Freeze-drying of human platelets: influence of intracellular trehalose and extracellular protectants

Freeze-drying is an ideal alternative for long-term preservation of platelets in blood banks. Intracellular trehalose is believed to be an effective lyoprotectant for preserving cells during freeze-drying. In this study, 13 mM intracellular trehalose was loaded into human platelets through fluid-phase endocytosis pathway. Bovine serum albumin and trehalose were used as extracellular protectants. The effects of intracellular trehalose and extracellular protectants on freeze-dried platelets were studied respectively. The results showed 13 mM intracellular trehalose was beneficial to freeze-dried human platelets, but only slightly enhanced the protection afforded by extracellular protectants. Loaded with 13 mM intracellular trehalose, platelets were freeze-dried in a formulation of 1 percent bovine serum albumin and 1 percent trehalose, 40 days later, the survival rate of rehydrated platelets was about 85 percent, the morphology of rehydrated platelets was intact and the aggregation percentage with thrombin (1 U/ml) was 97.3 percent.     

Evolution of DMSO concentration in garlic shoot tips during a vitrification procedure

In this paper, the evolution of dimethylsulfoxide (DMSO) concentration and moisture content (MC) of garlic shoot tips was studied during the course of a vitrification protocol using the PVS2 vitrification solution. DMSO concentration of shoot tips increased rapidly, reaching 34.1 mg per g fresh weight after 20 min of PVS2 treatment and remained stable afterwards, while moisture content decreased from 82 to 60 percent, reaching 53 percent after 60 min. A reverse process was observed during unloading. There was a highly significant negative correlation between shoot tip moisture content and DMSO concentration during the dehydration and unloading treatments. Using unloading solutions with osmolarities between 0.42 and 2.29 Osm led to very different shoot tip MCs, between 63.55 and 81.24 percent, while DMSO concentration was between 14.83 and 19.97 mg per g fresh weight. After 24 h on recovery medium, DMSO concentration of shoot tips had decreased to 3.2 mg per g fresh weight.     

Optical measurement of the viability of stored human platelets

An experimental study has been carried out to test the feasibility of optically assessing the viability of stored blood platelets. A red (633 nm) laser beam was passed through the platelet pack, and the scatter measured. Live platelets scattered the beam differently from dead ones, owing to their different shape.The results compared favourably with those from established in vitro tests of platelet viability.     

化学发光分析法测定绿原酸

本文基于碱性介质中绿原酸对鲁米诺-铁氰化钾化学发光体系的抑制作用,首次建立了绿原酸的化学发光分析法,并探讨了其作用机理。方便简便、迅速、重复性好、灵敏度高。采用该法可测定1.0×10-8g/ml～1.0×10-5g/ml范围内的绿原酸,其检出限达到5.2×10-9g/ml,回收率为104%.     

Applications of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds

Shoot-tips of Parkia speciosa, a recalcitrant seed producing tropical leguminous tree withstood cryopreservation using encapsulation-vitrification in combination with trehalose preculture. Differential scanning calorimetry (DSC) revealed that trehalose moderated the thermal characteristics of the shoot-tips. A 30 min PVS2 treatment had the lowest glass transition temperature (Tg) (-50.2 +/- 1.1 degree C) when applied in combination with 5% (w/v) trehalose. The Tg increased to -40.2 +/- 1.0 degree C as the sugar concentration was decreased to 2.5 percent (w/v). Tg heat capacity for shoot-tips treated with 2.5 percent and 5 percent (w/v) trehalose and exposed to PVS2 for 30 min increased from 0.17 +/ 0.05 to 0.23 +/- 0.01 J per gram, respectively. Enthalpies of the melt-endotherm varied in proportion to trehalose concentration, for the 30 min PVS2 treatment, whereas the melt enthalpy for control shoots was greater than 150 J per gram and decreased to ca. 60 J per gram with 2.5 percent (w/v) trehalose. For 5 percent and 10 percent (w/v) trehalose treatments, enthalpy declined to ca. 24 and 12 J per gram respectively and freezing points were depressed to -75 degree C and -85 degree C with 2.5 percent and 5 percent trehalose (w/v), respectively. DSC elucidated the critical points at which vitrification occurred in germplasm exposed to trehalose and PVS2. A 60 min PVS2 treatment supporting ca. 70 percent survival was found optimal for stable glass formation during cooling and on rewarming.     

用紫外分光光度测定水中硝苯柳胺的含量

硝苯柳胺是自己合成的酰胺类新杀螺剂、杀蚴剂 ,化学名称为N (4 硝基苯基 ) 5 氯水杨酰胺。本文介绍了一种用紫外分光光度法测定硝苯柳胺的含量的方法。本方法中 ,选择 372± 1nm作定量峰 ,在pH≥9 5的条件下 ,硝苯柳胺的水溶液浓度在 0 5～ 40mg·L-1范围内 ,吸收度与浓度的线性关系良好 ,相关系数0 9999。水溶液在室温条件下放置 48h仍稳定 ,平均回收率 99 8%。     

NOR expression increases in interphase lymphocytes of Down syndrome babies/children as AgNORs surface, according to the mitogen concentration in the culture medium

The extra chromosome 21 of Down syndrome (DS) or trisomy 21 patients contains an average of 40 extra copies of rRNA genes and the in vivo/in vitro regulation of the activity of these genes is not fully understood. The objective of this work was to compare the NORs expression pattern in interphase lymphocytes of DS patients with regular trisomy 21 and control individuals according to phytohemagglutinin (PHA) concentration (0.37, 0.75, 1.48 and 2.21 ml) per 100 ml of medium. Because the AgNOR staining is an indicator of the active rRNA genes, comparison of the image analysis values of the AgNOR area in 72 h cultivated lymphocytes for each concentration of PHA between DS patients (N=30) and controls (N=24) provided a plausible conclusion on the regulation of the extra rRNA genes in DS lymphocytes. The nucleolus organizer regions area/total nuclear area (NORa/TNa) was calculated using an in-house computer program. Fifty consecutive interphases per PHA concentration were analysed for each individual, for determination of the NORa/TNa. In contrast to healthy controls, NORa/TNa of lymphocytes from DS patient babies/children (0-8 years old) increased gradually in parallel with the PHA concentration in the culture medium: 10.44+/-1.72% for 0.37 ml of PHA, 11.74+/-1.93% for 0.75 ml of PHA, 13.25+/-2.03% for 1.48 ml of PHA and 13.43+/-2.08% for 2.21 ml of PHA per 100 ml of medium. Contrary to control cells (in which the NORa/TNa ratio according to PHA concentration in the culture medium remains constant), DS interphase lymphocytes in culture do not down-regulate their NOR expression. These results obtained from interphase NORs are consistent with the previous results obtained by evaluating the mean of AgNOR+ chromosome number in metaphase cells, also in relation to the mitogen concentration in the culture medium.     

猪主动脉血管冷冻干燥及烘干处理的微CT实验研究

以猪主动脉血管为研究对象,分别进行真空冷冻干燥及烘干两种干燥处理的方法,用称量法测得冻干及烘干血管的失水率分别为64.7%,71.3%。同时对两种干燥血管及复水后的血管进行微CT扫描和分析,得到微观孔隙率、灰度等数值。得出:冻干血管外观乳白色,且结构疏松多孔;烘干血管呈较深的黄色,结构致密透明。从复水状况来看,冻干血管复水容易,外观、孔隙率、灰度值等方面较烘干血管来说,更加接近新鲜血管。     

染料荧光增强苯的受激拉曼散射

本文报道若丹明6G荧光增强苯的受激拉曼散射一阶Stokes谱线的实验研究,在若丹明6G苯溶液浓度为～10-3mg/ml时,获得了有效的增强,浓度为13×10-3mg/ml时最大增强因子达到32。对增强机制进行了物理解释。     

液体火箭推力室面板发汗冷却与燃烧耦合数值模拟

对推力室的喷嘴多孔面板的发汗冷却和燃烧室内的燃料燃烧过程进行了耦合数值计算,建立了一个带燃烧的三维、真实气体、变物性的推力室CFD计算模型。利用UDF编写了CH₄、O₂、CO₂、H₂O气体的实际气体状态方程,并根据NIST物性数据拟合了不同温度和压力下各气体的比热容、扩散系数、黏性系数和导热系数等物性多项式。基于EDC模型建立了甲烷-氧燃烧的多步反应机理。计算了三种厚度的面板和多种燃料进口工况下的推力室内的发汗冷却和燃烧过程,研究了面板厚度、冷却剂进出条件等因素对发汗冷却和燃烧过程的影响规律。     

火焰原子吸收法测定蜂蜜中的铁

本文报道了用火焰原子吸收光谱法测定蜂蜜中的铁含量,经与其分分析测定方法比较,该方法具有样品预处理十分简便、无需干法或湿法消解的特点,测定结果令人满意。该方法样品加标回收率为９３．０％－１０９％,相对标准偏差为０．８０８％－２．６２％,检出限为０．０８ｍｇ／Ｌ。     

食品工业清洁生产测评技术与方法研究--废水中氨氮含量的测定

对肉类加工废水中的氨氮(NH3-N)含量的测评技术和方法进行了研究.采用纳氏试剂分光光度法测定了不同废水中的NH3-N含量.结果表明,屠宰和熟食加工产生的废水、熟食加工产生废水和混合废水经处理后的水样中氨氮含量分别为255mg/L、8.24mg/L、和13.4mg/L,平均回收率为93.0%,平均相对标准偏差为2.19%.所得数据可靠、准确,其方法快速、可行.探讨的测评废水中氨氮的方法,将为企业实施内部监测提供依据,为我国制定清洁生产测评标准提供参考.     

Palm cryobanking

We describe the development of an efficient cryopreservation protocol for proembryogenic masses (PEMs) of date palm variety 'Barhee'. Proembryos were induced by inoculating small pieces of juvenile leaves on MS medium supplemented with 0.3 mg per liter 2,4-D. Application of these in vitro conditions led to true-to-type plants as observed after plant fructification. When compared to the standard vitrification protocol, the ultra-rapid droplet-vitrification technique proved to be superior. Sucrose preculture considerably increased post-cryopreservation recovery. The highest regeneration after cryogenic exposure reached 63.3 percent when PEMs were treated with PVS2 for 30 min at 0 degree C and 56.7 percent when PVS2 treatment lasted for 15 min at 25 degree C. The first signs of regrowth of cryopreserved PEMs were observed after 2 to 3 weeks. Cryopreservation did not affect the morphogenetic capacities of the plant material. Moreover, highly proliferating suspension cultures could be established from the cryopreserved material. The overall production of somatic embryos from 500 mg cryopreserved PEMs reached 1030 +/- 50 units after 1 month. The morphological study of date palms regenerated from cryopreserved material confirmed the stability of clonal material following cryopreservation.     

钐的时间分辨激光荧光光谱分析方法研究

本文利用Ｓｍ－ＴＴＡ－ＥｔＯＨ液体荧光体系，采用时间分辨激光荧光检测手段，建立了散的时间分辨光荧光分析法，该法的线性范围为０．０１～１０μｇ／ｍｌ，检出限为３．３ｎｇ／ｍｌ，０．５μｇ／ｍｌ钐标样测定的相对标准差为３．７％（ｎ＝１２），结合混合稀土中多种元素共存的特点，实验确定了装置的有关参数，用标准加入方法对实际样品进行了测定，并与其他方法测定结果进行了比较，结果满意，本法测定实际样品中钐的相对     

Recovery of cryopreserved embryogenic cultures of maritime pine--effect of cryoprotectant and suspension density

Embryogenic cultures were obtained from seeds of open-pollinated maritime pine trees representing part of a breeding population. The aim of the present study was to develop and optimize a protocol for cryopreservation of Pinus pinaster somatic embryogenic tissue. The density of the embryogenic suspension and the type of pre-treatment significantly affected the recovery of cryopreserved embryogenic cultures, as evaluated by their survival and regrowth rate. An initial density of the suspension culture of 250 mg/ml improved the regrowth rate of the embryogenic lines. Pre-treatment with maltose 0.4 M significantly increased the regrowth rate when compared to the other tested carbohydrates. Also, the addition of DMSO in a mixture of PEG 4000 and sucrose (PSD solution), instead of DMSO alone, at the same final concentration, was clearly beneficial for recovery of cryopreserved tissues. This improved method for the cryopreservation of P pinaster embryogenic of cultures allowed the successful recovery of 97 percent of the lines stored in liquid of nitrogen.     

酶荧光毛细分析法测定血清中微量丙酮酸

提出了一种用于血清微量丙酮酸（PA）测定的酶荧光毛细分析法（P-LE-FCA）。优选的测定条件为：乳酸脱氢酶和还原型辅酶Ⅰ的浓度分别为100U/L和60μmol/L,反应时间5min。测定PA的线性范围4—80μmol/L,检出限0.73μmol/L,RSD〈1.3%。血清共存物质对PA测定无明显干扰。使用该方法对血清PA进行测定,其回收率在93.0%—106.5%之间;结果与常规紫外分光光度法一致。本方法的样品预处理过程简单、省时,省试剂,灵敏度高,重现性好。     

Morphology study of freeze-drying mononuclear cells of human cord blood

The research on haematopoietic stem cells of human cord blood has become more important recently. At present, cord blood is mainly preserved at ultra-low temperatures. In the former study, we compared the effects of preserving mononuclear cells (MNC) and whole human cord blood by freeze-drying. This time, a further study was conducted on freeze-drying mononuclear cells. Samples in the presence of PVP, sucrose, mannitol and FBS were firstly frozen to -38 degrees C. Afterwards, they were vacuum-dried at a selected shelf temperature of -30 degrees C for the main drying stage, and then vacuum-dried at 15 degrees C for the second drying stage. The entire time of freeze-drying process was 41 hours. Samples were stored at room temperature for 7 days prior to evaluation. Subsequently, the dried samples were resuspended in an isotonic phosphate-buffered saline solution. The residual moisture content was 6.5 +/- 0.87%. The recovery of the cells was tested by a haemacytometer, and the numerical cell count recovery of rehydrated MNC increased by 8%. Morphology of the fresh and rehydrated MNC was analyzed respectively using standard light microscopy, scanning electron microscope and transmission electron microscope. The results showed that karyons changed and cytoplasm decreased after rehydration, but it is still unknown that whether these changes will influence the proliferative ability of the stem cells.     

The effect of saccharides on the post-thaw recovery of cane toad (Bufo marinus) spermatozoa

The effect of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose, trehalose) as diluents, in cryoprotective additives containing 15% (v/v) DMSO or glycerol as cryoprotectants, were investigated on the recovery of sperm motility after cryopreservation of cane toad (Bufo marinus) spermatoazoa at low (approximately 5 degrees C/min(-1)) and high cooling rates (approximately 35 degrees C/min(-1)). The results show that: 1. recovery of percentage motility was higher with slow cooling than with high cooling rates (37.0 +/- 2.5%, 15.3 +/- 1.6%, P<0.001, respectively), 2. disaccharides were more effective than monosaccharides in protecting spermatozoa with slow cooling (43.9 +/- 1.2%, 26.8 +/- 2.5%, P<0.02, respectively), 3. glycerol was more effective than DMSO with fast cooling (18.3 +/- 2.2%, 12.6 +/- 2.3%, P<0.02, respectively), 4. trehalose with glycerol was the most effective cryoprotective additive with fast cooling (31.0 +/- 3.2%, P<0.05), and 5. overall the recovery of degree (vigour) of motility (range, 1.9 - 3.2) was more resilient to cryopreservation than recovery of percentage motility (range, 8.9 - 51.5 %). Comparison of post-thaw percentage and vigour of sperm motility up to 24 minutes after activation showed disaccharides supported greater duration sperm motility than monosaccharides This result and the recovery of spermatozoa immediately after freeze-thaw, show the main effect of saccharides are as cryoprotectants and not as exogenous energy substrates.     

Simplified technique of human ovarian tissue freezing: quick cooling from -36 degree C

Standard protocol of freezing of human ovarian tissue presupposes the very slow cooling (-0.3 C/min) from auto-seeding to -40 C, then slow cooling (-10 C/min) to -140 C and then direct plunging into liquid nitrogen. The aim of this investigation was to compare the -10 C/min cooling rate of human ovarian tissue from -40 C to -140 C with the -220 C/min cooling rate (direct plunging into liquid nitrogen) from -36 degree C. After post-thawing in vitro culture of tissue, hormonal activity as well as follicle viability was evaluated. After culture of fresh tissue pieces (Group 1), pieces after freezing and thawing with slow cooling (-10 C/min) from -40 C (Group 2) and pieces after freezing and thawing with direct plunging into liquid nitrogen (-220 C/min) from -36 C (Group 3), the supernatants showed estradiol 17-ss concentrations of 481, 441 and 459 pg per ml, respectively, and progesterone concentrations of 9.05, 5.06, 4.87 ng per ml, respectively. It is concluded that 94, 96, and 98 percent follicles for Groups 1, 2 and 3, respectively, were normal. Technique of human ovarian tissue cryopreservation with very slow cooling to -36 C and then direct plunging into liquid nitrogen with -220 C/min cooling rate is tolerated without apparent detriment.     

Effect of alcohol consumption on the liver detoxication capacity as measured by [13C]methacetin- and [methyl-13C]methionine-breath tests

The aim of this study was to investigate the hepatic microsomal and mitochondrial functions by using the 13CO2-breath test in healthy subjects either before or after the consumption of red wine. Fourteen adults received [13C]methacetin and [methyl-13C]methionine together with a standardised dinner. Expired air samples were taken over 6 h. After a wash-out period, the subjects consumed 0.4 ml ethanol/kg/day together with dinner over a 10-day period. Thereafter, 13C-tracer administration was repeated under identical conditions. The 13CO2-enrichments were measured by isotope ratio mass spectrometry. The mean cumulative percentage 13C-dose recovery (CPDR) after administration of [13C]methacetin and [methyl-13C]methionine either without or with red wine consumption amounted to 38.2+/-6.3 vs. 36.3+/-6.7% (p=0.363) and 9.5+/-3.3 vs. 8.8+/-2.5% (p=0.47), respectively. Moderate alcohol consumption does not induce significant short-term changes of the microsomal and the mitochondrial functions of the human liver in healthy subjects.