新型Kdo2-lipid A合成菌株构建及其免疫生化活性研究

脂多糖(LPS)是革兰氏阴性细菌外膜的主要组成部分,由疏水的Kdo2-lipid A和亲水的多糖长链组成,能激活先天性免疫系统。Kdo2-lipid A是LPS的生物活性中心,能被开发为新型疫苗佐剂。本研究在前期菌株基础上敲除与LPS庚糖合成相关waaC-waaF基因,构建了直接合成特殊结构Kdo2-lipid A、不含多糖长链的突变株BW003。通过薄层层析和电喷雾电离质谱技术鉴定了BW003合成的Kdo2-lipid A结构;HEK-Blue h TLR4细胞和THP-1细胞刺激实验证实突变株及其合成的Kdo2-lipid A的细胞毒性均明显减弱;生化表型分析结果表明BW003较野生型呈现出更高的外膜渗透性、抗生素敏感性、疏水性及自凝集能力。本研究为后续新型疫苗佐剂开发和产业化利用奠定了理论基础。     

加压素细菌内毒素分析方法学

目的：建立加压素细菌内毒素检测方法。方法：采用灵敏度为0.25EU/ml的鲎试剂,以凝胶法定性测定xxx的细菌内毒素水平,通过干扰初筛试验和干扰试验对其检测方法进行验证;结论：凝胶法适用于xxx的内毒素定性检测。     

注射用脑蛋白水解物细菌内毒素检查法的建立

目的建立检查注射用脑蛋白水解物细菌内毒素的方法。方法按照中国药典2010年版（二部）附录XIE细菌内毒素检查法,对3批样品进行干扰试验和细菌内毒素检查。结果注射用脑蛋白水解物的游离氨基酸质量浓度0.5 mg/mL时,供试品不干扰细菌内毒素检查。结论建立的注射用脑蛋白水解物细菌内毒素检查法可行,细菌内毒素限值确定为0.5 EU/mg。     

马来酸桂哌齐特注射液细菌内毒素检查方法学研究

目的建立马来酸桂哌齐特注射液的细菌内毒素检查方法。方法按照《中国药典》2010年版(二部)附录细菌内毒素检查法,用2个不同生产厂家的鲎试剂(TAL)对3批马来酸桂哌齐特注射液进行干扰试验和细菌内毒素检查。结果样品的细菌内毒素限值确定为1.0 EU/mg,供试品溶液低于或等于0.25 mg/mL时,对TAL与细菌内毒素之间的凝集反应无干扰抑制作用。结论建立的细菌内毒素检查方法可行,可用于马来酸桂哌齐特注射液的检查。     

动态显色法鲎试验用于果汁细菌内毒素活性检测及干扰分析

目的建立一种定量检测果汁细菌内毒素活性的方法,准确评估果汁潜在的内毒素污染风险。方法采用动态显色法东方鲎(Tachypleus amebocyte lysate, TAL)试验对常见的4种果汁(橙汁、桃汁、苹果汁和葡萄汁)细菌内毒素活性进行检测,对果汁样品中的干扰因素进行识别与排除,最后使用抗增液排除β-葡聚糖等类似物质带来的假阳性结果。结果市售果汁(n=36)的细菌内毒素活性范围为0.2～209.5 EU/mL,自榨果汁(n=12)的细菌内毒素活性范围为0.4～4.2EU/mL,加标回收率范围均在50%～200%之间,符合药典要求。结论动态显色法TAL试验适用于常见的4种果汁(橙汁、桃汁、苹果汁和葡萄汁)细菌内毒素活性的检测,为果汁的健康风险评价和卫生监管提供参考。     

硫酸软骨素注射液细菌内毒素检查方法研究

目的建立检查硫酸软骨素注射液细菌内毒素的方法。方法用中国药典2005年版附录细菌内毒素检查法。结果硫酸软骨素注射液浓度0.025mg/mL,用灵敏度0.125EU/mL的鲎试剂检测细菌内毒素无干扰因素的影响。结论硫酸软骨素注射液可用鲎试剂检查细菌内毒素。     

替硝唑注射液细菌内毒素检查方法的研究

目的建立替硝唑注射液的细菌内毒素检查法。方法按中国药典2000年版附录细菌内毒素检查法要求进行实验。结果替硝唑注射液1→2稀释后,不干扰鲎试剂与细菌内毒素的凝胶反应。结论可用细菌内毒素检查法替代热原检查法对替硝唑注射液进行热原检查。     

注射用盐酸甲砜霉素甘氨酸酯细菌内毒素检查法研究

目的建立注射用盐酸甲砜霉素甘氨酸酯细菌内毒素检查法。方法参照《中国药典》2010年版二部细菌内毒素检查法进行方法学研究。结果注射用盐酸甲砜霉素甘氨酸酯稀释至1.66 mg/mL后对细菌内毒素检查无干扰。结论细菌内毒素检查法替代热原检查法可行。     

硫酸软骨素注射液细菌内毒素检查方法研究

目的 建立检查硫酸软骨素注射液细菌内毒素的方法.方法 用中国药典2005年版附录细菌内毒素检查法.结果 硫酸软骨素注射液浓度0.025 mg/mL,用灵敏度0.125 EU/mL的鲎试剂检测细菌内毒素无干扰因素的影响.结论 硫酸软骨素注射液可用鲎试剂检查细菌内毒素.     

硫酸软骨素A钠注射液细菌内毒素检查方法研究

目的建立检查硫酸软骨素A钠注射液细菌内毒素的方法。方法采用中国药典2005年版附录细菌内毒素检查法。结果硫酸软骨素A钠注射液浓度为0.15 mg.mL-1,用灵敏度为0.25 EU.mL-1的鲎试剂检测细菌内毒素,无干扰因素的影响。结论硫酸软骨素A钠注射液可用鲎试剂检查细菌内毒素。     

Molecular control of luteal secretion of progesterone

Cholesterol provided by low- or high-density lipoprotein is the precursor for biosynthesis of progesterone. Once inside the cell, cholesterol can be used for steroidogenesis or esterified with long-chain fatty acids and stored as cholesterol esters in lipid droplets. When it is needed for steroidogenesis, free cholesterol is transported to the mitochondrion via a mechanism that involves cytoskeletal elements and sterol carrier proteins. Cytochrome P450 cholesterol side chain cleavage enzyme complex converts the cholesterol to pregnenolone, which is then converted to progesterone by 3beta-hydroxysteroid dehydrogenase/delta5,delta4 isomerase in the smooth endoplasmic reticulum. Transport of cholesterol from the cytoplasm to the inner mitochondrial membrane is both the rate-limiting step in progesterone biosynthesis and the step most acutely influenced by second messengers. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptors (PBR) are involved in this transport. StAR may bind cholesterol in the cytosol and transport it to the mitochondrial membrane where PBR is involved in transport from the outer to the inner mitochondrial membrane. Phosphorylation of StAR by protein kinase A (PKA) stimulates cholesterol transport, whereas phosphorylation by PKC may inhibit this process. Endozepine, the natural ligand for PBR, also appears to be involved in regulation of the rate of cholesterol transport to the inner mitochondrial membrane and to play a role in the stimulatory effects of PKA on steroidogenesis. Increased concentrations of endozepine were detected in large luteal cells, and may explain the increased progesterone secretion from this type of cell. Fluorescence energy transfer procedures indicate that StAR associates with PBR in mitochondrial membranes. A model is presented for the proposed interactions of StAR, PBR and endozepine in the transport of cholesterol from the outer to the inner mitochondrial membrane.     

The effect of endotoxin on functional parameters of mammary CID-9 cells

The effect of endotoxin on mammary CID-9 cells, which differentiate in culture and express beta-casein, was investigated. Cells in culture supplemented with lactogenic hormones and dripped with EMS-Matrix (EMS-drip), were treated daily with endotoxin (0.5-500 microg/ml). Endotoxin at concentrations of less or equal to 10 microg/ml did not affect cell growth and viability up to 5 days post endotoxin treatment. Endotoxin (0.01-10 microg/ml) was added to the culture medium, upon confluence, and functional parameters were examined within 48 h post endotoxin treatment. Nuclear factor-kappaB (NF-kappaB) (p52) increased in nuclear extracts from endotoxin-stimulated cells within 1 h of treatment, while beta-casein mRNA and protein expression decreased in a concentration-dependent manner at 24 and 48 h post treatment. Zymography showed that the 72 and 92 kDa gelatinase activity increased in cells at 24 and 48 h post endotoxin treatment at 10 and 50 microg/ml. At the latter concentration, the active form of 72 kDa gelatinase was induced at 48 h. Interleukin-6 and tumor necrosis factor-alpha levels increased at 1-3 h post endotoxin treatment and peaked at 6 h in cells on plastic and EHS-drip. Nerve growth factor (NGF) levels increased in control and endotoxin-treated cells in a time-dependent manner, and endotoxin increased NGF levels in culture at 6 and 9 h post endotoxin treatment. This study shows that endotoxin activated NF-kappaB, suppressed beta-casein expression and upregulated gelatinases, cytokines and NGF. This model could be used to investigate the role of mammary cells in initiating and propagating inflammation and to test candidate molecules for potential anti-inflammatory properties.     

Incorporation of amino acids by isolated submitochondrial fractions of lactating goat mammary gland in vitro.

Incorporation of amino acids by mitochondria and its subfractions from mammary gland of the goat during lactation was studied in vitro. Assessment of distribution of radioactivity incorporated by intact mitochondria into its subfractions revealed maximum specific activity in the inner membrane. During lactation the inner membrane of mitochondria exhibited further stimulation in such incorporation. Matrix was the next active fraction whereas outer membrane and peripheral space had negligible radioactivity. In isolated submitochondrial fractions from mammary gland incubated with radioactive amino acids under similar conditions of assay, the inner membrane was the most potent subfraction in incorporation. In mammary tissue during lactation this fraction was the most active site. The relative sequence of the subfractions appeared to remain unaltered in lactation and was in the order of inner membrane greater than matrix greater than outer membrane greater than peripheral space. The ratio of specific activity in inner to outer membrane appears to increase significantly during lactation.     

Localization of phosphatidylserine in boar sperm cell membranes during capacitation and acrosome reaction

One of the essential properties of mammalian, including sperm, plasma membranes is a stable transversal lipid asymmetry with the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), typically in the inner, cytoplasmic leaflet. The maintenance of this nonrandom lipid distribution is important for the homeostasis of the cell. To clarify the relevance of lipid asymmetry to sperm function, we have studied the localization of PS in boar sperm cell membranes. By using labeled annexin V as a marker for PS and propidium iodide (PI) as a stain for nonviable cells in conjunction with different methods (flow cytometry, fluorescence and electron microscopy), we have assessed the surface exposure of PS in viable cells during sperm genesis, that is, before and during capacitation as well as after acrosome reaction. An approach was set up to address also the presence of PS in the outer acrosome membrane. The results show that PS is localized in the cytoplasmic leaflet of the plasma membrane as well as on the outer acrosome membrane. Our results further indicate the cytoplasmic localization of PS in the postacrosomal region. During capacitation and acrosome reaction of spermatozoa, PS does not become exposed on the outer surface of the viable cells. Only in a subpopulation of PI-positive sperm cells does PS became accessible upon capacitation. The stable cytoplasmic localization of PS in the plasma membrane, as well as in the outer acrosome membrane, is assumed to be essential for a proper genesis of sperm cells during capacitation and acrosome reaction.     

快速分析食品包装复合膜材质结构的方法研究

目的研究快速分析食品包装复合膜材质结构的方法。方法将样品用夹具固定,切片机切片处理后获得材料横截面,并标记样品内外层,利用扫描电子显微镜仪(scanning electron microscope, SEM)对样品层数和由外到内厚度进行表征和测量。利用衰减全反射(attenuated total reflection, ATR)对材料的内外表层进行材质分析,切下的材料薄层采用傅里叶显微红外光谱仪(micro-Fourier transform infrared spectroscopy,Micro-FTIR)对各层树脂进行材质鉴定,结合ATR测试内外表层结果确定复合膜由外到内材质组成。结果该包装复合膜由7层结构组成,由外到内材质依次为聚对苯二甲酸乙二醇酯(polyethylene terephthalate,PET)/聚酰胺(polyamide,PA)/聚乙烯(polyethylen,PE)/PA/乙烯-乙烯醇共聚物(ethylene-vinyl alcohol copolymer,EVOH)/PA/PE,厚度依次为14, 18, 54, 17, 11, 18, 81μm。结论通过切片机切片获取样品横截面和薄层样品,SEM、Micro-FTIR联合进行分析,可以确认食品包装复合膜的整体组成结构及每层材质信息。该方法无需树脂包埋和有毒有机溶剂浸泡剥离,方法前处理简单,可以快速、准确地完成多层未知复合膜材料的结构分析。     

3种鲎试验在乳品内毒素活性定量检测中的应用评价

目的 比较3种内毒素活性定量检测方法在乳品中的应用情况。方法 采用2种传统东方鲎(Tachypleus amebocyte lysate, TAL)试验(动态浊度法/动态显色法)和1种便携式内毒素检测仪(portable test system, PTS)对牛奶、酸奶和奶粉产品内毒素活性进行检测, 评估3种方法在乳品内毒素活性检测中的应用情况。结果 3种方法用于乳品内毒素活性检测的加标回收率为50%~200%, 符合药典要求。对比动态浊度法, 动态显色法检测乳品内毒素活性的加标回收率范围更加接近100%。PTS方法的内毒素活性检测值分别与2种传统TAL试验的检测值有较好的线性关系。结论 3种方法均适用于乳品内毒素活性的检测, 但动态显色法更加适合乳品内毒素的检测。     

The biosynthesis of cellulose

To date, two basic cellulose synthesizing structures (TCs) have been described, the linear TC, typical of giant algae such as Boergesenia, Valonia and Eremosphaera and the rosette TC, characteristic of all vascular plants, ferns, mosses and certain algae such as Chara, Nitella and Moeugeotia. Linear terminal complexes are transported to the surface as subunits and later assembled into the linear structure. These linear structures continue to grow in length during microfibril assembly. The relationship of the TC structure to microfibril dimensions is discussed. Rosette TCs appear to be pre-assembled in the Golgi apparatus and transported to the surface intact. The topographical localization of TC structure in relation to patterns of microfibril deposition is described. The structure of the TC is discussed in relation to the synthesis of cellulose I and II. The biosynthesis of microbial cellulose by Acetobacter xylinum is summarized. Recent evidence for in vivo cellulose synthesis is given. The cellulose synthesizing complexes are located on the inner cytoplasmic membrane of Acetobacter and the regulatory proteins for controlling microfibril assembly may be located on the outer lipopolysaccharide membrane. Recent studies of in vitro synthesis indicated that cellulose II is made in vitro, whereas cellulose I is the more common allomorph produced in vivo.     

Endotoxin of Escherichia coli and permeability of the mammary gland of goats

Serial collections of milk were used to determine where in the mammary gland endotoxin of Escherichia coli was effective in altering the transfer of selected milk components into blood and blood components into milk. Lactating goats had half the gland infused with 1 microgram of endotoxin and the other half served as a control. Sodium-24 and 42K or [14C] lactose were included with 141Ce in the infusate in some experiments, whereas in others 99mTc-labelled albumin or 24Na and 42K were given intravenously 2 h after the endotoxin infusion. Milk was collected 3 h after endotoxin infusion. Endotoxin increased the loss of 24Na, 42K, and [14C] lactose from the mammary gland and increased the transfer of 24Na and 99mTc-albumin into the gland. The transfer in of 42K was reduced compared with control halves. Movement of stable Na and K was in accord with the movement of the 24Na and 42K. Endotoxin was effective in all parts of the gland but particularly from the mid-portion upward to the alveoli. For the control halves there was evidence that some 24Na and 42K crossed the ductal or cisternal epithelium into blood outside of the alveoli, whereas only 42K provided evidence for transfer from blood to milk in these same regions. There was no demonstrable transfer of lactose and albumin in regions other than the alveoli.     

Flavonoid concentrations in the inner leaves of head lettuce genotypes

Typical HPLC profiles of phenolics from head lettuce (butter lettuce) genotypes showed quercetin (Qu) compounds Qu 3-O-glucuronide, Qu 3-O-(6-malonylglucoside), an acidic luteolin (Lu) glycoside, chlorogenic acid and chicoric acid as the main compounds in outer and - except the Lu conjugate - inner leaves.The outer leaves of some genotypes additionally contained cyanidin 3-O-(6-malonylglucoside). The generally marginal flavonoid concentrations of the cores of lettuce heads increased between 10-fold and 20-fold during the illumination of opened inner leaves. This indicated the competence of inner leaves of lettuce heads to perform the light-dependent flavonoid biosynthesis. After hydrolysis of flavonoid extracts from 15 head lettuce varieties, Qu concentrations were detected between 19.4 and 152.1 µg/g dry plant material (DW) in inner leaves and from 417.3 to 2482.7 µg/g DW in outer leaves. While the Qu concentrations of the inner leaves of most genotypes, including the commercial varieties, amounted to about 3% of those found in outer leaves, these proportions were 15% and 19% in two genotypes. Two other varieties showed relatively high Qu values of cores in connection with maximum flavonoid levels of outer leaves. It is indicated that such genotypes may be useful in increasing the flavonoid concentrations in the inner leaves of commercial head lettuce varieties by conventional breeding methods.