Structural difference between the two forms of guinea-pig beta 2-microglobulin and their occurrence in inbred guinea-pig strains

The structural difference between two forms (basic and acidic) of guinea-pig beta 2-microglobulin (beta 2m) has been established. Both forms are present in urine from inbred guinea-pig strains. The beta 2m forms were each digested with carboxypeptidase Y and carboxypeptidase A contaminated with carboxypeptidase B. Released amino acids were separated from remaining protein, dansylated and analysed by 2-dimensional TLC on polyamide layer sheets. From the results it was concluded that the basic beta 2m form has lysine and the acidic beta 2m form has asparagine as their respective C-terminal amino acids. The acidic form is also 1 amino acid (lysine) shorter than the basic form, which is supported by electrophoretic studies on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of the 2 forms of beta 2m in urine from inbred guinea-pig strains 2 and 13, shown by gel filtration and ion exchange chromatography, makes it unlikely that the 2 forms are a result of genetic polymorphism.     

MUC1/Y基因体外冲击树突状细胞诱导抗瘤免疫反应

目的制备含MUC1/Y cDNA质粒转染的树突状细胞(DC),体外诱导杀伤细胞,研究其治疗消化道肿瘤的效果.方法构建MUC1/Y cDNA真核表达载体pIRES2-EGFP-MUC1/Y、pcDNA3.1-MUC1/Y.以pcDNA3.1-MUC/Y电转染8例HLA-A2(+)消化道肿瘤患者单个核细胞衍生的DC后,与自体T细胞混合培养,诱导CTL(T-pcDAN3.1-MUC1/Y).以SW620细胞[HLA-A2(+)、MUC1/Y(+)]为特异性靶细胞,Raji细胞[HLA-A2(-)、MUC1/Y(-)]和Lovo细胞[HLA-A2(-)、MUC1/Y(+)]为非特异性靶细胞,通过乳酸脱氢酶(LDH)释放实验测定杀伤活性,ELISA法检测基因修饰后DC刺激自体T细胞产生IFN-γ的能力,并以ANNEXIN V-FITC试剂盒检测特异性CTL诱导靶细胞凋亡情况.结果pIRES2-EGFP-MUC1/Y转染效率为8%左右.T-pcDAN3.1-MUC1/Y诱导的杀伤作用显著高于T-pcDNA3.1[pcDNA3.1(+)修饰DC诱导的CTL]和T-IL-2(IL-2刺激外周血单个核细胞产生的CTL),P＜0.05.而且T-pcDNA3.1-MUC1/Y对靶细胞的杀伤和诱导凋亡的能力显著高于对照组.基因修饰后的DC能刺激自体T细胞分泌高水平IFN-γ,与未转染的DC相比具有显著差异(P＜0.05).结论成功构建MUC1/Y全长cDNA真核表达载体.pIRES2-EGFP-MUC1/Y可用于真核细胞转染,通过观察转染效率,易于筛选阳性克隆;经pcDNA3.1-MUC1/Y修饰的DC可有效诱导特异性抗肿瘤免疫应答.     

浙江畲族DYS287、DYS440位点的多态性研究

目的　研究浙江畲族人群中DYS2 87和DYS44 0位点的多态性。方法　采用聚合酶链反应扩增DYS2 87和DYS44 0 ,PCR产物用 2 %的琼脂糖电泳分析 10 0名畲族个体的基因型。结果　 10 0名个体DYS2 87位点全部是YAP-;没有发现YAP ;10名为DYS44 0 3 ,占总人群数的 10 % ,其余 90名是DYS44 0 4。结论　浙江畲族人群DYS2 87和DYS44 0多态性与属于汉藏语系的其它民族之间存在着明显的不同 ,因此这两个基因位点对研究人类的进化是一种稳定的、重要的遗传标记。     

Fertilization in vitro increases non-disjunction during early cleavage divisions in a mouse model system

BACKGROUND: We have been studying an unusual mouse-the BALB/cWt (Wt) male-in which the Y chromosome is susceptible to high rates of mitotic non-disjunction, particularly at the first two cleavage divisions. As these are the same divisions that human embryos generated through assisted reproductive technology must complete in an artificial setting, analysis of the Wt Y chromosome allows us to examine the effect of fertilization and culture in vitro on mammalian chromosome segregation. METHODS: We performed standard mouse IVF, cultured embryos in 5% CO2 in air or in a lowered oxygen atmosphere, and used fluorescence in-situ hybridization to examine the sex chromosome constitutions of 2-, 4-, 8- and 16-cell stage Wt Y-bearing embryos. RESULTS: We observed a significant increase in mosaic sex chromosome aneuploidy at each embryonic stage in embryos cultured in 5% CO2 in air, but under lowered oxygen conditions mosaicism returned to control (in-vivo) levels. CONCLUSIONS: Our results demonstrate that slight alterations in in-vitro conditions may have a considerable impact on the genetic quality of assisted reproductive technology-derived embryos and suggest that the genetic quality of embryos should be a fundamental concern in the development of new culture systems for clinical use.     

交感神经化学切除后大鼠心窦房结区神经递质的变化

用６－羟多巴胺化学切除大鼠心内交感神经后，有免疫组织化学结合图像分析法和放免测定观察窦房结区神经肽Ｙ及降钙素基因相关肽的变化；同时应用组织化学方法结合图像分析、生物化学测量观察了ＳＡＮ区去甲肾上腺素及乙酰胆碱酯酶的变化，实验发现，静脉注射６－ＯＨＤＡ５０ｍｇ／ｋｇ一周后，ＳＡＮ区ＮＡ及ＮＰＹ含量明显减少，ＡＣＨＥ活性显著增加，ＣＧＲＰ含量增加不明显。     

大鼠小脑皮质NPY能神经元的形态与分布

目的　研究大鼠小脑皮质内NPY免疫阳性神经元的形态与分布。方法　SD大鼠 10只 ,雌雄不限 ,升主动脉灌注固定 ,开颅取小脑 ,SP免疫组织化学染色。结果　大鼠小脑内NPY免疫阳性神经元分布在皮质的Purkinje细胞层 ,胞体呈梨形或烧瓶状 ,细胞核清晰不染 ,轴突伸向颗粒层 ,树突呈网状伸向分子层 ,突起未见染色。分子层和颗粒层未见阳性细胞 ,但可见神经纤维的存在。结论　小脑Purkinje细胞层内存在NYP阳性神经元。     

Galanin-positive nerves of trigeminal origin innervate rat cerebral vessels

Galanin (GAL)-positive nerve fibers in rat cerebral vessels were demonstrated by immunohistochemistry, and their origin in the trigeminal ganglia and pathway in the nasociliary nerve to the vessels was shown by retrograde tracer technique and nerve transection. Some fibers in the vertebrobasilar system appear to originate in extracranial sources. With the antiserum used only few GAL fibers could be seen in the vessels, mostly in the vertebrobasilar system. In neonatally sympathectomized animals a rich network could be visualized in most pial arteries - still particularly in the vertebrobasilar system - probably as a result of a diminished competition for nerve growth factor. No vasomotor effect of GAL could be detected in isolated segments of pial arteries, neither in normal nor in sympathectomized animals, which rules out a direct postsynaptic effect on vascular tone. GAL did not display prejunctional modulatory action on the adrenergic nerves present in the vascular preparations. A sensory function of GAL is discussed.     

μ and δ opioid receptor activation inhibits ω-conotoxin-sensitive calcium channels in a voltage- and time-dependent mode in the human neuroblastoma cell line SH-SY5Y

 Ca²⁺ channel modulation by the μ opioid agonist [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAGO) and the δ opiate agonists [d-Pen², d-Pen⁵]-enkephalin (DPDPE) and [d-Ala², d-Leu⁵]-enkephalin (DADLE) in cultured human neuroblastoma SH-SY5Y cells was investigated using the whole-cell variant of the patch-clamp technique. In SH-SY5Y cells, differentiated in vitro with retinoic acid, all agonists reversibly decreased high-voltage-activated, ω-conotoxin-sensitive Ba²⁺ currents in a concentration-dependent way. Inhibition was maximal with a 1 μM concentration of opiate agonists (76% with DAGO and 63% with δ agonists, when measured at 0 mV) and was characterized by a clear slow down of Ba²⁺ current activation at low test potentials. Both inhibition and slow down of activation were attenuated at more positive potentials, and could be partially relieved by strong conditioning depolarizations. Current suppression operated by both μ and δ agonists was prevented by pre-treatment of the cells with pertussis toxin. No sign of additivity was observed when δ agonists were applied to cells that were maximally activated by DAGO, suggesting that a common mechanism, involving the same type of modulating molecule, is responsible for Ca²⁺ channel inhibition promoted by activation of μ and δ opioid receptors in SH-SY5Y cells. Received: 10 October 1996 / Received after revision and accepted: 18 November 1996     

Aneuploidy in recurrent spontaneous aborters: the tendency to parental nondisjunction

To test the hypothesis that parental aneuploidy, particularly involving sex chromosomes, indicates an increased risk of meiotic nondisjunction, we analyzed chromosome numbers from 283 parents who had had 2 or more spontaneous abortions and, if they had children, no abnormal offspring. A sample of 15 lymphocyte metaphases was examined per individual for a total of 4,245 metaphases. Two or more cells with the same abnormal chromosome complement were found in 19 individuals: 4 males with a minor number of 45, X cells and 15 females with minor numbers of 45, X and/or 47, XXX cells (p< 0.001). The most logical explanation is that mitosis may, in part, reflect meiosis. This group of parents appears predisposed to chromosome errors in meiosis leading to recurrent spontaneous abortion.