Mapping protease susceptibility sites on the Escherichia coli transcription factor sigma70.

N-terminally and C-terminally histidine-tagged versions of Escherichia coli RNA polymerase initiation factor sigma70 were subjected to limited proteolysis and electrophoretic separation. The protein fragments were transferred to nitrocellulose, and biotinylated nitrilotriacetic acid was used to detect the His-tagged ladder that resulted. Using size markers of known lengths derived from chemical cleavage of the same His-tagged sigma70, we were able to map the sites of proteolysis for sigma70 free in solution, bound to core RNA polymerase, and in the Mg2+-dependent open complex with lambdaPR promoter DNA. Numerous sites of changed susceptibility were mapped. Most of these sites mapped near residues 100 and 500. In addition, the highly acidic region around residue 190 became susceptible to cleavage in the open promoter complex. These results suggest that sigma70 undergoes significant conformational changes upon binding to core RNA polymerase and upon open promoter complex formation.     

Drosophila ERCC1 is required for a subset of MEI-9-dependent meiotic crossovers

Drosophila MEI-9 is the catalytic subunit of a DNA structure-specific endonuclease required for nucleotide excision repair (NER). The enzymatic activity of this endonuclease during NER requires the presence of a second, noncatalytic subunit called ERCC1. In addition to its role in NER, MEI-9 is required for the generation of most meiotic crossovers. To better understand the role of MEI-9 in crossover formation, we report here the characterization of the Drosophila Ercc1 gene. We created an Ercc1 mutant through homologous gene targeting. We find that Ercc1 mutants are identical to mei-9 mutants in sensitivity to DNA-damaging agents, but have a less severe reduction in the number of meiotic crossovers. MEI-9 protein levels are reduced in Ercc1 mutants; however, overexpression of MEI-9 is not sufficient to restore meiotic crossing over in Ercc1 mutants. We conclude that MEI-9 can generate some meiotic crossovers in an ERCC1-independent manner.     

Effects of biological and non-biological immunomodulatory therapies on the immunogenicity of vaccines in patients with rheumatic diseases

Vaccinations are administered to patients to induce a protective immune response, resulting in immunological memory. Preventing infection through the use of vaccines is particularly important in immunocompromised and immunosuppressed individuals given their increased frequency and severity of infections relative to healthy individuals. Recent surveys show that the vaccination rate is still alarmingly low in patients with rheumatic disease. In this review we briefly discuss the different types of vaccines and then critically examine evidence related to vaccination efficacy in patients with autoimmune disease and the effects of immunomodulatory therapy, with an aim to provide guidance and optimize the administration of vaccines in such individuals.     

尿毒症皮肤瘙痒患者采取腹膜透析与血液透析治疗的效果探究

目的：探究尿毒症皮肤瘙瘁患者采取腹膜透析与血液透析治疗的临床效果,并为该病的临床治疗提供经验积累。方法：选取我院肾内科于2005年1月-2012年12月收治的52例尿毒症皮肤瘙痒患者,利用随机数字表法进行分组,分别设为研究组和对照组。其中对照组实施血液透析治疗,而研究组开展腹膜透析治疗。记录两组在治疗前和治疗后第8周末血生化客观指标及皮肤瘙痒程度,并做好对比。结果：两组在治疗前的皮肤瘙痒评分、β2-MG、PTH、p3,BUN、SCr值差异无统计学意义（P〉0．05）;治疗后,研究组皮肤瘙瘁评分为（3．4±0．8）分,对照组为（5．8±0．9）分,差异有统计学意义（P〈0．05）。治疗后,研究组β2-MG、PTH及Ps．均低于对照组,差异有统计学意义（P〈0．05）。结论：尿毒症皮肤瘙痒患者的主要致敏因子为大中分子,采取腹膜透析能够有效清除大分子物质,进而达到瘙痒程度的有效改善。     

Molecular systematics of the Awaous banana complex (River gobies; Teleostei: Oxudercidae)

Diadromous fishes can exhibit interesting evolutionary and population-level patterns given their use of freshwater and marine environments as part of their life histories. The River goby genus Awaous are prominent members of riverine ichthyofaunas and occur throughout Atlantic and Pacific slopes of the Americas from the southern United States to Ecuador and Brazil. Here we study the widespread and polymorphic Awaous banana complex to assess phylogeographic patterns and test previous hypotheses that all populations of this species in the Americas belong to the same species. Analysis of sequence data based on the mitochondrial cytochrome oxidase I gene shows multiple clades within the Atlantic and Pacific basins, which correspond to previously described species. Additionally, haplotype analysis demonstrates unique and unconnected networks between these species. Within these clades we document biogeographic patterns that are congruent with results of other co-occurring diadromous species, as well as a novel biogeographic pattern for the region. Our results support the recognition of distinct species of Awaous in the Atlantic (A. banana and A. tajasica) and Pacific (A. transandeanus) basins. These results are concordant with previously established morphological characters permitting the separation of these species.     

重组人MASP2蛋白在大肠杆菌中的表达和纯化

目的:获得酶原形式的重组人甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MASP2)。方法:在大肠杆菌中诱导表达重组人MASP2全长蛋白,包涵体裂解后,经复性、透析、浓缩、考马斯亮蓝染色、SDS-PAGE及Western印迹,鉴定纯化结果及酶活性。结果:复性后的MASP2蛋白经考马斯亮蓝染色未见杂带。自激活实验表明,当MASP2浓度在1μmool/L以下时,无论在4℃还是37℃,都能较稳定地保持酶原形式;蛋白浓度为3.5μmool/L时只能在4℃保持稳定,37℃发生自激活;蛋白浓度达到12μmool/L后,在4℃时已不能稳定存在。结论:获得了较纯的重组人MASP2蛋白,且具有自激活活性。     

Age-related instability in spermatogenic cell nuclear and mitochondrial DNA obtained from Apex1 heterozygous mice

The prevalence of spontaneous mutations increases with age in the male germline; consequently, older men have an increased risk of siring children with genetic disease due to de novo mutations. The lacI transgenic mouse can be used to study paternal age effects, and in this system, the prevalence of de novo mutations increases in the male germline at old ages. Mutagenesis is linked with DNA repair capacity, and base excision repair (BER), which can ameliorate spontaneous DNA damage, decreases in nuclear extracts of spermatogenic cells from old mice. Mice heterozygous for a null allele of the Apex1 gene, which encodes apurinic/apyrimidinic endonuclease I (APEN), an essential BER enzyme, display an accelerated increase in spontaneous germline mutagenesis early in life. Here, the consequences of lifelong reduction of APEN on genetic instability in the male germline were examined, for the first time, at middle and old ages. Mutant frequency increased earlier in spermatogenic cells from Apex1(+/-) mice (by 6 months of age). Nuclear DNA damage increased with age in the spermatogenic lineage for both wild-type and Apex1(+/-) mice. By old age, mutant frequencies were similar for wild-type and APEN-deficient mice. Mitochondrial genome repair also depends on APEN, and novel analysis of mitochondrial DNA (mtDNA) damage revealed an increase in the Apex1(+/-) spermatogenic cells by middle age. Thus, Apex1 heterozygosity results in accelerated damage to mtDNA and spontaneous mutagenesis, consistent with an essential role for APEN in maintaining nuclear and mtDNA integrity in spermatogenic cells throughout life.