Approaches to functional genomics: potential of matrix-assisted laser desorption ionization--time of flight mass spectrometry combined with separation methods for the analysis of DNA in biological samples

MALDI-TOF MS has potential as a valuable technique in DNA mapping studies and may well be complementary to other approaches to DNA analysis such as gel electrophoresis and sequencing. This study used 2,6-dihydroxyacetophenone (DHAP) mixed with diammonium hydrogen citrate (DAHC) as the matrix. In addition, recent technical advances such as time lag focussing (TLF) and better selection of matrices (such as 3-hydroxypicolinic acid (3 HPA) and picolinic acid (PA)) extended the range of DNA fragments that can be studied by this approach. The following samples were investigated: Poly-T mixture (dT 15, 19, 20, 25, 74 and 75), plasmid pBR322 derived oligonucleotides (10, 11, 12, 13, 14, 15, 19, 20 and 50 nucleotides long) and DNA fragments of 25, 36 and 37 base pairs corresponding to a fragment in the restriction map for the gene corresponding to the hexon protein of Adenovirus 2 and 5. The results were contrasted with similar analyses performed by ion-paired reversed-phase HPLC coupled to on-line electrospray mass spectrometry.     

巢蛋白和阶段特异性胚胎抗原-1在大鼠2型星形胶质细胞中的表达

目的 观察1型和2型星形胶质细胞(T1A、T2A)是否表达神经干细胞的标志物、是否具有神经干细胞的特性.方法 取新生大鼠脑皮质,体外培养纯化的O-2A祖细胞、T1A和T2A,应用激光共焦双重免疫荧光标记技术检测巢蛋白和阶段特异性胚胎抗原-1(SSEA-1)的表达;观察O-2A祖细胞、 T1A和T2A在碱性成纤维生长因子(bFGF)和表皮生长因子(EGF)的培养液中生长方式的改变.结果 巢蛋白在O-2A祖细胞和T2A中表达,T1A不表达;SSEA-1仅在T2A中表达.在干细胞培养基中培养10d,T2A形成能增殖和连续传代的细胞球,细胞球巢蛋白标记阳性,贴壁后分化细胞具有神经元、星形胶质细胞和少突胶质细胞样形态;但相同培养条件下的O-2A祖细胞和T1A生长方式无改变.结论 巢蛋白和SSEA-1在两型星形胶质细胞中的表达存在差异,T2A具有神经干细胞的某些生物学特性.     

Bone-particle-impregnated bone cement: an in vivo weight-bearing study

To evaluate an experimental inorganic-bone-particle-impregnated bone cement, canine hip prostheses were implanted in dogs using a regular bone cement on one side and the experimental bone cement on the other. In a preliminary feasibility study, bone ingrowth into the resorbed bone-particle spaces was established 3 months after implantation in three dogs. In a more detailed study, twenty-eight (28) dogs were divided in four groups to delineate the effects of time on the phenomena of bony ingrowth. One month after implantation, active bone ingrowth into the bone cement was obvious. By 3 months postimplantation, the ingrowth appeared to have traversed the thickness of the bone-particle-impregnated cement. By the fifth month, most of the interconnected inorganic bone particles were replaced by new bone. At the end of a year, the ingrown bone was mature and negligible new bone activity was present. Biomechanical pushout tests closely corroborated the histologic observations. The maximum shear strength of the cement/bone interface of the experimental side reached 3.6 times that of the control side at 5 months postimplantation. No further improvements were seen at 12 months postimplantation. A viable bone/cement interface may result in a better orthopedic implant fixation system by combining the advantages of both cement for immediate rigidity and biological ingrowth for longterm stability.     

The lack of effect of histamine on spontaneous activity in the isolated human myometrium

The effects of histamine on the spontaneous activity of the isolated human myometrium were studied. Both the frequency and force of contractions of the muscle strips were not significantly altered by histamine. The presence of either a histamine H₁-or H₂-receptor antagonist in the organ bath did not significantly change the responses of the uterine muscle to histamine. These findings suggest that histamine has negligible effects on the human myometrium, possibly due to the absence, or paucity, of histamine receptors.     

钴60放射法制备免疫功能抑制大鼠模型

目的通过系统的钴60放射计划,对大鼠免疫功能抑制的合适剂量进行评估,为干细胞移植研究提供合适的的动物模型。方法SD大鼠随机分为12.5Gy、10Gy、7.5Gy、5Gy4个剂量组,10只/组。计算放射后2个月的死亡率。检测白细胞数量,评价各剂量组动物的白细胞参考值范围。结合死亡率和白细胞参考值范围,确定合适的放射剂量。通过对外周血中性粒细胞(PMN)吞噬实验和外周血白细胞移行抑制实验(MIT),对细胞免疫功能进行检测。结果行12.5Gy照射的大鼠,3d内全部死亡;10Gy放射后的大鼠在1个月内全部死亡;7.5Gy照射的大鼠2个月内死亡1只;5Gy照射的大鼠没有死亡情况发生。设定95%作为可信区间,计算白细胞数量参考值范围(109个/L),未放疗组:16.978+1.96×6.46;5Gy放疗组:4.93+1.96×0.72;7.5Gy放疗组:2.313+1.96×0.782;10Gy放疗组:1.03+1.96×0.507。t检验表明,随着放疗剂量的增加,各实验组的白细胞数量显著减少。对于细胞免疫功能的检测,外周血中性粒细胞(PMN)吞噬实验结果表明,机体全身免疫功能降低;外周血白细胞移行抑制实验(MIT)表明,机体T细胞免疫功能降低。结论7.5Gy可以一定程度上抑制大鼠的免疫功能,为合适的放疗剂量。     

Detection of turkey rhinotracheitis virus in turkeys using the polymerase chain reaction

Six-week-old turkey poults were infected with the virulent UK/3B/85 strain of TRTV. Tracheal and oesophageal swabs were made every 2 to 3 days from groups of five poults and the RNA extracted. The TRTV RNA was then reverse-transcribed into complementary DNA (cDNA) using an oligonucleotide complementary to the 3' end of the fusion protein (F) mRNA. The cDNA was then used in a polymerase chain reaction (PCR) with an upstream primer to generate a product of approximately 0.5 kbp which was detected by ethidium bromide staining after electrophoresis. In this way, TRTV was detected in both types of swab for 17 to 19 days post-infection, nearly 2 weeks after the peak titres of infectious virus. Swabs which were allowed to dry completely before RNA extraction were as successful as swabs kept wet and extracted almost immediately, useful for when samples are collected in the field. The oligonucleotides amplified the 0.5 kbp product from TRTV strains isolated in six countries over a 13-year period, indicating that they might be usable as 'universal' oligonucleotides for TRTV detection.     

Significance of cytogenetic and fluorescence in situ hybridization analysis in evaluating antichronic myeloid leukemia efficacy of different immune effector cells

We report on the antileukemia effect of interleukin 2 (IL2) on different immune cells from 22 patients with chronic myeloid leukemia (CML). Bone marrow cells from these patients were first cultured in modified long-term bone marrow culture medium for several days, then separately cultured with lymphokine activated killer cells (LAK), cytokine-induced killer cells (CIK), and dendritic cell cocultured CIK (DC-CIK) for another 1-2 days. They were then detected for presence of the Philadelphia chromosome (Ph) by cytogenetic analysis and fluorescence in situ hybridization (FISH). The percentage of Ph-chromosome-positive cells in the bone marrow mononuclear cells after culturing with CIK and DC-CIK was significantly lower than that after culturing with IL2 or LAK. Our results demonstrate that cytogenetics and FISH are useful techniques for the evaluation of the anti-CML effect of immune cells and that CIK or DC-CIK can be appropriate candidates for adoptive immune cell therapy in vivo or for leukemia cell purging ex vivo.     

路氏乳杆菌JCM1081黏附相关蛋白的初步鉴定

目的研究路氏乳杆菌(Lactobacillus reuteri,也称罗伊氏乳杆菌)JCM1081菌体表面蛋白对其黏附HT-29细胞的影响。方法将路氏乳杆菌JCM1081菌体进行胰蛋白酶、蛋白酶K处理;用氯化锂和盐酸胍对乳杆菌表面的蛋白进行抽提,进行SDS-PAGE后与黏蛋白受体进行Western blot,并对杂交阳性蛋白进行质谱分析鉴定。结果路氏乳杆菌JCM1081菌体经胰蛋白酶、蛋白酶K处理后,其对HT-29细胞的黏附力显著下降(P<0．01);用氯化锂去除路氏乳杆菌JCM1081菌体外表面的S层蛋白后,路氏乳杆菌JCM1081对HT-29细胞的黏附力无显著变化;Western blot结果显示相对分子质量(Mr)为29×103和14×103的两种菌体表面蛋白与黏蛋白受体杂交中出现了强阳性;质谱分析结果显示29×103蛋白与路氏乳杆菌ATCC55730的h0793蛋白相似性高达71．1％。结论路氏乳杆菌JCM1081菌体表面的蛋白参与了乳杆菌的黏附,其中29×103和14×103的两种胞壁表面蛋白能够特异地识别黏蛋白受体并与之结合,29×103蛋白属ABC转运蛋白家族。     

Ⅱ期高血压病高胰岛素血症对血液粘度及血脂的影响

以２１例Ⅱ期高血压患者、２１例Ⅱ期高血压病高胰岛素血症患者及２０例正常人为研究对象，以全血比粘度、全血还原比粘度、血浆比粘度、红细胞压积及血浆甘油三酯、总胆固醇、高密度脂蛋白为指标；结果表明Ⅱ期高血压病患者全血比粘度、全血还原比粘度较正常人高，Ⅱ期高血压病高胰岛素血症患者亦较单纯高血压病患者进一步增高，同时其血浆甘油三酯较单纯高血压病患者增高，高密度脂蛋白降低．而血浆比粘度、红细胞压积、总胆固醇三组间无显著性差异。单纯高血压病患者与正常人组比较血脂无差异。因而认为高血压病患者的高胰岛素血症可使高血压病患者增高的血粘度进一步增高，且血粘度的增高主要是红细胞机能、代谢的改变所致。单纯高血压病患者血脂改变不明显，高血压病患者血脂的改变主要是由高胰岛素血症所致。