrial and microsomal fractions. Note some P450 reductase activity in the top (soluble) fractions representing proteolytically cleaved enzyme. (Reprinted with permission from Feyereisen, R., 1983. Polysubstrate monooxygenases (cytochrome P-450) in larvae of susceptible and resistant strains of house flies. Pestic. Biochem. Physiol. 19, 262-269 Elsevier.) Figure 4 Sucrose density centrifugation separation of subcellular fractions of housefly larval homogenates showing the distribution of marker enzyme activities between mitochondrial and microsomal fractions. Note some P450 reductase activity in the top (soluble) fractions representing proteolytically cleaved enzyme. (Reprinted with permission from Feyereisen, R., 1983. Polysubstrate monooxygenases (cytochrome P-450) in larvae of susceptible and resistant strains...

Sugar cane Saccharum officinarum Poaceae

The process of producing unrefined sugar has been known in the Far East and India for several millennia. The Ottoman Turks produced refined white sugar in the 1300s. The Crusaders took sugar cane from Israel to various Mediterranean islands and Iberia. From this launching point, it spread rapidly as colonists traveled to Madeira and the Canary Islands, then to the West Indies and the Americas, finally arriving in the islands of the Indian Ocean. Now grown throughout the tropical world, especially in the West Indies and Hawaii, almost all cultivars are derived from S. officinarum, a complex aggregate of hybrids. Nearly all sugar cane is vegetatively propagated by means of stem cuttings. One of the reasons for the successful establishment of the cane industry worldwide is that this method of propagation allows for the harvesting of one crop per year.

High Intensity Sweeteners

The search for sweet-tasting plants and their extracts has a long history, but the most intensive search has occurred in the last 75 years. Several plant-derived compounds of the protein, terpenoid, and phenolic types have commercial use as sweeteners. Most of these new compounds are prototype high-intensity sweeteners ranging from 30 to 2,000 times sweeter than sucrose that may be worthy targets for chemical synthesis or for semisynthetic modification to produce substances with enhanced sweetness properties.

Tissue Sectioning and XGal Reaction

Organs are cryoprotected by immersion in 30 sucrose solution (in phosphate buffer) overnight. Sections are cut using a cryostat (10 m) or a freezing microtome (100-200 m) and washed 3X in washing buffer, at 20 min intervals. Incubation in X-gal reaction solution is best carried out overnight in a 37 C oven (see Note 7). To ensure that the sections do not dry out, an adequate volume of reaction solution should be added (e.g., 1 mL for a 35-mm Petri dish). Gentle shaking on a rocking platform is optional this ensures even penetration of the substrate. An intense blue reaction product should be evident by 12 h. Sections are then rinsed in wash buffer and mounted on slides for dehydration in graded ethanols, cleared, and cover-slipped using Permount. The reaction product is stable in mounted sections. A light bluish background stain is sometimes seen in certain tissues (see Note 8).

Treatment of Iron Deficiency

For some patients iron supplements are ineffective. We do not know why some people fail to absorb supplementary iron. These patients may require intravenous iron. The kind of intravenous iron formerly used in the United States, iron dextran, caused death in approximately 0.7 percent of patients due to allergic reactions, making its use very dubious however, iron sucrose, which has been used safely in Europe for decades, is now available here. So is ferric gluconate, another form of intravenous iron. Recently, the effectiveness of iron sucrose, given intravenously weekly for five weeks, was compared with the same treatment plus erythropoietin injections in 90 anemic predialysis patients. Hematocrit rose rapidly in both groups and often could be maintained with iron treatments alone.

Isolation and Use of Rafts

This unit describes methods for isolating and analyzing rafts by detergent insolubility. To distinguish these rafts from raft-like membranes isolated by other methods (see Background Information), they are referred to here as detergent-resistant membranes (DRMs). DRMs can be isolated by flotation on sucrose density gradients (see Basic Protocol) or by pelleting after detergent extraction (see Alternate Protocol). DRM proteins can be analyzed by SDS-PAGE and immunoblotting (see Basic Protocol). Additionally, radiolabeled DRM proteins can be analyzed (see Support Protocols 1 and 2), and lipids can be quantitated by high-performance thin layer chromatography (HP-TLC see Support Protocol 3). Support protocols needed for the lipid analysis are also provided (see Support Protocols 4 and 5). Finally, protocols for raft disruption by cholesterol removal (see Support Protocol 6) and measuring the kinetics of such removal (see Support Protocol 7) are included together with a method that...

Proteins By Immunoblotting

Cells are lysed on ice with Triton X-100 following several possible procedures, outlined below. These vary depending on whether nonadherent or adherent cells are used, and whether comparisons are to be made between different cell lines. Lysates, which contain solubilized proteins, insoluble cytoskeleton and nuclear remnants, and insoluble DRMs, are adjusted to a high density with sucrose and placed in a centrifuge tube. A step gradient is formed over the lysate. After centrifugation for the appropriate time, low-density DRMs float to their position of equilibrium density, high-density cytoskeletal proteins sediment to the bottom, and cytoplasmic proteins and mixed micelles remain near their original position. DRMs can be isolated by this method from virtually any mammalian cell type. The sucrose gradient can be fractionated to determine the distribution of proteins of interest by immunoblotting (steps 14a and 15a). Alternatively, the floating DRM band can be harvested from the...

Preparation Of Detergentresistant Membranes By Centrifugation

This procedure is faster than isolation of DRMs by sucrose-gradient centrifugation, and allows one to determine whether a DRM protein is insoluble in Triton X-100. Triton-solu-bilized DRM proteins can be isolated (though not as cleanly as by sucrose-density gradients) with an additional step that takes advantage of the solubility of DRMs in SDS or octyl glucoside. This allows comparison of the Triton-soluble fraction with Triton-insoluble, SDS- or octyl glucoside-soluble fractions. The SDS-soluble fraction contains mostly cytoskeleton, and the octyl glucoside-soluble fraction contains mostly DRM proteins. SDS should be used for proteins that are stable or can refold after brief exposure to 1 SDS. Because volumes are small, this procedure is useful for studying DRM association of radioactively labeled proteins (for instance, by pulse-chase analysis see Support Protocol 1) or by immunoblotting. A disadvantage is that this procedure does not separate low-density DRMs from high-density...

Mechanisms of action

Hypoglycemia in acarbose- or miglitol-treated subjects must be treated with simple carbohydrates found in milk, juices, or glucose tablets. Disaccharides or polysaccharides (sucrose (table sugar), candy, and soft drinks) cannot be used because the a-glucosidase inhibitory effects delay their hydrolysis and absorption.

Disorders of the Kidney

Diabetes and hypertension (high blood pressure) are the two leading causes of kidney disease. In diabetes, blood flow through the kidneys increases, causing the kidneys to enlarge, and the excess sugar in the blood damages the glomeruli (tiny blood vessels that are part of the nephrons). High blood pressure can cause kidney disease by damaging the small blood vessels needed for filtering and reabsorption of fluids. Conversely, hypertension can result from kidney disease if blood flow through the kidneys is obstructed or slowed, resulting in the release of hormones that cause blood pressure to rise. See page 365 for more information about diabetes and page 217 for more information about hypertension.

Thaumatin Thaumatococcus daniellii Marantaceae

The thaumatins are a class of intensely sweet proteins (1,600 times as sweet as sucrose) isolated from the aril of the fruit of the tropical west African species Thaumatococcus daniellii. Thaumatin is approved for use in many countries, and it serves both as a flavor enhancer and a high-intensity sweetener. The supply of naturally occurring thaumatin is limited, which has prompted extensive research into its synthesis via transgenic organisms. The gene encoding thaumatin has been introduced into various microorganisms. The unique properties of thaumatin as a food additive could well be exploited by the food and beverage industry. Or, we might yet see the thaumatin gene engineered directly into fruit and vegetable crops to improve their flavor and sweetness.

Load and run gradients

Use a gradient-forming device to form a continuous sucrose gradient on top of the loaded samples. To form the gradient, place 0.25 M sucrose solution in the back reservoir of the gradient-forming device and 1.02 M sucrose solution in the mixing 5. After the gradients are made, insert the tubes into the buckets of the swinging-bucket rotor. Weigh buckets plus tops and balance in pairs by adding a small amount of the lowest-density sucrose medium to the lighter tube of each pair. If the deceleration is too rapid, the layers of sucrose (e.g., between the load zone and the gradient) rotate relative to one another, which causes vortexing into the overlying sucrose layer(s) and decreases the resolution. With older ultracentrifuges (Beckman L5-50, L5-65, and older), the rotor coasts to a stop from maximal speed, taking 30 min from 28,000 rpm. As most of the disturbance of gradients occurs below the final 1000 rpm of deceleration, newer instruments decelerate to 800 rpm and then coast to a...

Assays for the Mannan Binding Lectin Pathway

As discussed in previous units of this chapter, the complement system is part of the innate immune system and contributes to the establishment of adaptive immune responses. The mannan-binding lectin (MBL) pathway of the complement system is activated when MBL binds to specific carbohydrates (e.g. mannose, glucose, L-fucose, and N-acetylglu-cosamine) expressed on the surfaces of microorganisms. Such sugar residues are usually present at the terminal nonreducing position. High-affinity binding with MBL requires that these sugars be clustered to activate the MBL pathway. The affinity towards a single monosaccharide is in the order of 10-3 M, whereas the avidity can reach 10-9 M when multiple bindings are achieved. In the circulation, MBL is found in a complex with serine protease zymogens MASP-1 (MBL-associated serine protease-1), MASP-2 (MBL-as-sociated serine protease-2), and MASP-3 (MBL-associated serine protease-3). The MBL-MASP complex also contains a nonenzymatic protein MAp19. The...

Recent Studies on the Possible Identity of Microsomal CATa

Possible Detection of CATa by Reaction with DNP-Etomoxiryl-CoA. Incubation of rat liver microsomes (purified by sucrose-density gradient centrifugation) with DNP-coupled etomoxiryl-CoA, followed by SDS polyacrylamide gel electrophoresis of membrane proteins and probing with an anti-DNP primary antibody, showed that DNP-etomoxiryl-CoA inhibited CATa activity by > 95 and labelled several protein bands.11 Of these, it was particularly noteworthy that one had a Mr of 88,000, similar to liver mitochondrial CPTi,12 and another had a Mr of 49-50,000, similar to that assigned to CATa by Murthy & Pande.6 The abundance of the 88kDa polypeptide in both mitochondrial outer membranes and microsomes was increased 2-3 fold on fasting (24 hours)

Gap Junctions Composed of Cx32 and Cx26 from Spodoptera Transfected Cultured Cells

Resuspend pellets in 12 mL of NaHCO3 buffer and place in a 100-mL flask. Place a large stirbar in the flask of sample, and, with spinning, slowly add 67 buffered sucrose until the sample has a sucrose density of 42 . Use a hand refractometer to confirm this, such as a Reichert-Jung 10431 hand refractometer. 6. Prepare six tubes (one sixth of the sample in each) of discontinuous sucrose gradients in ultraclear disposable tubes for the ultracentrifuge rotors, such as a SW-40 Ti or SW-28 rotor, by first adding 4-5 mL of the sample. Add 5 mL of 30 sucrose in buffer and finally top with NaHCO3 buffer. Centrifuge at 100,000g for 100 min. Stop without braking. 7. Collect bands at both the 42 30 and the 30 NaHCO3 interfaces. Also collect any material in the 30 sucrose bulk.

Measurement of Bacterial Ingestion and Killing by Macrophages

This unit presents fairly simple assays for measuring the binding of bacteria to macrophages, internalization of bacteria (also called ingestion or phagocytosis Fig. 14.6.1), and bacterial killing by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria. Because it is critical to remove residual extracellular organisms, the protocol presents two alternative steps to accomplish this a washing procedure and a more stringent method in which cells are sedimented through sucrose. In addition, it is important to distinguish those bacteria truly ingested by a macrophage from those that are bound to, but not internalized by, the cell. A simple but effective way to do this is described as the first alternate protocol.

Determination Of Stokes Radius By Hplc Gel Filtration

This protocol describes the use of HPLC gel filtration to rapidly determine the Stokes radius of proteins (also see unit 8.3, Basic Protocol 3). Protein standards may be ordered individually or as part of a kit. Table 8.3.8 provides a list of commercially available standards. It is important to note that a Stokes radius is a measure of molecular size and not molecular mass effectively, it is the radius of an equivalent hydrated sphere (Cantor and Schimmel, 1980). Thus, an approximate molecular mass may be obtained only when the protein of interest and the protein standards that are used in calibration have the same shape. As most standards are roughly spherical, the protein of interest should also be spherical. A better estimate of molecular mass (independent of shape) may be calculated from the Stokes radius combined with a sedimentation coefficient, which can be obtained using analytical ultracentrifugation or sucrose gradients (Seigel and Monty, 1966 unit 7.5).

Koichiro Murashima and Roy H Doi 1 Introduction

Commonly, cellulase activity is measured by a liquid assay, in which reducing sugars liberated from cellulosic substrates by cellulases are measured (8). The substrates for the liquid assay are Avicel for crystalline cellulose degradation activity, and carboxymethyl cellulose (CMC) for endoglucanase activity. Although the liquid assay is accurate and well-established, this method requires a complicated procedure, including boiling and dilution of the reaction mixture (8). Therefore, the liquid assay is not suitable for high-throughput screening of cellulases from DNA shuffled gene libraries.

Overview of Protein Purification and Characterization

As more proteins, and particularly enzymes, were purified and crystallized, they started to be used increasingly in diagnostic assays and enzymatic analyses, as well as in the large-scale food, tanning, and detergent industries. Many enzymes used in industry are not in fact very pure, but as long as they do the job, that is sufficient. Process enzymes such as a-amy-lase, proteases, and lipases are produced in ton quantities, mainly as secretion products in bacterial cultures, and may undergo only limited purification processes to minimize costs. Enzymes have long been used commercially in the food industry. Products such as cheese and high-fructose corn syrup could not be produced without the addition of enzymes. Rennet, used in cheesemaking, was one of the first commercially available bioengineered proteins. Partially purified protein preparations, such as whey or soy protein concentrates or isolates, are added as functional ingredients to a variety of food systems. At the other...

Preparation Of Periplasmic Extracts

This protocol describes a small-scale procedure to confirm the expression of proteins that are designed to be localized to the periplasmic space of E. coli. It is based on subjecting cells to an osmotic shock in a concentrated sucrose solution, which mechanically forces the contents of the periplasm through the outer membrane and cell wall. 20 (w v) sucrose 10 mM Tris-Cl (pH 7.5), ice-cold 0.5 M EDTA (pH 8.0) Ice-cold water

Chemical Additive Vitrification

Prepare a solution of standard liquid medium containing 1.2 M sucrose (see Note 23). PVS3 is an alternative solution comprising 50 (w v) sucrose and 50 (w v) glycerol prepared in standard liquid culture medium (8). 4. Remove the 100 PVS2 and replace with unloading solution containing 1.2 M sucrose in standard liquid medium. Perform two to three washes in fresh unloading solution and maintain in this solution for up to 30 min.

Encapsulation Vitrification

Suspend shoot-tips meristems following an appropriate pregrowth or hardening treatment (see Subheadings 3.2. and 3.3.), in Ca2+-free 2 or 3 (w v) Na-alginate loaded with a final concentration of 0.4 Msucrose, or a mixture of 2 Mglycerol and 0.4 M sucrose. 2. Dispense as droplets into 0.1 MCaCl2 solution made up in liquid culture medium containing a final concentration of 0.4 Msucrose, or a mixture of 2 Mglycerol and 0.4 M sucrose. e. Proceed as in steps 6-10, but omitting the application of PVS2 and sucrose unloading stages.

Sugar Chemistry Terminology

Household commodity many of us use to sweeten our coffee. In chemistry, sugar is the name given to any one of a large group of carbohydrate compounds that share common structural and chemical properties. In addition to cane sugar, known chemically as sucrose, other more familiar sugars are glucose and fructose. The sugars group has different subgroups based on molecular structural configurations or on other chemical properties. For the purpose of this chapter, sugars will be referred to as either monosaccharides or disaccharides. Monosaccharides are sugars that have the chemical formula C6H12O6 and are all isomers of each other. In other words, they have the same chemical formula but differ in their molecular configurations. Examples of monosaccharides are glucose and fructose. Monosaccharides are able to combine with each other to form chain molecules of any number of monosaccharide units, which are called polysaccharides. However, of interest to us at present are the disaccharides,...

Preparation Of Chicken Chromatin By Hypotonic Lysis Of Chicken Erythrocyte Nuclei

Company http cat no. C51153) Buffer A 0.08 M NaCl 0.02 M EDTA, pH 7.5 1.5 (v v) Triton X-100 in buffer A 1.7 M and 2.25 M sucrose (gradient quality) in buffer A 0.1 M EDTA 2. Prepare two cold discontinuous sucrose gradients in separate Beckman polyallomer 25 x 89-mm tubes, consisting of 2 ml of 2.25 M sucrose in buffer A (bottom of tube)

Identifying Adulterants and Diluents

Adulterants commonly encountered in heroin include quinine, procaine, acetaminophen, caffeine, diphenhydramine, aspirin, phenobarbital, and lidocaine. Adulterants commonly encountered in cocaine include procaine, benzocaine, and lidocaine. Diluents found in heroin include different kinds of starches. It is not uncommon to find in heroin substances such as calcium carbonate which had been added during the morphine extraction processes. Diluents found in both cocaine and heroin include lactose, mannitol, sucrose, and dextrose.

What Is Sugar Inversion

Sucrose is a disaccharide of combined glucose and fructose and has the chemical formula C12H22On. The chemical bond that Acidhydrolysis of sucrose results in combines the glucose and the fructose can be broken in several its conversion into glucose and ways that will result in the sucrose splitting up into its two fructose-monosaccharides, glucose and fructose. One way that this can come about is by hydrolysis, the chemical process in which a molecule of water breaks a bond between two parts of a molecule of another compound. In a sucrose solution, this hydrolysis reaction takes place under acidic conditions and is, therefore, called acid hydrolysis. The lower the pH, the faster the reaction proceeds. Heat will also increase the speed of the reaction. This hydrolysis of sucrose can be presented in the following chemical reaction. I indicated the molecular weights of the compounds, as these will be referred to shortly. Sucrose + Water Glucose + Fructose (6.1) Figure 6.1 describes the...

Diabetes and Insulin Resistance

The majority of data for animal studies thus far suggest that adiponectin acts as an insulin-sensitizing hormone. Adiponectin-knockout mice develop insulin resistance either independently of diet or only after high-fat and high-sucrose diet, and treating these mice with adiponectin ameliorates their insulin resistance (35,42). The insulin resistance in adiponectin-deficient lipoatrophic and obese mice can partially be reversed via adiponectin administration and fully restored with both leptin and adiponectin supplementation (29). Furthermore, in a longitudinal study analyzing the progression of type 2 diabetes in obese monkeys, decrease in adiponectin closely parallels the observed reduction in insulin sensitivity, and the obese monkeys with greater plasma levels of adiponectin had less severe insulin resistance (43). Although not entirely known, the cellular and molecular mechanisms linking adiponectin to improved insulin sensitivity are also likely multifactorial. In rodents,...

Why Is It Called Sugar Inversion

Sucrose + Water Glucose + Fructose (6.2) This shows that sucrose and glucose are dextrorotatory sugars, while fructose is highly levorotatory. In the acid hydrolysis of a sucrose solution, if allowed to go to full completion, the resultant solution would be a 50 50 glucose fructose mixture. This mixture would have an angle of rotation that would be the sum effects of the two monosaccharides. As the levorotatory rotation of the fructose is higher than the dextrorotatory rotation of the glucose, the solution of the two monosac-charides will display a levorotatory rotation. The net effect of this hydrolysis of sucrose is that a sugar solution that was at first dextrorotatory has now become levorotatory. As the polarized light rotation of the original sucrose solu- , . , will be sweeter than the original sucrose solution due to the fact Honey is a form of natural invert that fructose, which is about 1.6 times sweeter than sucrose, r J sugar is found in nature. In fact, the honey that bees...

Sugar Inversion And Soft Drink Technology

Sucrose is the major natural sweetener in soft drinks. Though in some parts of the world high-fructose corn syrup (HFCS) is being used as a preferred alternative natural sweetener, sucrose is still widely used in beverages. It is in these naturally sweetened beverages that sugar inversion plays an important role, and they are the subjects of this chapter on inversion. The sugar inversion process takes place under acidic conditions and speeds up with a decrease in pH. As acidic conditions promote the inversion process, it will take place in sucrose-sweetened soft drinks due to their usually relatively highly acidic nature. Regardless of the exact acid content of such beverages, they can all be considered, to a lesser or greater degree, as acidic solutions. As such, some inversion of the sucrose in these beverages will take place. As we have seen, one of the consequences of inversion is an increase in Brix. How and to what degree does this potential change in Brix impact on soft drink...

Myocardial Disease in Stimulant Abusers

A heterogeneous group of agents can cause toxic myocarditis, and since cocaine can be adulterated with a very long list of agents, implicating cocaine as the cause of eosinophilic myocarditis is difficult. A review paper published in 1988 listed sugars (lactose, sucrose, and manitol) as the most common cocaine adulterants, followed by stimulant drugs (caffeine, amphetamines) and local anesthetic agents as the most common agents found mixed with cocaine samples.64 Eosinophilic infiltrates in the myocardium could be in response to any of these agents, alone or in combination. Furthermore, most adult drug abusers are polydrug abusers, which further enlarges the list of possible offenders.

Control issues Clostridium botulinum

Glycerol, sucrose) or degree of cooking or baking resulting in adjustment of aW, must be considered carefully and tested. Thus the reduction in salt in certain preserved products may reach values that cannot prevent the growth of the organism, e.g. in cold-smoked fish levels of less than 3.5 salt on water have been found.42 In general the tendency towards more lightly preserved, lower thermal processing of foods could readily lead to more outbreaks of botulism as more reliance is placed on low temperatures (less than 3 C) alone to prevent or limit growth. Temperature abuse, in the distribution chain or in the domestic refrigerator, can occur readily.

Genes encoding enzymes for the biosynthesis of compatible compounds

Under stress, plants accumulate some osmolytes, such as sugars (mainly sucrose and fructose), sugar alcohols like glycerol, methylated inositols (D-ononitol), mannitol, complex sugars (fructans, threalose, raffinose), charged metabolites (proline, glycine, betaine, ectoine, dimethyl sulfonic propionate, polyamines) and ions (K+) (see review by Hasegawa et al. 2000). It is believed that their accumulation facilitates osmotic adjustment, contributing to stress tolerance. Transgenic plants (see review by Bajaj et al. 1999), overexpressing one or more osmolytes did not always result, as expected, in resistance to stresses such as cold or high salt concentration. In other cases the transgenes became resistant only with low concentration of osmolytes and were unable to play a role in osmotic adjustments (Zhang et al. 1999). This evidence suggests a different function for osmolytes, or their efficacy should depend on the presence around the organs (i.e. thylakoids and plasma membrane) to...

Strategic Planning

Basic Protocol 1 makes use of the fact that one of the products resulting from the PGase-catalyzed reaction is a reducing sugar (Figure C1.2.1). Thus, in the simplest case, a determination of the number of reducing ends generated during a given reaction period will directly correspond to the number of catalytic events during that period. The assay requires a measurement of the number of reducing ends present at the initiation of the reaction and at a sufficient number of time points to establish an initial velocity. The assay is based on taking measurements at predetermined time points rather than continuously monitoring the extent of the reaction, because of the relatively harsh conditions necessary for the detection of reducing sugars. The assay is equally sensitive to endo- and exo-acting PGases, because both enzymes generate a single reducing end per catalytic event.

Stepwise Summary and Review of TSF

Liposomes are centrifuged through an isoosmolar density gradient (TSF gradient) formed from the urea solution and a more dense solution in which the urea is osmotically replaced by sucrose (sucrose has greater density than urea). 7. Liposomes that do not contain functioning connexin channels are not permeable to urea and sucrose. Their density remains low due to the entrapped urea solution, and they remain in the upper part of the gradient. 8. Liposomes that contain functioning connexin channels are permeable to urea and sucrose, and equilibrate these two osmolytes. These liposomes effectively have the (greater) density of the phospholipid membrane, and move to a lower position in the gradient.

Blood glucose homeostasis

Glucose is produced by glycogenolysis and gluconeogenesis. Dietary glucose intake can be from glucose itself, glucose-containing polysaccharides (starch and glycogen), glucose-containing disaccharides (sucrose, maltose, lactose), sugars readily converted to glucose (fructose), gluconeo-genic amino acids and the glycerol moiety of triglycerides.

Mouse Immunization With rVV rFPV OR rAd

The construction, amplification, and purification of rVV are described in detail in Earl et al. (1998). All viruses mentioned in this unit can be obtained through Dr. Nicholas Restifo (email restifo at the Surgery Branch, NCI, NIH. This protocol describes the use of recombinant vaccinia virus (rVV), recombinant focolpox virus (rFPV), and recombinant adenovirus (rAd) in vivo. Make sure to contact the animal facility safety personnel to discuss safety and regulations for the use of viruses in a particular facility. Since rVV and rFPV are used identically, procedures are described for rVV only. The use of sucrose-purified preparations of rVV and rFPV at a concentration > 1 x 109 pfu ml is recommended since use of crude, unpurified, or low-titered preparations can result in the injection of relatively large and potentially lethal quantities of cellular debris and contaminants originating from the amplification cell line.

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