Type II antifreeze protein from herring and smelt (AFPII) exhibits thermal hysteresis (TH) activity when the protein is saturated with Ca^<2+>. The ice-binding site of AFPII consists of Thr96, Thr98, and two Ca^<2+>-coordinating residues. Here we examined TH activity of AFPII from Japanese smelt (Hypomesus nipponensis) and its mutant proteins, T96S, T96V, T96A, T98S, T98V, and T98A. Note that T96S denotes a mutant whose Thr96 is replaced with Ser. It appeared that substitution of Thr96 with Ser and Val retained 90% and 45% of TH activity, respectively, while it becomes inactive when substituted with Ala. For Thr98, Ser substitution had no significant influence on TH, whereas it becomes less than 20% by Val and Ala substitutions. These results suggest that hydroxyl group rather than methyl group of Thrs are essential for TH activity of AFPII, which is quite distinct from the other types of AFP.

View full abstract

The NIAS Genebank project has been implemented as a national program in Japan to collect, characterize, evaluate, rejuvenate, conserve and use plant, micro organism and animal genetic resources for food and agriculture since 1985. NIAS has functioned as a central bank of the project in cooperation with other agricultural public and private sectors. This national network on genetic resources for food and agriculture currently preserves approximately 240,000 accessions of crops germplasm and wild relatives as the base collection of about 1,450 species involving 45,000 accessions of clonal crops. Cryopreservation at super-low temperatures, below -135℃, appears to be the logical choice for the long-term storage of plant germplasm and materials. Because this method requires a minimum of space, labor, medium, and maintenance. In NIAS Genebank, a practical cryopreservation project was started in 2004 using two cryopreservation protocols that had been developed. These cryopreservation procedures contribute and advance to establishment of the National Cryo-genebank for a back up of plant germplasm according to our priority of collections to be cryopreserved.

View full abstract

Cryopreservation in liquid nitrogen is the most reliable method for long-term storage and permits the maintenance of many cultures with little labor. Because biological time virtually stops at liquid nitrogen temperature, cell viability is independent of the period of storage. Recent developments have opened the possibility of cryopreservation for marine macroalgae. In this article the principle of cryopreservation is reviewed, and the practical procedure and equipment of cryopreservation are summarized.

View full abstract

Cryopreservation of mouse spermatozoa has been widely applied for maintenance of genetically modified mouse strains. Although cryopreservation of mouse spermatozoa is simpler, less time-consuming, and less costly than of embryos for preservation of the genetic materials, maintenance of cryopreserved spermatozoa still has a high running cost because of the need for a constant supply of liquid nitrogen. In 1998, it has been reported that freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injection into oocytes, and so they have attracted a great deal of attention as storable gene resources. However, attempts of freeze-dry spermatozoa are not new. Successful conception and full-term development using freeze-dried spermatozoa were reported in the cow and rabbit prior to 1960. Unfortunately, these preliminary results have not been confirmed. Spermatozoa become defective in motility after freeze-drying and are unable to fertilize eggs both in vivo and in vitro. Since freeze-drying of mammalian spermatozoa was demonstrated without loss of genetic or reproductive potential, recent investigations of freeze-dried sperm have focused on the factors affecting freeze-drying spermatozoa in an effort to store the male gamete at ambient temperature. It is particularly essential to assure long-term preservation for several decades or centuries. Recent findings indicate that the pressure levels at primary drying of freeze-dried spermatozoa, in addition to the components of the suspending solution, appears to be an important factor for long-term preservation of freeze-dried spermatozoa in mice.

View full abstract

To develop cryopreservation techniques for teleost fish, the effects of various cryopreservation conditions on post-thaw motility were examined in several species. Spermatozoa diluted with 10% methanol or 10% DMSO as a cryoprotectant showed the highest post-thaw motility. Fetal bovine serum was the best extender of the cryoprotectant for all species examined. Spermatozoa of all species cooled to -50℃ and then immersed in liquid nitrogen (LN) showed higher post-thaw motility than those cooled to -30℃, -40℃, -60℃, or -70℃ before the immersion. The spermatozoa were cooled to -50℃ at various cooling rates by changing the height of the sample above the LN surface. The height of the sample yielding the highest post-thaw motility of spermatozoa varied in a species-dependent manner. These results indicated that methods for the cryopreservation of spermatozoa in teleost fish that yield the highest post-thaw motility are similar except for the nature of the cryoprotectant and the cooling rate.

View full abstract

Seed plants, such as rice, wheat, soybean, and corn, are very important staple for people around the world. Thus, as the world's population continuously swell, there is a pressing need to increase and improve food production and quality. Selective breeding of plants requires seed preservation at low temperature. Seed life-span is dependent on relative humidity, so that longer shelf-life is attained at low temperature and humidity states. In this report, thermal analysis of rice, watermelon, pumpkin, and cucumber seeds was conducted. Changes in the freezing and non-freezing water of unpolished rice grains at each growth stage after flowering were measured by differential scanning calorimetry (DSC). The moisture content of unpolished rice grains rapidly increased until 10 days and decreased 20days after flowering. The length of unpolished rice grains remained unchanged after 10 days. The non-freezing water content peaked at the 10^<th> day and reached constant value after 20 days. The glass transition temperature was constant at about -100℃ up to 40 days after flowering. However the glass transition temperature abruptly increased after the 40^<th> day, when the freezing (unbound) water in the rice grains was no longer observed in the unpolished rice. For the unpolished rice, therefore, the glass transition temperature seemed to be dependent on the grain moisture content. The glass transition temperature of melon, cucumber, pumpkin, and watermelon seeds in full grown stage was at about 100℃. Fully ripe seed was stable at room temperature and germinated for high temperature above the glass transition of seed

View full abstract

High-sensitive biological calorimetry for living organisms is described. A commercial isothermal calorimeter (LKB 2277-201) was modified to measure thermogenesis less than 0.1 μW. Baseline was stabilized to be ±10 nW over a week. Glass ampoule with a screw cap made high sensitive measurement possible in an hour after loading a sample. Calorimetric studies of post-dormant development of Artemia franciscana and spore germination of Dictyostelium discoideum Ax-2 are presented as examples.

View full abstract

Trehalose is potentially a useful cryo- or anhydro-protectant molecule for cells and biomolecules such as proteins and nucleotides. A major obstacle to application is that cellular membranes are impermeable to trehalose. We recently isolated a novel trehalose transporter (TRET1) from an anhydrobiotic insect, Polypedilum vanderplanki. Transport activity of TRET1 was stereochemically specific for trehalose and independent of extracellular pH (between 4.0 and 9.0), indicating that TRET1 is a trehalose-specific facilitated transporter and that the direction of transport is reversible depending on the concentration gradient of trehalose. The extraordinarily high values for apparent Km (≧100 mM) and Vmax (≧500 pmol/min/oocyte) for trehalose both indicate that TRET1 is a high-capacity transporter of trehalose. Lastly I discuss possible applications of TRET1 for anhydro-preservation technology in the future.

View full abstract

The inert fluid perfluorocarbon (PFC) has been used since about 1960 in liquid respiration and artificial blood for mammals. PFC has been used to successfully resuscitate tardigrades that had been dried and exposed to a high barometric pressure of 6,000 atmospheres. Next, scientists attempted to experimentally preserve organs that had been removed from animals, dried, and immersed in PFC. Since 1998 preservation and resuscitation experiments have been conducted with mammalian hearts using 2,015 rats and 70 pigs. Among those experiments, the maximum time after desiccation until successful resuscitation was 26 days for a rat heart and 37 days for a pig heart. However, these results could not be reproduced. Finally, in 2005, this laboratory demonstrated that, a rat heart removed under of 2 atmospheres pressure and a CO_2 partial pressure of 400 hPa, followed by desiccation for 24 hr, could be revived and heterotypically transplanted. Moreover, these results were reproducible. The preservation time can be extended to 72 hr if, after immersing isolated rat hearts in PFC, they are dried by air exposure under a CO_2 partial pressure of 100 hPa. The present report documents the resuscitation of this heart after 48-120 hr of preservation followed by heterotrophic transplantation.

View full abstract