质粒超螺旋比例对其细胞转染效率及表达的影响

检测质粒开环、闭环比例不同时对其细胞转染效率及表达的影响。构建携带人肝细胞生长因子基因的质粒(pcDNA3-HGF)及携带LacZ报告基因的质粒(pcDNA3-LacZ),用核酸纯化试剂盒提取开环、闭环比例不同的质粒,然后用LipofectAMINE介导它们分别转染NIH3T3细胞,采用X-Gal染色和ELISA法分别观察转染效率和表达活性。结果表明,用超螺旋比例分别为85.45%和48.44%的质粒pcDNA3-LacZ转染生长旺盛的NIH3T3细胞,48h检测转染效率分别为23.4%±3.8%和9.3%±2.5%,前者约是后者的2.5倍。用超螺旋比例分别为93.28%和40.53%的质粒pcDNA3-HGF转染1×106NIH3T3细胞,ELISA法检测48h细胞培养上清中HGF的表达量分别为46.5±6.3ng和25.6±4.2ng,前者是后者的1.8倍。初步认为质粒开环、闭环比例不同对其细胞转染效率及表达有一定影响,超螺旋比例高时其转染效率及表达量较高。     

Differentiating proteomic biomarkers in breast cancer by laser capture microdissection and MALDI MS

We assessed proteomic patterns in breast cancer using MALDI MS and laser capture microdissected cells. Protein and peptide expression in invasive mammary carcinoma versus normal mammary epithelium and estrogen-receptor positive versus estrogen-receptor negative tumors were compared. Biomarker candidates were identified by statistical analysis and classifiers were developed and validated in blinded test sets. Several of the m/ z features used in the classifiers were identified by LC-MS/MS and two were confirmed by immunohistochemistry.     

蒙古国喀尔喀部蒙古族4项群体指趾遗传学特征研究

在内蒙古民族大学调查了160例(男66例,女94例)来自蒙古国的喀尔喀蒙古族留学生的拇指类型、环食指长、指甲形状、足趾长4项群体指趾遗传学特征。研究结果:1)直型拇指率为44.38%,环指长率78.13%,指甲形状长型率58.75%、方型率6.88%、扁型率34.38%,足趾类型拇趾长率35.00%;2)指甲形状出现率性别间差异具统计学意义,两两特征间均未表现出明显相关关系;3)与我国10个蒙古族族群相比,多数差异具有统计学意义。     

Functional integrity of human neutrophils following 24 hour incubation with hydroxyapatite and fluoride

We have previously shown that hydroxyapatite (HA) priming of human neutrophils to a second stimulus of formyl-methionyl-leucyl-phenylalanine (fMPL) is influenced by a bisphosphonate and fluoride. The purpose of this study was to investigate the long-term effects of low concentrations of NaF (10⁻³–10⁻¹¹ mol/L F) on HA-mediated neutrophil chemiluminescence (CL) as a measure of oxidative function. CL assays were conducted following extended time periods of incubation (30 min, 3 h, 18 h and 24 h). Results were calculated as integrals of total energy output and expressed as the difference between the experimental (NaF/HA) and control (cells alone) assays. Transmission electron microscopy (TEM) was used to estimate cellular integrity and confirm HA phagocytosis. CL inhibition was observed at all fluoride concentrations at 30 min incubation. No significant difference compared to the control was observed in the CL output at 3 or 18 h. However, at 24 h the response showed a significant increase in activity at all NaF concentrations. The TEM results confirmed the functional integrity of the neutrophils, particularly those phagocytosing HA particles up to 24 h. Based on these results we demonstrate that human peripheral blood neutrophils can be maintained in a fully functional state with respect to the respiratory burst and morphology for at least 24 h. © 1997 John Wiley & Sons, Ltd.     

ERRATUM: New Body Mass Estimates for Omomys carteri,a Middle Eocene Primate From North America. Am J Phys Anthropol 109:41–52.

Payseur BA, Covert HA, Vinyard CJ, Dagosto M. 1999. New Body Mass Estimates for Omomys carteri, a Middle Eocene Primate From North America. Am J Phys Anthropol 109:41–52. This article included an incomplete Table 2. The final two columns, showing “Intercept” and “SEE” data were omitted. The complete Table 2, with these two columns included, is provided below.     

Validation and use of centrifugal ultrafiltration-dialysis in the measurement of percent free oestradiol in serum

The validity of the centrifugal ultrafiltration-dialysis method in the measurement of the percentage of oestradiol and testosterone which is not bound to protein (free) in human serum was assessed. In an initial validation, the values of % free steroid obtained for oestradiol and testosterone in protein-free ultrafiltrates of human serum were higher than expected. Filter paper discs used in the system were shown to adsorb [3H]oestradiol from protein-free solution. Their replacement with glass-fibre discs, which did not adsorb oestradiol, decreased but did not eliminate the discrepancy between observed and expected results for % free oestradiol in ultrafiltrates. The use of polyethylene rather than polypropylene vials then reduced the discrepancy to insignificant levels. The estimates for % free oestradiol in serum were reduced by these modifications.     

The expression of theta-like antigen by rat peripheral lymphocytes: serologic and functional studies.

AKR/Cum mice (Thy-1b = thetaC3H) immunized with nucleated cells from WF rat thymus, Peyer's patches, peritoneal exudate, mesenteric lymph nodes, blood, bone marrow, or spleen produced antibodies cytotoxic for ADR/J (Thy-1a = thetaAKR) but not for AKR/Cum thymocytes. The specificity of these antibodies for the Thy-1.1 (theta-AKR) antigen was confirmed by tests using thymocytes from backcross mice segregating at the Thy-1 locus. This result suggested that the rat lymphocyte antgen cross-reactive with Thy-1.1 was expressed by at least some members of each of the rat lymphoid cell populations tested. AKR/Cum mice immunized with killed rat cells also produced anti-Thy-1.1 antibodies; thus indicating that further differentiation of the injected cells was not a prerequisite for the anti-Thy-1.1 response. Unexpectedly, about 9% of unimmunized adult AKR/Cum males were found to be producing antibodies against Thy-1.1. To our knowledge, natural antibodies of this specificity have not been previously reported. Finally, it was found that peritoneal exudate cells taken from WF rats previously immunized with EL-4 mouse leukemia cells were neither killed nor functionally inactivated by treatment with anti-Thy-1.1 antibodies and complement.     

Inhibition of germination of dormant barley (Hordeum vulgare L.) grains by blue light as related to oxygen and hormonal regulation

Germination of primary dormant barley grains is promoted by darkness and temperatures below 20 °C, but is strongly inhibited by blue light. Exposure under blue light at 10 °C for periods longer than five days, results in a progressive inability to germinate in the dark, considered as secondary dormancy. We demonstrate that the inhibitory effect of blue light is reinforced in hypoxia. The inhibitory effect of blue light is associated with an increase in embryo abscisic acid (ABA) content (by 3.5‐ to 3.8‐fold) and embryo sensitivity to both ABA and hypoxia. Analysis of expression of ABA metabolism genes shows that increase in ABA mainly results in a strong increase in HvNCED1 and HvNCED2 expression, and a slight decrease in HvABA8′OH‐1. Among the gibberellins (GA) metabolism genes examined, blue light decreases the expression of HvGA3ox2, involved in GA synthesis, increases that of GA2ox3 and GA2ox5, involved in GA catabolism, and reduces the GA signalling evaluated by the HvExpA11 expression. Expression of secondary dormancy is associated with maintenance of high embryo ABA content and a low HvExpA11 expression. The partial reversion of the inhibitory effect of blue light by green light also suggests that cryptochrome might be involved in this hormonal regulation.     

RevSex duplication-induced and sex-related differences in the SOX9 regulatory region chromatin landscape in human fibroblasts

It was recently shown that duplications of the RevSex element, located 0.5 Mb upstream of SOX9, cause XX-disorder of sex development (DSD), and that deletions cause XY-DSD. To explore how a 148 kb RevSex duplication could have turned on gonadal SOX9 expression in the absence of SRY in an XX-male, we examined the chromatin landscape in primary skin fibroblast cultures from the index, his RevSex duplication-carrier father and six controls. The ENCODE project supports the notion that chromatin state maps show overlap between different cell types, i.e., that our study of fibroblasts could be of biological relevance. We examined the SOX9 regulatory region by high-resolution ChIP-on-chip experiments (a kind of “chromatin-CGH”) and DNA methylation investigations. The RevSex duplication was associated with chromatin changes predicting better accessibility of the SRY-responsive TESCO enhancer region 14–15 kb upstream of SOX9. Four kb downstream of the TESCO evolutionary conserved region, a peak of the enhancer/promoter-associated H3K4me3 mark was found together with a major dip of the repressive H3K9me3 chromatin mark. Similar differences were also found when three control males were compared with three control females. A marked male/female difference was a more open chromatin signature in males starting ~400 kb upstream of SOX9 and increasing toward the SOX9 promoter. In the RevSex duplication-carrier father, two positions of DNA hypomethylation were also found, one corresponding to the H3K4me3 peak mentioned above. Our results suggest that the RevSex duplication could operate by inducing long-range epigenetic changes. Furthermore, the differences in chromatin state maps between males and females suggest that the Y chromosome or X chromosome dosage may affect chromatin conformation, i.e., that sex-dependent gene regulation may take place by chromatin modification.