力竭运动大鼠心室肌蛋白质组表达特征

目的:采用蛋白质组学技术,建立安静和递增运动负荷训练后力竭大鼠心室肌蛋白质组的差异性表达谱,初步筛选出心室肌对力竭运动产生反应的目标蛋白质。方法:实验于2007-03在湖南师范大学生命科学学院蛋白质化学与蛋白质组学国家教育部重点实验室和省级运动人体科学实验室完成。①实验分组:10只SD雄性大鼠随机分为对照组和运动组,每组5只。②实验方法:运动组经过7周的大强度递增运动负荷训练后(最后一次力竭),对两组心室肌组织的全蛋白进行双向凝胶电泳分离。结果:经图像分析,在运动组的电泳图谱上共展现蛋白质点(338±17)个,对照组展现蛋白质点(352±17)个。运动后差异表达的蛋白质点共有99个。对其中差异表达的9个蛋白质点进行质谱鉴定,共鉴定出7个蛋白质,Stress-70protein,NADH-ubiquinone oxidoreductase Mr75000subnunit,Long-chain specific acyl-CoA dehydrogenase,Tropomyosin-1alphachain在运动后"缺失",Nitrilase family,member2在运动后表达上调在5倍以上,一个相对分子质量为21000的未知蛋白在运动后表达下调在5倍以上,另外有两个点经鉴定均为Myosin-6,在运动后表达量相反。这些蛋白质属于收缩蛋白、能量代谢酶、分子伴侣等。结论:递增运动负荷训练后力竭时,大鼠心室肌蛋白质组明显地发生了反应。运动后"缺失"和下调的蛋白质点与心肌收缩的调控和能量代谢的方式转变以及细胞的应激反应有关,其中,成功筛选出6种在运动医学领域尚未涉足的、具有运动应激特点的目标蛋白质。     

Whole-exome sequencing identifies ALMS1, IQCB1, CNGA3, and MYO7A mutations in patients with Leber congenital amaurosis

It has been well documented that mutations in the same retinal disease gene can result in different clinical phenotypes due to difference in the mutant allele and/or genetic background. To evaluate this, a set of consanguineous patient families with Leber congenital amaurosis (LCA) that do not carry mutations in known LCA disease genes was characterized through homozygosity mapping followed by targeted exon/whole-exome sequencing to identify genetic variations. Among these families, a total of five putative disease-causing mutations, including four novel alleles, were found for six families. These five mutations are located in four genes, ALMS1, IQCB1, CNGA3, and MYO7A. Therefore, in our LCA collection from Saudi Arabia, three of the 37 unassigned families carry mutations in retinal disease genes ALMS1, CNGA3, and MYO7A, which have not been previously associated with LCA, and 3 of the 37 carry novel mutations in IQCB1, which has been recently associated with LCA. Together with other reports, our results emphasize that the molecular heterogeneity underlying LCA, and likely other retinal diseases, may be highly complex. Thus, to obtain accurate diagnosis and gain a complete picture of the disease, it is essential to sequence a larger set of retinal disease genes and combine the clinical phenotype with molecular diagnosis.     

Characteristics of human large granular lymphocytes and relationship to natural killer and K cells

Recent evidence, has demonstrated an association between a subpopulation of peripheral blood mononuclear cells, morphologically identified as large granular lymphocytes (LGL), and natural killer (NK) activity. We have now evaluated more directly the role of LGL in both NK activity and antibody- dependent cellular cytotoxicity (ADCC), by using highly enriched populations of LGL, obtained by centrifugation of peripheral blood mononuclear cells on Percoll discontinuous density gradients. Both spontaneous and interferon- augmented NK and ADCC activities were exclusively associated with the LGL- enriched, low density fractions. The majority of LGL formed conjugates with NK-susceptible and antibody-coated target cells. Approximately 20 percent of small conventional lymphocytes also formed conjugates with the target cells for NK, but this was not associated with cytotoxic activity. Virtually all LGL were found to have receptors for the Fc portion of IgG (FcγR). The frequency of LGL among blood leukocytes was 2-6 percent. LGL could be enriched to an average purity of 95 percent by combining discontinuous density gradient centrifugation with subsequent adsorptions of the low density fractions on monolayers of immobilized immune complexes. About 50 percent of LGL were found to be FcγR-bearing T cells (T(G)), forming low affinity rosettes with sheep erythrocytes at 4 degrees C. Only 10-20 percent of LGL formed high affinity rosettes with sheep erythrocytes at 29 degrees C. LGL could be enriched to a purity of more than 90 percent by depleting high affinity rosette-forming cells from low density Percoll fractions. LGL were only a subpopulation of T(G) cells, because some lymphocytes with conventional morphology also adhered to the immobilized immune complex monolayers and formed high affinity rosettes with sheep erythrocytes. Separation of these cells from LGL by discontinuous density gradient centrifugation indicated that they are not cytotoxic, suggesting a morphological and functional subdivision of T(G) cells. The verification in this study that virtually all human NK and K cells have a characteristic morphology adds a useful parameter to the monitoring of human lymphocytes, and the ability to purify these cells by simple physical procedures should be invaluable in their further characterization.     

Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation

Yoo E‐H, Woo H, Ki C‐S, Lee HJ, Kim D‐K, Kang I‐S, Park P, Sung K, Lee CS, Chung T‐Y, Moon JR, Han H, Lee S‐T, Kim J‐W. Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation. Marfan syndrome (MFS) is an autosomal dominant disorder of the fibrous connective tissue caused by mutations in the fibrillin‐1 (FBN1) gene. Although clinical and genetic analyses have been performed in various populations, there have been few studies in Korea. The aim of this study was to investigate the clinical characteristics and genetic background of Korean patients with MFS. In 39 Korean patients with MFS who met the Ghent criteria, the most common clinical finding was aortic dilatation and/or dissection (94.9%), whereas only 35.9% of patients had ectopia lentis. The majority of MFS patients had fewer than four of the skeletal findings required to fulfill the major skeletal Ghent criterion for MFS. Only 21% of Korean patients had major skeletal abnormalities and most cases showed only minor skeletal involvement. FBN1 gene mutations were detected in 35 out of 39 patients (89.7%), which is similar to rates presented in the previous reports. These results suggest that some clinical features in Korean patients with MFS differed from those reported in Western MFS patients.     

Identification of Intellectual Disability Genes in Female Patients with a Skewed X‐Inactivation Pattern

Intellectual disability (ID) is a heterogeneous disorder with an unknown molecular etiology in many cases. Previously, X‐linked ID (XLID) studies focused on males because of the hemizygous state of their X chromosome. Carrier females are generally unaffected because of the presence of a second normal allele, or inactivation of the mutant X chromosome in most of their cells (skewing). However, in female ID patients, we hypothesized that the presence of skewing of X‐inactivation would be an indicator for an X chromosomal ID cause. We analyzed the X‐inactivation patterns of 288 females with ID, and found that 22 (7.6%) had extreme skewing (>90%), which is significantly higher than observed in the general population (3.6%; P = 0.029). Whole‐exome sequencing of 19 females with extreme skewing revealed causal variants in six females in the XLID genes DDX3X, NHS, WDR45, MECP2, and SMC1A. Interestingly, variants in genes escaping X‐inactivation presumably cause both XLID and skewing of X‐inactivation in three of these patients. Moreover, variants likely accounting for skewing only were detected in MED12, HDAC8, and TAF9B. All tested candidate causative variants were de novo events. Hence, extreme skewing is a good indicator for the presence of X‐linked variants in female patients.     

A girl with duplication 17p10-p12 associated with a dicentric chromosome

We report a 7 1/2-year-old girl with an approximately 9.5 Mb duplication of proximal 17p. Her clinical features include moderately severe developmental delay, absence of speech, talipes, congenital dislocation of the hips, premature adrenarche, dysmorphic facial features, deep palmar creases, and signs and symptoms of peripheral neuropathy consistent with Charcot-Marie-Tooth disease type 1A (CMT1A). Chromosome analysis revealed a partially duplicated 17p with two centromeres on the derivative chromosome. Fluorescence in situ hybridization (FISH) analysis demonstrated the tandemly duplicated segment spans 17p10-p12, including the entire Smith-Magenis syndrome (SMS) critical region and a portion of the CMT1A critical region. One breakpoint mapped within the centromere and the second breakpoint mapped within the CMT1A critical region, distal to the PMP22 gene. Microsatellite polymorphism studies showed that the duplicated chromosome is of maternal origin. We compare the clinical features of our patient to those of individuals with partial trisomy of proximal 17p to further delineate the genotype-phenotype correlation associated with segmental duplication of this chromosomal region.