No abstract available.
Glucose* ; Hantaan virus* ; Lactose* ; Trehalose*

Now the clinical preservation methods of human red blood cells mainly include hypothermic storage (4 degrees C) and cryopreservation (-80 degrees C or -196 degrees C). The preservation time of hypothermic storage of red blood cells is relatively short and it is easy to be contaminated by microbes. Cryopreservation greatly prolongs the storage time, but it needs heavy storage equipments. Because the protective solutions in cryopreservation contain glycerol, red blood cells need complicated washing in order to remove glycerol. These shortage methods limit their application to some special conditions, such as war or natural disasters. Compared with conventional preservation methods of red blood cells, lyophilization has many advantages such as less weight, convenient transportation, room temperature preservation, prone to be rehydrated. In this review, the progress and challenge in the development of lyophilization of red blood cells, especially application of trehalose and its mechanism in the lyophilization of red blood cells were systematically discussed. This review can provide some theoretic guidance for developing a safe, simple and efficient preservation approach of red blood cells by lyophilization.
Blood Preservation ; Cryopreservation ; Erythrocytes ; Freeze Drying ; Humans ; Trehalose ; pharmacology

Selaginella tamariscina is a typical resuscitation medicinal plant with extreme drought tolerance. Trehalose plays an important role in the resurrection process, and the trehalose-6-phosphate synthase(TPS) is the key enzyme to synthesize trehalose in plants. In this study, the sequence of TPS was obtained by splicing from the transcriptome data of S. tamariscina. After the synthesis of cDNA based on the template of total RNA, the sequence was cloned by RT-PCR for verification and then analyzed by bioinformatics methods. The results indicated that the full-length coding sequence of StTPS was 2 799 bp (GenBank accession no. MH155231), and the encoded protein contained 932 amino acids. StTPS could be located in the chloroplastid according to subcellular localization prediction. There were two conserved domains belonging to glycogen phosphorylase glycosyltransferase (GPGTF) family but no signal peptide or transmembrane domain in StTPS. The expression of StTPS was determined by qRT-PCR and the variation of trehalose content was measured by HPLC-ELSD during the resurrection process of S. tamariscina. Meanwhile, the correlation between them was analyzed. The results showed that both the expression level of StTPS and the trehalose content increased associated with the extension of dehydration time, and declined associated with the extension of rehydration time which proved a significant positive correlation between the StTPS expression level and the trehalose content. The results suggested that the StTPS probably plays a central role in recovery process in S. tamariscina.
Amino Acid Sequence ; DNA, Complementary ; Glucosyltransferases ; Selaginellaceae ; Trehalose

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.
Biocatalysis ; Cloning, Molecular ; Escherichia coli ; Glucosyltransferases ; Streptomyces coelicolor ; Trehalose

Preservation methods on the physiological and brewing technical characters in bottom and top brewing yeast strains were investigated. The preserved yeasts were reactivated after 24 months storage and grown up to stationary phase. The samples of filter paper storage indicated a higher cell growth and viability during propagation than those of nitrogen and lyophilization storage independent on propagation temperature. In addition, the filter paper storage demonstrated a faster absorption of free amino nitrogen and a highest level of higher aliphatic alcohols production during propagation than other preservation methods, which can be attributed to intensive cell growth during propagation. Moreover, the filter paper storage showed a faster accumulation for glycogen and trehalose during propagation, whereas, in particular, lyophilization storage noted a longer adaptation time regarding synthesis of glycogen and trehalose with delayed cell growth. In beer analysis, the filter paper storage formed an increased higher aliphatic alcohols than control. In conclusion, the preservation of filter paper affected positively on yeast growth, viability and beer quality independent on propagation temperature. In addition, in this study, it was obtained that the HICF and Helm-test can be involved as rapid methods for determination of flocculation capacity.
Absorption ; Alcohols ; Beer ; Fermentation* ; Flocculation* ; Freeze Drying ; Glycogen ; Nitrogen ; Trehalose ; Yeasts*

OBJECTIVE: The aim of this study was to compare the survival and developmental rate of two vitrification solutions for the vitrification of mouse expanded blastocysts. METHODS: Mouse embryos were obtained at 2 cell stage and cultured to expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% serum substitute supplement (SSS). The vitrification solutions used were EFS40 and VS. EFS40 consisted of 40% ethylene glycol, 18% ficoll, and 0.5 M sucrose while VS consisted of 20% ethylene glycol, 20% DMSO, and 10% 1,3-butanediol diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% calf serum (CS). Toxicity was tested by exposing expanded blastocysts to vitrification solution. The vitrification procedure used for EFS40 was performed in three steps, after which they were warmed in 5 steps with 0.5 M sucrose. VS was performed in two steps, after which they were warmed with 1.0 M trehalose. Recovery, survival and hatching rate per expanded blastocysts recovered were compared between two groups. RESULTS: In toxicity test, survival and hatching rate of EFS40 group were 95% and 100%, respectively. In contrast, survival and hatching rate of VS group were 100% and 87.5%, respectively. After vitrification and warming in solution, recovery rate for EFS40 group was 73.7% whereas recovery rate for VS group was 66.5%. After 24 h culture, survival and hatching rate were 80.5% and 20.7% for EFS40 and 66% and 0% for VS group, respectively. After 48 h culture, survival and hatching rate were 69% and 33.3% for EFS40 and 58.3% and 1.9% for VS group, respectively. Survival and hatching rate in EFS40 group were significantly higher than those found in VS group. CONCLUSION: The EFS40 solution was better than VS solution for vitrification of mouse expanded blastocysts.
Animals ; Blastocyst* ; Dimethyl Sulfoxide ; Embryonic Structures ; Ethylene Glycol ; Ficoll ; Humans ; Mice* ; Sucrose ; Toxicity Tests ; Trehalose ; Vitrification*

Trehalose, as a nonreducing disaccharide, plays a role in protecting the cytoactivity when the cells is freezing, drying or lyophilization. It has been a biomembrane protectant applied to lyophilization of human blood cells (platelets and erythrocytes), and from which astonishing results have been obtained. Having powerful hydration, distinctive vitrification transform and crystal transform and unique resistance of high temperature and humidification, trehalose is thought of a preferred protectant in the study of cell preservation. In recent years, people concerned trehalose on its protective mechanism, experimental means of transit trehalose to mammal cells and the mechanism of loading in red blood cells. The above aspects were briefly summarized in this article.
Blood Preservation ; methods ; Cryoprotective Agents ; pharmacology ; Erythrocytes ; cytology ; drug effects ; Freeze Drying ; Humans ; Trehalose ; pharmacology

This study was purposed to evaluate the effect of different lyophilizing protectants including human albumin, glucan, polyvinyl pyrrolidone and glycerine on lyophilized trehalose-loading red blood cells (RBC), then to screen the optimal lyophilizing protectant. The RBC were incubated in 800 mmol/L concentration of trehalose solution at 37°C for 7 hours, and washed 3 times with PBS solution to obtain the trehalose-loading RBC. The trehalose-loading RBC in control group were directly lyophilized without lyophilizing protectants, the trehalose-loading RBC in the experimental group were mixed with Lyophilizing protectants. The samples of 2 groups were kept at room temperature for 30 minutes, pre-frozen at -80°C for 24 hours, then lyophilized in freeze-dryer for 24 hours. Finally the samples were quickly rehydrated by 6% HES at 37°C. The recovery rate and hemolysis rate of hemoglobin were detected by using cyanohemoglobin detection kit. The water content of unhydrated samples were detected at the same time. The results showed that when the moisture content of sample was 3% - 5%, the recovery rate of hemoglobin in control group was 33.57 ± 2.89%, and that in experimental group was 51.15 ± 1.98%, there was statistically significant difference between the control and experimental group (P < 0.05). When the different concentration of dextran solution was chosen as protectants, the recovery rate of hemoglobin of lyophilized RBC was obviously lower. The higher concentration of dextran, the better the recovery rate. The recovery rate of hemoglobin was 22.15 ± 4.12% when the concentration of dextran was 36%, there were statistically significant difference between the two groups (P < 0.05). When the different concentration of polyvinyl pyrrolidone (PVP) solutions was chosen as protectants, especially the concentration below 40%, the recovery rate of hemoglobin of lyophilized RBC was significantly belower than the control group, there was statistically significant difference between the two groups (P < 0.05). When 10% glycerol was used as protectants, the recovery rate of hemoglobin was 3.93 ± 1.80%. There was also statistically significant difference between the two groups (P < 0.05). It is concluded that human serum albumin shows an important protective effect on the lyophilization of the trehalose-loading red blood cells. The dextran and PVP at the concentration lower than 40% can decrease the protective effect of trehalose in cells. Glycerol can not be chosen as protectant for lyophilized trehalose-loading red blood cells.
Blood Preservation ; methods ; Cryoprotective Agents ; pharmacology ; Erythrocytes ; drug effects ; Freeze Drying ; methods ; Humans ; Trehalose ; pharmacology

After freeze-drying, the ultrastructure of boar sperms was observed by optical and electron microscopy. The in vitro development ability of the sperm was also examined with intracytoplasmic sperm injection (ICSI). The rate of male pronuclear formation was (68.52%), for cleavage (59.17%) and for blastocyst formation (19.16%) of the trehalose group (0.2 mol/L), significantly higher than those of the 50 mmol/L EDTA group (64.59%, 56.26% and 15.62%) and the control group (35.36%, 52.33% and 8.60%) (P < 0.05). After storage for 60, 120 and 180 d at 4 degrees C, no significant difference in the in vitro development was observed (P > 0.05). The male pronuclear, cleavage and blastocyst formation after ICSI with freeze-dried spermatozoa incubated for 1 h was superior than those incubated for 2 h (P < 0.05). No significant differences in the structures after stored at 4 degrees C or -20 degrees C (P > 0.05) were observed between the trehalose group and EDTA group. The percent of B grade freeze-dried boar spermatozoa in the trehalose group was higher than that of the EDTA group (P < 0.05). Based on the ultrastructure observation, main cryogenic damage in freeze-dried boar spermatozoa was swelling, damage or rupture in the sperm acrosome, neck and tail.
Animals ; Freeze Drying ; Male ; Semen Preservation ; methods ; veterinary ; Sperm Injections, Intracytoplasmic ; veterinary ; Spermatozoa ; Swine ; Trehalose ; pharmacology

Trehalose, a compatible solute, is widely used in food, cosmetics, pharmaceutical products and organ transplantation. Nowadays, trehalose is mostly produced by enzymatic synthesis with many secondary products and lowpurity. In this study, high amount of trehalose was produced by recombinant E. ccli fermentation. First, a bifunctional trehalose gene TPSP was amplified from genome of C. hutchinscoii. Second, an expression vector pTac-HisA containing TPSP was constructed and transformed into the host E. coli. Expression of this bifunctional enzyme-TPSP converted glucose to trehalose. The result suggested that TPSP from C. hutchinsonji has been successfully expressed in E. ccoi. High amount of extracellular trehalose generated from glucose by whole-cell catalysis and After optimization, the production of trehalose in shake flasks was improved to 1.2 g/L and the relative conversion rate reached 21%. The production in bioreactor reached 13.3 g/L and the relative conversion rate reached 48.6%. It is the first time to realize the functional expression of the bifunctional enzyme-TPSP of C. hutchinsonii in E. coli and achieved the conversion form glucose to trehalose. This study laid a foundation for industrial large-scale production of trehalose.
Bioreactors ; Catalysis ; Escherichia coli ; genetics ; Glucose ; Glucosyltransferases ; Industrial Microbiology ; Organisms, Genetically Modified ; Trehalose ; biosynthesis