Beneficial effect of microinjected trehalose on the cryosurvival of human oocytes.

OBJECTIVE: To determine the effectiveness of trehalose as an intracellular cryoprotectant for the cryopreservation of human oocytes. DESIGN: In vitro comparative study. SETTING: Clinical and academic research environment at a medical school teaching hospital. PATIENT(S): Women undergoing in vitro fertilization (IVF). INTERVENTION(S): Discarded human oocytes, obtained from IVF patients, were randomly distributed into three groups: control group (no trehalose), extracellular trehalose group (0.5 M extracellular trehalose), and intracellular trehalose group (0.15 M intra- and 0.5 M extracellular trehalose). Trehalose was introduced into oocytes by microinjection. The oocytes in each group were cooled to different temperatures (i.e., -15 degrees C, -30 degrees C, and -60 degrees C) at rate of 1 degrees C/minute and thawed at ambient air temperature. Survival was examined after overnight culture. MAIN OUTCOME MEASURE(S): Survival of human oocytes cryopreserved in the presence and absence of trehalose. RESULT(S): The majority of oocytes in the intracellular trehalose group survived cooling to -15 degrees C (63%), -30 degrees C (53%), and -60 degrees C (66%). In contrast, only a small number of oocytes in both the control (13%) and extracellular trehalose group (22%) survived cooling to -15 degrees C, while all oocytes degenerated when cooled to -30 degrees C and -60 degrees C. CONCLUSION(S): Small amounts of intracellular trehalose in the absence of any other cryoprotectant provide a significant protection against freeze-associated stresses. Our results suggest that sugars such as trehalose should be considered as intracellular cryoprotectants for cryopreservation of human oocytes.     

Role of trehalose in resistance to macrophage killing: study with a tps1/tps1 trehalose-deficient mutant of Candida albicans

Accumulation of trehalose by yeast is an important protective mechanism against different stress conditions. This study examined the effect of trehalose on several growth features, as well as its association with the intracellular survival of yeasts exposed to macrophages. A tps1/tps1 mutant and its parental counterpart, CAI4, exhibited similar growth rates and preserved their dimorphic conversion and agglutination ability. However, electron-microscopy of cell-wall architecture showed a partial loss of material from the outer cell-wall layer in the tps1/tps1 mutant. Flow-cytometry revealed that the mutant had lower auto-fluorescence levels and a higher fluorescein isothiocynate staining efficiency. When co-cultured with macrophages, a slight reduction in binding to macrophages and slower ingestion kinetics were revealed for the tps1/tps1 mutant, but these did not interfere significantly with the amount of yeast ingested by macrophages after co-incubation for 2 h. Under the same conditions, CAI4 cells were more resistant to macrophage killing than was the tps1 null mutant, provided that the macrophages had been stimulated previously with interferon-gamma. Measurement of trehalose content and the anti-oxidant activities of yeast cells recovered after phagocytosis revealed that the trehalose content and the glutathione reductase activity were increased only in CAI4 cells, whereas levels of catalase activity were increased similarly in both strains. These results suggest that the presence of trehalose in Candida albicans is a contributory factor that protects the cell from injury caused by macrophages.     

Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplanki

Anhydrobiosis is an extremely dehydrated state in which organisms show no detectable metabolism but retain the ability to revive after rehydration. Thus far, two hypotheses have been proposed to explain how cells are protected during dehydration: (i) water replacement by compatible solutes and (ii) vitrification. The present study provides direct physiological and physicochemical evidence for these hypotheses in an African chironomid, Polypedilum vanderplanki, which is the largest multicellular animal capable of anhydrobiosis. Differential scanning calorimetry measurements and Fourier-transform infrared (FTIR) analyses indicated that the anhydrobiotic larvae were in a glassy state up to as high as 65 degrees C. Changing from the glassy to the rubbery state by either heating or allowing slight moisture uptake greatly decreased the survival rate of dehydrated larvae. In addition, FTIR spectra showed that sugars formed hydrogen bonds with phospholipids and that membranes remained in the liquid-crystalline state in the anhydrobiotic larvae. These results indicate that larvae of P. vanderplanki survive extreme dehydration by replacing the normal intracellular medium with a biological glass. When entering anhydrobiosis, P. vanderplanki accumulated nonreducing disaccharide trehalose that was uniformly distributed throughout the dehydrated body by FTIR microscopic mapping image. Therefore, we assume that trehalose plays important roles in water replacement and intracellular glass formation, although other compounds are surely involved in these phenomena.     

外源海藻糖对NaCl胁迫下甘草幼苗生长及总黄酮含量的影响

目的 研究外源海藻糖对NaCl胁迫下甘草Glycyrrhiza uralensis幼苗的生长及总黄酮含量的影响。方法 研究了NaCl胁迫对甘草幼苗生理生长、酶活性、离子含量、渗透调节及总黄酮含量的影响,采用Microsoft Excel 2010对数据进行处理分析与整理,以SPSS19.0统计软件对数据进行方差分析。结果 10～20 mmol/L海藻糖能够显著降低NaCl对甘草幼苗的伤害,且外源海藻糖浓度为15 mmol/L时的作用效果最好。NaCl胁迫下施加外源海藻糖浓度为15 mmol/L时甘草幼苗的生长最旺盛其生长量增加最明显,且影响甘草幼苗渗透调节的K⁺、K⁺/Na⁺浓度增加最明显,Na⁺和Cl^-的浓度相对于无NaCl胁迫时有所降低;外源海藻糖浓度为15 mmol/L时可使NaCl胁迫下甘草幼苗内的抗氧化酶活性提高,并且可以增加NaCl胁迫下植物细胞内光和色素叶绿素的含量,外源海藻糖浓度为15 mmol/L可以降低NaCl胁迫下甘草幼苗内可溶性糖、脯氨酸以及细胞调节物质丙二醛（MDA）的含量。结论 甘草在NaCl胁迫下施加适宜浓度的外源海藻糖能够促进甘草幼苗生长和有效成分的积累,减少了盐害对甘草生长的危害,增强NaCl胁迫下甘草的生长能力。     

人红细胞冻干前负载海藻糖最佳化研究

为更好的实现海藻糖在红细胞冻干保存中的保护作用,关键是克服质膜对海藻糖的非渗透性,使胞质内海藻糖达到有效浓度。本研究的目的是通过对人红细胞负载海藻糖的规律性研究,筛选出海藻糖负载的最佳负载条件并评价海藻糖负载对红细胞各项理化指标的影响。在不同孵育温度(4、22和37℃)、孵育时间(0、2、4、6、8、10小时)、不同负载缓冲液浓度(0、200、400、600、800、1000mmol/L)条件下检测新鲜红细胞对海藻糖的成功负载量及红细胞各项理化指标;在固定负载条件下,对新鲜红细胞和4℃保存72小时红细胞海藻糖负载、游离血红蛋白(FHb)、血红蛋白(Hb)和红细胞平均体积(MCV)进行了比较。结果表明:红细胞对海藻糖的负载与孵育温度、时间及负载缓冲液海藻糖浓度密切相关。随着温度的升高、时间的延长和负载缓冲液海藻糖浓度的增加,红细胞对海藻糖的摄取量也随之增加。在海藻糖负载最佳条件下,新鲜红细胞和4℃保存72小时红细胞的胞内海藻糖浓度、FHb浓度分别为65.505±6.314mmol/L、66.2±5.002mmol/L和6.567±2.568g/L、16.168±3.922g/L。结论:红细胞负载海藻糖的最佳条件是采用新鲜红细胞,在37℃条件下、海藻糖浓度为800mmol/L的负载缓冲液中孵育8小时,这一条件可使胞内海藻糖达到有效浓度,并保持红细胞细胞理化性质稳定和膜完整性。     

基于离子色谱法的海藻糖检测方法研究

目的建立符合药用辅料检测要求的海藻糖检测分析方法。方法采用离子色谱高效阴离子交换层析分离与脉冲安培检测器联用技术进行检测。结果海藻糖与杂质的色谱分离度R>1.5;检测限为2μg·L-1(S/N=3.5),定量限为10μg.L-1(S/N=13.3);检测线性范围为62.5～10 000μg·L-1(进样量25μL,标准曲线Y=0.0026 X+0.1265,r=0.9999);精密度试验RSD=0.42%;回收率为98.09%～100.0%,RSD=0.58%。结论本文建立的海藻糖检测分析方法达到中国药典对药物检测分析方法的相关要求,可作为海藻糖药用辅料的检测方法。     

新型血小板添加液对血小板低温液态保存效果的实验研究

目的探讨使用改良的血小板添加液（PAS）替代70％自体血浆在低温液态条件下对血小板的保存效果。方法采集6单位单采血小板,每一单位单采血小板平均分为3组,A组加入70％PAS-Ⅲ M／30％血浆10℃保存;B组加入100％血浆22℃保存;C组加入100％血浆和海藻糖-85℃保存。A、B组在第1、5、7、9天取样检测,C组在20d后复苏取样检测。分别检测PLT计数、PDW、MPV、CD62p、HSR、PAgT、pH值、LDH以及GLU的变化。结果保存期内随保存时间延长,A组和B组CD62p表达增加,HSR和最大聚集率降低;A组的LDH释放、GLU消耗、CD62p表达、HSR、血小板最大聚集率与B组比较,差异有统计学意义（P〈0．05）。B组pH值在第5天起明显降低（P〈0．05）。C组LDH释放明显较其他两组增多,但CD62p表达较少（P〈0．05）。C组PDW显著升高,除与B组第5天结果相似外,均高于A、B组各时间点结果（P〈0．05）。各组PLT计数相差不显著。结论改良的PAS-ⅢM能够很好的替代血浆用于血小板的保存,同时在低温条件下能够很好地保护血小板的功能,保存效果好于血浆常温保存。     

Analysis of genome sequence and trehalose lipid production peculiarities of the thermotolerant Gordonia strain

Gordoniae are one of the most promising hydrocarbon-oxidizing actinobacteria. Here we present the genome sequence analysis of thermotolerant strain Gordonia sp. 1D isolated from oil-refinery soil. It is capable of alkane consumption and biosurfactant production at temperatures of up to 50°C. Gordonia sp. 1D demonstrates maximum biosurfactant production when grown on hexadecane, and at 40°C it was slightly higher than at 27°C: 35 and 39 mN/m, respectively. For the first time, it was experimentally confirmed that the carbohydrate component of extracellular biosurfactants produced by strain 1D is trehalose. In addition, genes for the production of trehalose lipid biosurfactants were identified. The genetic determinants for two different pathways for trehalose synthesis were found. The strain carries genes otsA and otsB involved in de novo trehalose biosynthesis. Moreover, the genes treY and treZ responsible for trehalose biosynthesis from maltooligosaccharides and starch or glycogen were identified.     

Rhythmic change of adipokinetic hormones diurnally regulates locust vitellogenesis and egg development

Adipokinetic hormones (AKHs), the neurohormones synthesized in the insect corpora cardiaca are known to mobilize lipids and carbohydrates for energy‐consuming activities including reproduction. However, both inhibitory and stimulatory effects of AKHs on insect reproduction have been reported, and the underlying mechanisms remain elusive. Using the migratory locust, Locusta migratoria, as a model system, we report here that AKHs are expressed in response to rhythmic diel change, and AKH III expression increases markedly at photophase. Diurnal injection of AKH III but not AKH I or AKH II in adult females stimulates vitellogenesis and egg development. In contrast, AKH treatment at scotophase represses female reproduction. RNA interference‐mediated knockdown of AKH receptor (AKHR) results in significantly reduced vitellogenin (Vg) expression in the fat body at photophase along with reduced Vg deposition in the ovary. AKHR knockdown also leads to decreased expression of Brummer, triacylglycerol lipase and trehalose transporter, accompanied by suppressed mobilization of triacylglycerol and trehalose. We propose that in addition to stimulating Vg expression at photophase, AKH/AKHR signalling is likely to regulate ovarian uptake of Vg via triacylglycerol mobilization and trehalose homeostasis. This study provides new insights into the understanding of AKH/AKHR signalling in the regulation of insect reproduction.