脂多糖结合蛋白的分离、纯化及其多抗的制备

目的 分离纯化大鼠脂多糖结合蛋白（LBP），并制备兔抗大鼠LBP多抗。方法 大鼠血清先通过硫酸铵盐析，再依次以Bio-Rex70阳离子交换层析和MonoQ阴离子交换层析进行纯化，纯化产物采用SDS－PAGE鉴定。用提纯的LBP免疫制备兔抗大鼠LBP的抗血清，用饱和硫酸铵盐析纯化后，经Westernblot鉴定其纯度。结果 分离纯化到较高纯度的LBP。用纯化到LBP免疫制备的兔抗LBP多抗与LBP具有良好的结合活性。结论 分离纯化到高纯度的LBP并制备出兔抗大鼠LBP多抗。     

人红细胞膜衰变加速因子的分离纯化及其生物学活性鉴定

目的　获得纯化并具有生物学活性的人红细胞膜衰变加速因子。方法　经胰蛋白酶消化、正丁醇抽提及DE32和Sepharose 6B色谱分离等步骤从人红细胞膜影中分离纯化人红细胞膜衰变加速因子 (DAF)。以SDS PAGE及免疫印迹实验检测纯度及抗原特异性 ,以C3转化酶体外组装实验及促衰变活性实验检测其补体抑制活性。结果　 4 0 0mL人全血最终可得纯化DAF约 370 μg ,回收率达 10 .4 % ;比活性为每毫克 2 .2 4× 10 5units;纯化产物在SDS PAGE表现为 70 0 0 0u的单一蛋白条带 ,在免疫印迹实验中可与抗人DAF单抗特异性结合。在生物学活性实验中 ,纯化产物既可促进C3转化酶衰变 ,也可抑制C3转化酶形成。结论　采用本方法可获得纯化的具有生物学活性的人衰变加速因子     

幽门螺杆菌脂多糖生物学活性的研究

应用酚水法提取了幽门螺杆菌９０２９０５株的脂多糖（ＬＰＳ）。该ＬＰＳ在０．５ｎｇ／ｍｌ浓度时即能凝固鲎血液变形细胞溶解物，注入家兔后可使动物出现Ｓｈｗａｒｔｚｍａｎ现象和发热反应，其热曲线呈双峰型。该ＬＰＳ注入家兔后０．５小时动物血压明显下降，至注后１小时血压降为零，外周血中白细胞数则先升后降。小鼠体内注入该ＬＰＳ后，可导致动物死亡，其内脏出现严重的毛细血管扩张并伴有少量出血。与Ｅ．ｃｏｌｉＬＰＳ对比实验结果证实幽门螺杆菌ＬＰＳ具有典型的内毒素活性。     

附子多糖FI的分离纯化及生物学活性研究

附子是毛莨科乌头属植物的侧根,是一种常用的中药材,具有回阳救逆、温补肾阳、祛寒止痛之功能。近年来研究表明,附子提取液尚具有强心、抗癌、抗衰老和增强免疫机能的作用。因此,对附子中所含多糖的研究与利用具有重要的医学价值。我们将白附片经热水抽提、Sevag法脱蛋白、乙醇沉淀、DEAE-C32柱层析分离,     

人重组TNF-α突变体471蛋白的初步纯化及生物学活性

目的：在利用原核表达系统表达重组人突变体471 TNF—α蛋白的基础上，对该蛋白进行初步纯化及生物学活性检测。方法：利用最佳发酵和表达条件，诱导基因重组的人突变体471 TNF—α工程菌表达目的蛋白。收集菌体，经超声破碎，分离人突变体471 TNF—α的包涵体，并观察变性剂及蛋白浓度对蛋白折叠的影响。采用MTT比色法检测并比较人野生型及突变体471 TNF—α的生物学活性。结果：在适当的变性与复性条件下，已成功地将突变体471 TNF—α折叠并聚合形成具有生物学活性的三聚体。突变体471 TNF—α对L929的细胞毒活性高于野生型TNF—α的15倍。结论：原核表达系统中表达的人突变体471 TNF—α经复性处理后具有显著的生物学活性，为进一步进行动物实验及临床研究奠定了基础。     

人β-NGF基因在CHO细胞中表达的生物学活性及分离纯化

目的：检测人β—NGF基因转染CHO细胞后培养上清中所表达的β—NGF的生物学活性及分离纯化。方法：采用脂质体介导的真核细胞转染，运用形态学观察和MTT法进行检测做定性定量分析，蛋白观察用SDS—PAGE电泳。结果：转染β—NGF基因的CHO细胞可分泌能促进PC12细胞生长的活性β—NGF，且可透过血脑屏障。结论：转染β—NGF基因的CHO细胞所分泌的β—NGF，接近自然合成的蛋白，具有较高生物学活性，为NGF的生物制药奠定了实验基础。     

香菇蛋白LFP91-3-A的分离纯化及其抗肿瘤机制

香菇是食用兼药用真菌，其子实体、菌丝体及发酵液具抗癌、保肝、降胆固醇、提高机体免疫力的作用。国内外学者对香菇的抗肿瘤活性进行了大量的研究，认为起主要作用的有效物质是多糖。我们对香菇中蛋白质的生物学活性进行了初步研究。     

艾蒿花粉主要变应原的分离、纯化与鉴定

目的　对我国蒿属花粉中常见、重要的变应原艾蒿花粉进行分离、鉴定与纯化。方法采用不同的提取液得到艾蒿花粉粗浸液 ,经饱和 (NH4 ) 2 SO4 分级沉淀后用聚丙烯酰胺凝胶电泳 (SDS PAGE)分离蛋白质组分 ,并用凝胶成像系统测定各组分的相对分子质量 (Mr) ;采用Westernblot鉴定其主要及次要变应原 ;通过DEAE CelluloseDE 32离子交换层析 (ionexchangechromatography ,IEC)和SephadexG 75凝胶层析 (gelchromatography)对艾蒿花粉变应原进行纯化。结果　分离后得到 2 0多种蛋白质组分 ,其中Mr 为 5 8× 1 0 3、38× 1 0 3、2 5× 1 0 3、2 0× 1 0 3、1 6× 1 0 3等 5个条带蛋白含量最丰富 ;分离到的蛋白质组分中有 9种蛋白能与确诊的蒿属花粉过敏患者血清中蒿属花粉特异性IgE结合 ,其中Mr 为 6 2× 1 0 3、4 3× 1 0 3、38× 1 0 3的蛋白条带的结合率最高 ;经纯化后仅得到Mr 为 6 2× 1 0 3的主要变应原。结论　艾蒿花粉的主要变应原Mr 分别为 6 2× 1 0 3、4 3× 1 0 3和 38× 1 0 3,层析技术可以对Mr 为6 2× 1 0 3的主要变应原成分进行纯化。     

黄粉虫抗菌肽Tenecin蛋白的纯化及其抑菌活性研究

目的 纯化黄粉虫抗菌肽Tenecin蛋白,并检测其抑菌活性.方法用1 mmol/L IPTG大量诱导表达Tenecin蛋白,纯化后检测其抑菌活性,包括金黄色葡萄球菌(Staphylococcus aureus)ATCC 29213,大肠埃希菌(Escherichia coli)ATCC 25922,白色念珠菌(Candida albicans)ATCC 10231和痢疾志贺氏菌(Shigella dysenteriae)CMCC 51252等4种标准菌.结果 SDS-PAGE电泳检测表明已获得纯化的Tenecin蛋白;体外抑菌试验结果表明,浓度为120、60、30、15 μg/ml的Tenecin与4种标准菌共培养18 h后,对金黄色葡萄球菌的抑制作用最强,而对白色念珠菌的抑制作用最弱.对同一菌种而言,浓度为60和30 μg/ml两组间无统计学意义(P＞0.05),而其他各浓度的组间均有显著性差异(P＜0.01);对同一浓度的Tenecin而言,其对白色念珠菌和痢疾志贺氏菌的抑菌效果组间无统计学意义(P＞0.05),其余各组之间均有显著性差异(P＜0.01).结论 获得的Tenecin蛋白可明显抑制病原菌,为进一步研究其抑菌机理和后期研发奠定了基础.     

可溶性DC-SIGN的表达、纯化及其生物学活性分析

目的　在大肠杆菌中表达并纯化DC SIGN融合蛋白并对该融合蛋白的抗原特异性和生物学活性进行分析。方法　以重组质粒pcDNA3 .1 DC SIGN为模板 ,进行PCR扩增出带KpnⅠ和SacⅠ酶切位点的人DC SIGN凝集素cDNA ,经相应酶切后插入原核表达载体pET 3 2a( ) ,转化大肠杆菌AD494(DE3 ) ,经IPTG诱导表达DC SIGN融合蛋白 ,用Ni2 NTA树脂对融合蛋白进行纯化 ,以Westernblot试验进行鉴定 ,通过HIV与DC SIGN的亲和实验研究融合蛋白的生物学活性。结果　酶切鉴定证实DC SIGN凝集素基因已插入原核表达载体pET 3 2a( )。重组表达质粒pET 3 2a( ) DL在大肠杆菌AD494(DE3 )中成功表达了DC SIGN融合蛋白 ,其相对分子质量 (Mr)约为 3 5× 10 3。Ni2 NTA树脂纯化后 ,融合蛋白的纯度可达 90 %以上。Westernblot试验显示DC SIGN融合蛋白与鼠抗人DC SIGN抗体有特异性免疫反应。结论　在大肠杆菌中表达DC SIGN融合蛋白 ,用亲和层析的方法对其进行初步纯化 ;HIV与DC SIGN的亲和实验表明 ,可溶性DC SIGN融合蛋白能抑制R5和X4HIV与DC SIGN受体结合     

Altered expression of insulin-like growth factor -1 and insulin like growth factor binding proteins-2 and 5 in the mouse mutant Hypodactyly (Hd) correlates with sites of apoptotic activity

Insulin-like growth factor-I (IGF-I) mediated signalling has been implicated to be of significant importance during vertebrate embryonic development. IGF-I signalling has also been shown to be modulated by a number of IGF binding proteins that are thought to act as either agonists or antagonists of IGF activity. IGF-I has been implicated in a number of cellular processes, including cell division and programmed cell death (apoptosis). We have used the mouse mutant Hypodactyly (Hd) as a tool to determine the role of IGF-I and two key IGF binding proteins (IGFBP-2 and IGFBP-5) during embryonic development. The Hd mutant is a good model with which to study developmental cascades, since it has a distinct phenotype in the limb where cellular and molecular circuits have been thoroughly investigated. The distinctive pointed limb buds observed in Hd mutant embryos have been shown to be the result of a massive increase in apoptosis. We show that all three genes, IGF-1, IGFBP-2 and IGFBP-5, display restricted expression patterns during limb development. Indeed, IGFBP-5 shows a remarkable similarity to the expression of Engrailed-1, which is the vertebrate homologue of the Drosophila selector gene Engrailed. We show that there is downregulation in the expression of IGFBP-2 in the entire apical ectodermal ridge (AER) in homozygous Hd/Hd limb buds, whereas IGFBP-5 is downregulated in specific regions in the mutant AER. IGF-I expression is downregulated in Hd limb buds in regions undergoing high levels of cell death, consistent with its proposed role as an anti-apoptotic factor, while IGFBP-5 is found at higher levels in regions of cell death, consistent with reports of its association with apoptosis in adult tissues. We propose that these three components of the IGF axis could be involved in the manifestation of the mutant phenotype in Hypodactyly, and that this is probably a result of their ability to regulate cell survival and cell death. Accepted: 1 February 2000     

Herpes simplex virus type 1 DNA is present in specific regions of brain from aged people with and without senile dementia of the Alzheimer type.

We have investigated the possible involvement of viruses, specifically Herpes simplex virus type 1, in senile dementia of the Alzheimer type (SDAT). Using the highly sensitive polymerase chain reaction, we have detected the viral thymidine kinase gene in post-mortem brain from 14/21 cases of senile dementia of the Alzheimer type and 9/15 elderly normals. The temporal cortex and hippocampus were usually virus-positive; in contrast, the occipital cortex was virus-negative in 9/9 SDAT cases and 5/5 elderly normals. Temporal and frontal cortex from younger normals (five infants and five middle-aged) were negative. Thus, the presence of Herpes simplex virus type 1 DNA is a region-dependent feature of the aged brain.     

In vitro activity of imipenem/relebactam,meropenem/vaborbactam and comparators against Enterobacterales from patients with intra-abdominal infections: Results of the study for Monitoring Antimicrobial Resistance Trends (SMART) in Taiwan, 2020

BackgroundMulti-drug resistant Enterobacterales is a growing health threat. Imipenem/relebactam and meropenem/vaborbactam, are not clinically used in Taiwan and the susceptibility is lack from routine laboratory tests.MethodsBroth microdilution method was used to determine the minimum inhibitory concentrations (MICs) and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints criteria. Isolates that were not susceptible to imipenem (MIC ≥ 2 mg/L), imipenem/relebactam (MIC ≥ 2 mg/L), or ceftolozane-tazobactam (MIC ≥ 4 mg/L) were selected for further molecular testing for genes encoding extended-spectrum beta-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases by multiplex PCR assays.ResultsA total of 290 Enterobacterales isolates from 9 participating hospitals were collected in 2020. Escherichia coli (n = 135, 46.6%) and Klebsiella pneumoniae (n = 88, 30.3%) were two leading pathogens of all Enterobacterales isolates. The antimicrobial agents with susceptibility rates more than 90% included amikacin (99.3%, 288/290), ertapenem (90.0%, 261/290), meropenem (97.2%, 282/290), imipenem/relebactam (94.8%, 275/290) and meropenem/vaborbactam (99.3%, 288/290). K. pneumoniae isolates were less susceptible to ertapenem, imipenem, meropenem, piperacillin-tazobactam and ceftozolane/tazobactam than E. coli (all p < 0.05). ESBL, AmpC, and carbapenemase were detected in 40.5% (17/42), 45.2% (19/42) and 11.9% (5/42) among tested isolates, respectively. The 5 carbapenemase genes included 4 bla_KPC and 1 bla_IMP. The imipenem-non-susceptible isolates (n = 30) had higher susceptibility rates to meropenem/vaborbactam (93.3%, 28/30) than imipenem/relebactam (50%, 12/30) (p < 0.05).ConclusionsImipenem/relebactam and meropenem/vaborbactam had excellent efficacy against Enterobacterales isolates. Meropenem/vaborbactam allowed better salvage therapy for carbapenem-resistant Enterobacterales infections.     

Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. I. Isolation and characterization of factor H: the serum cofactor for the C3b/C4b inactivator (factor I)

Factor H, purified from mouse EDTA-plasma using a 4-step procedure, consists of a single polypeptide chain of Mr 150,000 on SDS-PAGE. Mouse H (Hmo) was required for the cleavage of fluid-phase mouse C3b by mouse I (Imo). The final product of degradation of fluid-phase mouse C3b was iC3b, consisting of fragments of the alpha'-chain (alpha'-70, alpha'-43) linked by disulfide bonds to an intact beta-chain. Imo alone was capable of cleavage of membrane-bound mouse C3b and of generating iC3b. The addition of Hmo nevertheless had an enhancing effect on Imo activity, but cleavage did not proceed beyond iC3b. These observations suggest that one important function of Hmo is to permit the inactivation of fluid-phase C3b, and to inhibit irreversibly its activity. The concentration of H in the plasma of male and female BALB/c mice was not significantly different. Among different inbred strains of mice, large differences were observed in the plasma levels of H, and plasma H levels were positively correlated with the plasma levels of C3. This observation, taken together with the well known role of H in the control of the activation of the alternative pathway, suggests that the turnover of C3 is controlled to some extent by H.     

Cytoplasmic localization of p27 (cyclin-dependent kinase inhibitor 1B/KIP1) in colorectal cancer: inverse correlations with nuclear p27 loss, microsatellite instability, and CpG island methylator phenotype

Cytoplasmic mislocalization of p27 (CDKN1B/KIP1) is caused by activated AKT1 and has been associated with poor prognosis in various cancers. CIMP in colorectal cancer is characterized by extensive promoter methylation and is associated with MSI-MSI-H and BRAF mutations. We have recently shown a positive correlation between MSI/CIMP and loss of nuclear p27. However, no study has examined cytoplasmic p27 mislocalization in relation to CIMP and MSI in colorectal cancer. Using MethyLight assays, we quantified DNA methylation in 8 CIMP-specific gene promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) in 853 colorectal cancer samples obtained from 2 large prospective cohorts. We assessed expressions of nuclear and cytoplasmic p27 and nuclear p53 by immunohistochemistry. Cytoplasmic p27 expression was inversely associated with loss of nuclear p27 (P < .0001), CIMP-high (P < .0001), MSI-H (P < .0001), and BRAF mutations (P < .0001). The inverse association of cytoplasmic p27 with CIMP-high (or MSI-H) was independent of MSI (or CIMP) status. In addition, the inverse association of cytoplasmic p27 with CIMP-high was independent of KRAS/BRAF status. BRAF and CDKN2A (p16) methylation were not correlated with cytoplasmic p27 after stratification by CIMP status. The inverse associations of cytoplasmic p27 with MSI-H and CIMP-high were much more pronounced in p53-negative than p53-positive tumors. In conclusion, cytoplasmic p27 expression is inversely associated with MSI-H and CIMP-high, particularly in p53-negative tumors, suggesting interplay of functional losses of p27 and p53 in the development of various molecular subtypes of colorectal cancer.