Dextran phosphate

The preparation of dextran phosphate (Fig. 9) is simply achieved by treatment of the polysaccharide with polyphosphoric acid in formamide yielding products with up to 1.7% phosphorus. 

These pure dextran phosphates exhibit immunostimulatory effects independent of the molecular weight. It was shown that the mitogenic response of murine splenocytes can be enhanced [101]. Moreover, dextran phosphate (Mw 40 000 g mol-1) increases the survival rate of mice infected with influenza A2 virus (H2N2). Intraperitoneal administration of dextran phosphate, an interferon inducer, shows a 1-day delay in the virus growth in lung, and production of HAI antibody, when compared to the non-treated or dextran-treated mice as controls. More significant was the 2-day delay in the development of lung consolidation, which led to 40% survival of the treated mice [102]. 

Dextran phosphates with additional long chain aliphatic ester functions such as palmitoyldextran phosphates exhibit antitumour activity (Fig. 10). 

In contrast to dextran phosphate, which is only of scientific interest up to now, the dextran sulfuric acid half ester and its sodium salt, usually simple referred to as dextran sulfate, is a commercially available product today. Its high purity, water solubility and reproducible quality commend it for many applications in molecular biology and the health care sector. 

Suzuki M, Mikami T, Matsumoto T, Suzuki S (1977) Preparation and antitumor activity of O-palmitoyldextran phosphates, O-palmitoyldextrans, and dextran phosphate. Carbohydrate Res 53 223-229... 

M. Suzuki, T. Mikami, T. Matsumoto, and S. Suzuki, Preparation and Antitumor Activity of 6>-Palmitoyldextran Phosphates, 0-Palmitoyldextrans, and Dextran Phosphate, Carbohydr. Res. 53 ... 

Bare W, Nordmeier E (1996) Studies of polyelectrolyte solutions VI. Effects of counterion binding by dextran sulfate and dextran phosphate in aqueous/organic solvents. Polym J 28 712 726... 

Elution Proteins 0.3 M NaCl in 0.1 M (0.05 M for SWxl columns) phosphate buffer, pH 7 dextrans and PEOs distilled water. 

FIGURE 4.2 Polyethylene oxide, dextran, and protein calibration curves for TSK-GEL SW Columns. Column TSK-GEL SW, two 7.S mm x 60 cm columns in series. Sample , proteins Q, polyethylene oxides O, dextrans. Elution dextrans and polyethylene oxides distilled water proteins 0.3 A1 NaCI in 0.1 M phosphate buffer, ph 7. Flow rate 1.0 ml/min. Detection UV at 220 nm and Rl. 

Elution Polyethylene glycols and oxides distilled water, dextrans and proteins 0.2 M phosphate buffer, pEl 6.8. 

Classical gene transfer methods still in use today are diethylamino ethyl (DEAE)-dextran and calcium phosphate precipitation, electroporation, and microinjection. Introduced in 1965, DEAE-dextran transfection is one of the oldest gene transfer techniques [2]. It is based on the interaction of positive charges on the DEAE-dextran molecule with the negatively charged backbone of nucleic acids. The DNA-DEAE-dextran complexes appear to adsorb onto cell surfaces and be taken up by endocytosis. 

The calcium phosphate method was first used in 1973 to introduce adenovirus DNA into mammalian cells [3]. DNA-Calcium-phosphate complexes are formed by mixing DNA in a phosphate buffer with calcium chloride. These complexes adhere to the cell membrane and enter the cytoplasm by endocytosis. Disadvantages of DEAE-dextran and calcium phosphate transfection are a certain level of cytotoxicity, a complicated transfection procedure, and the fact that not all cell types can be transfected using these methods. 

Gel Filtration. The lyophilized protein was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 m NaCl 0.013 % sodium azide and loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fi actions were collected. Fractions with pectin lyase activity were combined, dialyzed against distilled water and used in the next step. To estimate the molecular mass of PNL, the column was calibrated with standard proteins (Sigma MW-GF-70 Albumin, 66,000 Da Carbonic Anhidrase, 29,00 Cytochrome, 12,400 and Aprotinin, 6,500). The proteins were eluted in the conditions described above and their volumes (F ) were calculated fi om the peak maximum of the absorbance at 280 nm. The partition coefficient was obtained fi om the relationship where F, represents the bed volmne of column and F the void volume (which was calculated using blue dextran. Sigma). The molecular mass was determined using a standard curve of vs the logarithm of the molecular masses of the standards [28, 29]... 

To make an amine derivative of dextran, dissolve ethylene diamine (or another suitable diamine) in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of 3 M. Note Use of the hydrochloride form of ethylene diamine is more convenient, since it avoids having to adjust the pH of the highly alkaline free-base form of the molecule. Alternatively, to prepare a hydrazide-dextran derivative, dissolve adipic acid dihydrazide (Chapter 4, Section 8.1) in the coupling buffer at a concentration of 30 mg/ml (heating under a hot water tap may be necessary to completely dissolve the hydrazide compound). Adjust the pH to 7.2 with HC1 and cool to room temperature. 

Procedure The chromatographic procedure may be carried out at room temperature using (a) a column (1 M x 25 mm) packed with a cross-linked dextran suitable for fractionation of globular proteins in the range of molecular weights from 5,000 to 350,000 (Sephadex G-150 is suitable), (b) mixed phosphate buffer pH 7.0 with azide as the mobile-phase with a flow rate of about 20 ml (4 ml per square centimetre) of column cross-sectional area) per hour, and (c) a detection wavelength of 280 nm. 

Figure 4.20 Calibration curves for size-exclusion liquid chromatography. Column, TSK GEL G3000SW, 120 cm x 7.5 mm i.d. eluent, 0.2 m sodium phosphate buffer pH 6.8 flow rate, 1.0 ml min-1. Standards 1, protein-, 2, dextran, and 3, polyethylene glycol.

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