Sucrose loading

Figure 2. Peak serum insulin levels vs postprandial urine calcium excretion after a sucrose load.

The characteristics of the phloem loading system can be summarized as follows. Sucrose loading is (1) dependent on metabolism (2) carrier-mediated (3) selective for sucrose (4) maintains a high concentration inside the phloem which is the basis for the osmotically-driven mass flow of solutions and (5) dependent on the factors which control assimilate supply to the loading sites (e.g., photosynthesis, sucrose synthesis, and sucrose movement between leaf cells, and within subcellular compartments such as the cytoplasm and vacuole) ((>, 7 ). 

Dissolve pellet in 20 pL TE buffer. Add 5 pL sucrose loading buffer (use frozen aliquots). 

McLaughin and coauthors have developed a straightforward method to assess the binding of proteins or peptides to liposomes (Buser and McLaughlin, 1998). They use sucrose-loaded hposomes obtained by extrusion through 0.4 fim pore size filter. When such hposomes are mixed with proteins in an aqueous buffer, they can be readily pelleted by ultracentrifugation. By comparing the amount of protein in the pellet and in the... 

Buser, C. A., and McLaughlin, S. (1998). Ultracentrifugation technique for measuring the binding of peptides and proteins to sucrose-loaded phospholipid vesicles. Methods Mol. Biol. 84, 267-281. 

Binding Assay. 10 /ig sucrose-loaded liposomes were added to 10% of S-methionine labeled in vitro translated reaction product (5 /il) in 50 /il... 

Figure 6. Effect of hyperosmolality on FMLP-induced calcium transients in indo-l-loaded neutrophils. Cells were incubated at 37°C in either regular buffer (A) regular buffer plus 0.15-ilf sodium sulfate, 662 mosmol/kg (B) 0.3-M sodium HEPES, 645 mosmol/kg (C) or 0.45-jy sucrose, 870 mosmol/kg (D). All were treated with 5 M8/ > cytochalasin B and stimulated with 10 M FMLP. Excitation at 340 nm and emission at 400 nm.

Fig. 2.5.9 COSY spectra acquired at 600 MHz with an eight-coil probe along with the chemical structures of the compounds used. Each sample was a 10 mM solution in D20 loaded into the coil via the attached Teflon tubes, with the samples being (A) sucrose, (B) galactose, (C) arginine, (D) chloroquine, (E) cysteine, (F) caffeine, (G) fructose and (H)...

The sucrose content of oral liquids may cause significant problems when these products are prescribed for long-term therapy (e.g., asthma, seizure control, recurrent infections). Oral liquid preparations can represent a substantial carbohydrate load to children with labile diabetes, particularly if a child is ingesting more than one liquid medication with a high sugar content. 

Bring to a final volume of 1 ml with lysis buffer and carefully load the lysate on 10 to 50% sucrose gradient without heparin. 

Carefully load the sample on a sucrose gradient containing heparin and centrifuge as in Section 3.2. 

In a slightly different form, Eq. (6) is commonly referred to as the Warren spring equation. Representative yield loci determined utilizing the simplified shear cell are shown in Fig. 7 for spray-dried lactose, bolted lactose, and sucrose. The yield locus for each material relates the shear strength to the applied load. 

The range of application of shear cell testing methodology is seen in Tables 2-6. Table 3 relates the flow properties of mixtures of spray-dried lactose and bolted lactose. These mixtures, in combination with the excipients tested, cover a broad range of flow. Tables 4 and 5, for example, show lot to lot variations in the flow properties of several materials, and Table 6 shows the variation in flow properties of bolted starch, sucrose, and phenacetin at different relative humidities (RH). Figure 8 presents the yield loci of sucrose at four different consolidation loads. Also shown in the figure are the shear indices determined at each consolidation load. 

The influence of consolidation load on the flowability of sucrose is shown in Fig. 8. For this material, the effective angle of internal friction is nearly constant yet the shear index is seen to change with state of consolidation. Apparently, for sucrose, increased consolidation results in a somewhat more free flowing although still cohesive material. As such, sucrose can be considered a complex powder [49] with perhaps somewhat better flow characteristics when consolidated (as might occur in a hopper). 

This experiment requires a horizontal electrophoresis apparatus, power supply, and a gel-pouring tray with a comb to form sample wells. The FB 1001 apparatus available from Fisher Biotech and an apparatus available from EdVotek both are appropriate. The gel-pouring tray with combs is also available from EdVotek. Directions for preparing the tray accompany the unit. The Fisher apparatus includes a cooling unit that allows running at a constant temperature. In the EdVotek apparatus, the gel is under buffer. Since samples are loaded after the gel is immersed in buffer, sucrose must be added to increase sample density. 

If the expression of the mRNA results in a clear signal, a linear sucrose gradient (6-20% sucrose, 5 mM EDTA, 0.25% (w/v) sarcosyl, 15 mM PIPES-NaOH, pH 6.4) is prepared in SW27 tubes or equivalent and the mRNA is carefully loaded on top. After centrifugation (19 h, 80,000 xg), about 33 fractions of 1 ml each are collected, and the mRNA is precipitated using sodium acetate and ethanol. Prior to injection, the RNA of each fraction is pelleted, washed and dissolved in RNAse-free water as described above. Upon injection of 50 ng fractionated mRNA, a two- to fivefold increase in transport activity is expected for the positive mRNA-fraction compared with unfractionated mRNA. All other fractions should not show any specific functional activity. In cases where two neighbouring fractions induce transport activity, pooling of these should be considered. 

Using pyranine (8-hydroxy-1,3,6-pyrene trisulfonate) as intraliposome pH indicator, the liposomes were prepared as above (as in section Preparation of 100 nm SSL Loaded with DOX via Transmembrane AS Gradient ) with the exception that pyranine (0.5 mM) was included in the hydration solution. Removal of untrapped pyranine was achieved by gel filtration on a Sephadex G-50 column, preequilibrated with either NaCl, KCl, sucrose or AS solution (according to need). All these solutions also contained lOmM Hepes buffer at the desired pH (usually pH 7.5). 

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