Cryo-protection

Henderson, R. (1990). Cryo-protection of protein crystals against radiation damage in election and X-ray diffraction. Proc. R. Soc. Land. B 241, 6-8. 

This enzyme exhibits no hydroxylase activity and is involved in the final synthesis of many naturally occurring /t-quinoncs. e.g. the naphthaquinone juglone in walnut (1.58) and the benzoquinone arbutin (hydroquinone-(3-D-glucopyranoside 2.46). Arbutin is a plant cryo-protectant that stabilizes membranes (Hincha et al., 1999). This compound has medicinal properties and has, for example, been used to treat urinary tract infections in humans. It is also used to lighten skin color, because it inhibits tyrosinase and hence the formation of melanin. The derivative deoxyarbutin (2.47 note the difference in the sugar molecule) was recently reported to be considerably more effective as a skin-lightening compound (Boissy et al., 2005). 

The freezing process will be discussed with model substances, which will be used as cryo-protective agents (CPAs). If a solution of water and glycerol is cooled quickly,... 

Fig. 18. Diagrams illustrating the differences and difficulties during freezing of cells in suspension (a) and on surfaces (b, c and d). In both cases, large ice crystal formation must be avoided, this means that freezing must be rapid and often involves the use of cryo-protectants. In suspension, the use of hypertonic solutions to shrink cells by osmosis helps to avoid membrane rupture. But with cells fixed to surfaces, shrinkage can lead to rupture of the filopodia or to parts of cytoskeleton or cell membrane (c). Additionally, animal cells under stress (including this kind of osmotic stress) tend to build up into a spherical shape. This means they would lose many of their surface contacts before freezing and disappear into solution after re-thawing. Cryo-con-servation of adhered cells in defined positions requires very precise control of the conditions...

One parameter, the kinetic energy of bovine spermatozoa motion (arbitrary units) in a viscous medium (standard cryo-protecting medium after a freeze-thaw cycle) was chosen to estimate bio-activity (Figure 2). Data corresponding to the admixture of nanocomposites (l)-(4) from Table 1 were measured using DLS in compared with the control cell suspension, and averaged over a wide concentration range of nanocomposite admixture. 

In the past, we have ascribed these differences in protein stabilization to Timasheff s thermodynamic mechanism. The only protein for which the needed thermodynamic parameters have been measured in the presence of all three cryo-protectants is chymotrypsinogen [83,84] Table 1. Although these data are not directly applicable to LDH, the general trends shown should be relevant to any protein. The increase in chymotrypsinogen chemical potential, (6 i2/Sm3), in the presence of either of two different molecular weights of PEG (e.g., = 400 or... 

K. Izutsu, S. Yoshioka, and T. Teroa, Decreased protein-stabilizing effects of cryo-protectants due to crystallization. Pharm. Res. 10 X232-X231 (1993). 

In brief, freeze-drying (lyophilization) consists of preparing the drug solution (with buffers and cryo-protectants), filtering through a bacteria-proof filter, dispensing into containers, removing water in a... 

Salts of weak organic acids that are soluble in lipids are also injurious to thylakoids. Examples are the salts of phenylpyruvic add (Figure 3) and caprylic acid (58). These salts, even if present at very low concentrations, cause extensive membrane inactivation during freezing, if cryo-protectants are absent. At 0°C, moderate concentrations of these salts will slowly inactivate thylakoids. 

A timesaving approach, which certainly is useful when there is reason to believe that antigenicity may be lost during the buffer rinses, is to rinse only briefly, section the tissue, and rinse the sections thoroughly. This drastically reduces the time in buffer. If the tissue is to be cryosectioned, one may even add cryo-protectant to the fixative and section the tissue taken directly from the fixative. 

Gonzalez, A., Thompson, A., and Nave, C. 1992. Cryo-protection of protein crystals in intense x-ray beams. Rev. Sci. Instrum. 63(1) 1177-1180. 

Incubate with 2.3 M sucrose or with a mixture of 1.8 M sucrose/20% polyvinylpyrrolidone (PVP) at room temperature for 30-120 min for cryo-protection. 

Bacterial isolates were maintained on working and short-term back-up nutrient agar slopes stored at 4 C. Cultures were plated out every 2 months to ensure purity, and fresh slopes made. Long-term storage was at -70 C in glass embroidery beads with 15% glycerol in nutrient broth as cryo-protectant (Jones et aL, 1991 [20]). 

Varisli, O., Uguz, C., Agca, C., and Agca, Y. 2009. Motility and acrosomal integrity comparisons between electro-ejaculated and epididymal ram sperm after exposure to a range of anisosmotic solutions, cryo-protective agents and low temperatures. Awim. Reproduc. Sci. 110 256-268. 

The use of hydroxyethyl starch as a cryoprotective agent for human erythrocytes has been examined in detail. The pertinent molecular properties of hydroxyethylstarch are described and conditions for its optimal use in cryo-protection summarized. 

Anomalous-scattering experiments usually need to collect diffraction data under several wavelengths, so the time for data collection should be longer, but radiation damage may become severe. In such cases, cryo-protection of the sample becomes necessary, due to the very strong synchrotron radiation beam. 

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