Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Multiple origins for phenylketonuria in Europe

Phenylketonuria (PKU), a disorder of amino acid metabolism prevalent among Caucasians and other ethnic groups, is caused primarily by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). PKU is a highly heterogeneous disorder, with more than 60 molecular lesions identified in the PAH gene. The haplotype associations, relative frequencies, and distributions of five prevalent PAH mutations (R158Q, R261Q, IVS10nt546, R408W, and IVS12n1) were established in a comprehensive European sample population and subsequently were examined to determine the potential roles of several genetic mechanisms in explaining the present distribution of the major PKU alleles. Each of these five mutations was strongly associated with only one of the more than 70 chromosomal haplotypes defined by eight RFLPs in or near the PAH gene. These findings suggest that each of these mutations arose through a single founding event that occurred within time periods ranging from several hundred to several thousand years ago. From the significant differences observed in the relative frequencies and distributions of these five alleles throughout Europe, four of these putative founding events could be localized to specific ethnic subgroups. Together, these data suggest that there were multiple, geographically and ethnically distinct origins for PKU within the European population.     

Association of a polymorphism of the angiotensin I-converting enzyme gene with essential hypertension.

Angiotensin I-converting enzyme (ACE) is responsible for production of angiotensin II and breakdown of kinins, leading to increased blood pressure (BP). Furthermore, ACE inhibitors are effective antihypertensive agents. A 287 bp insertion/deletion polymorphism in intron 16 of the ACE gene (ACE) was examined by PCR in a cross-sectional study of 80 hypertensive (HT) and 93 normotensive (NT) subjects whose parents had a similar BP status at age greater than or equal to 50. The frequency of the insertion allele was 0.56 in HTs and 0.41 in NTs, and the difference between observed alleles in all subjects in each group was significant (chi 2 = 7.6, P less than 0.01). The data thus provide evidence in favour of an association of HT with a polymorphism at the ACE locus (17q23), so implicating this locus, and possibly a genetic variant of ACE itself, in human essential hypertension.     

The ends and the middle: putting chromosomes together

The three known classes of eukaryotic telomeres share the requirement for an RNA template in their replication. This RNA-templated replication is subject to species-specific differences, such as telomere length and its regulation, which suggest that telomeres may have acquired different additional functions in different organisms. Centromeres show less conservation than do telomeres.     

四种动物病毒的细胞培养及血凝检测的比较研究

四种动物病毒的细胞培养及血凝检测的比较研究李天宪,赵林,罗怡珊,冯锋（中国科学院武汉病毒研究所,武汉４３００７１）关键词细小病毒,细胞培养,细胞病变,血凝试验云豹肠炎病毒（ＬＰＶ）、水貂肠炎病毒（ＭＥＶ）、犬肠炎病毒（ＣＰＶ）和猫泛白细胞减少症病毒（...     

卡介苗对枯否细胞生物特性影响的免疫细胞化学研究

卡介苗(BCG)是增强肝枯否细胞(KC)抗肝癌细胞作用的最佳免疫制剂之一。本实验是给大鼠BCG后,应用溶菌酶免疫细胞化学法显示KC,以图象分析系统测定KC的数量、分布和面积,以细胞分光光度计测定KC溶菌酶活性;分离KC体外受BCG作用后,用ELISA法测定RC的Fc受体表达。结果显示:BCG使KC数量增多一倍,尤以肝小叶周边带为显著;KC体积明显增大,细胞面积增大55.71％,周边带KC面积增大一倍上;细胞溶菌酶光密度增大64.35％;Fc受体表达增强64.82％。上述结果是BCG增强KC抗肝癌细胞作用有关细胞学机制。     

抑制CD36与Nogo-B表达抑制三阴性乳腺癌细胞增殖与迁移

分化聚类36（cluster of differentiation 36,CD36）是一种位于细胞表面的膜蛋白受体,可以结合并转运脂肪酸。内质网膜蛋白4B （Nogo-B）在肝脏中调控脂肪酸代谢而影响肝癌的发展。目前并不清楚CD36和Nogo-B的相互作用是否能够影响乳腺癌细胞的增殖和迁移。本研究在三阴性乳腺癌（triple-negative breast cancer,TNBC）细胞中同时干预CD36与Nogo-B的表达来探索它们对细胞增殖与迁移的影响。结果表明在三阴性乳腺癌细胞中,单独抑制CD36或Nogo-B的表达都能够抑制细胞的增殖与迁移;同时抑制CD36与Nogo-B的表达时,这种抑制效果更加明显,且Vimentin、B细胞淋巴瘤-2（B-cell lympoma-2,BCL2）和增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）的表达受到抑制。在小鼠移植瘤模型中,E0771细胞转染CD36或Nogo-B的siRNA后成瘤能力降低;同时敲减CD36和Nogo-B时,肿瘤生长速度显著减慢。机制研究发现,抑制CD36和Nogo-B表达能够抑制脂肪酸结合蛋白4（fatty acid binding protein 4,FABP4）和脂肪酸转运蛋白4（fatty acid transport protein 4,FATP4） mRNA的含量,同时CD36和Nogo-B过表达刺激的细胞增殖被FABP4的siRNA降低,预示着抑制乳腺癌细胞中CD36与Nogo-B的表达可能通过抑制脂肪酸的吸收和转运而抑制细胞的生长和迁移。此外,抑制CD36与Nogo-B的表达可激活P53-P21-Rb信号通路,参与抑制CD36与Nogo-B表达而抑制的细胞增殖与迁移。本研究证明同时抑制CD36和Nogo-B的表达能够协同抑制三阴性乳腺癌细胞的增殖和迁移,为临床抗三阴性乳腺癌药物的开发提供了新的靶点。     

Lipopolysaccharide-induced cytokine production in peripheral blood mononuclear cells: intracellular localization of tumor necrosis factor α and interleukin 1β detected with a three-color immunofluorescence technique

 Lipopolysaccharide (LPS) can induce monocytes to produce various cytokines such as tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). In the present study, the kinetics of both intracellular and extracellular accumulation of TNFα and IL-1β in LPS stimulated mononuclear cell (MNC) cultures has been determined. A three-color-immunofluorescence technique was used to detect intracellular accumulation of cytokines. Intracellular accumulation of TNFα in monocytes starts shortly after initiation of the culture; i.e., TNFα is detectable after 1 h, reaching a peak level after 3–4 hours with 50–65% of monocytes staining positive. In parallel with its increased intracellular presence, TNFα was also found in the culture supernatant. The intracellular accumulation of IL-1β in monocytes became detectable after 2 h of culture in the presence of LPS. After 4 h, a plateau was reached, with 90% of the monocytes being positive. In parallel, but with a little delay, IL-1β could be detected in the culture supernatant. TNFα and IL-1β can be produced simultaneously in the same monocytes as was shown by a three-color-immunofluorescence technique. It is concluded that TNFα and IL-1β are good parameters for the early measurement of monocyte activation and that both the intracellular accumulation in monocytes and the amount of secreted cytokines can be used for such a purpose. The intracellular accumulation in monocytes can be measured by the three-color-immunofluorescence technique described. Accepted: 27 August 1996     

Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).     

高邮杂交鲫（鲫♀×白鲫）及其亲本血清生化组成的比较研究

本文对高邮杂交鲫（鲫♀×白鲫♂）及其亲本血清生化组成的定量分析。结果表明,在测定的血清总蛋白、白蛋白、胆固醇、尿素氮、葡萄糖、乳酸脱氢酶、ａ－羟丁酸脱氢酶、ａ－淀粉酶和谷草转氨酶等九个生化指标中,大部分指标高邮杂交鲫与母本无显著差异,而与父本则有极显著差异。这从代谢方面论证了高邮杂交鲫是一个偏向于母本的杂交种。