人染色体11p15.5 BWS致病位点新基因C11orf21的初步功能分析

目的研究人染色体11p15.5Beckwith-Wiedemann综合征致病位点新基因C11orf21的生物学功能。方法用RT-PCR和Northern印记方法分析C11orf21基因的表达;研究C11orf21蛋白功能用Western印记和细胞培养方法。结果检测到C11orf21基因在成人和胎儿心脏和肝脏等组织转录,在心脏有较强的表达并有0.9和3.1kb两个转录物。C11orf21蛋白在COS-1和Hela细胞细胞质表达,蛋白相对分子质量为14kDa。结论初步分析显示C11orf21是11p15.5Beckwith-Wiedemann综合征致病位点的一个非印记基因,在成人心脏有较强表达的局限细胞质蛋白。     

B cell antigen receptor cross-linking induces tyrosine phosphorylation and membrane translocation of a multimeric Shc complex that is augmented by CD19 co-ligation

The SH2 domain-containing transforming Shc protein has been implicated in mitogenic signaling via several surface receptors through p21^ras. Following tyrosine phosphorylation by either receptor or non-receptor tyrosine kinases, Shc may interact with the adaptor protein Grb2, which is linked to Sos1, a guanine nucleotide exchange factor for human ras. Ligation of the antigen receptor complex on B cells (BCR) is known to activate various intracellular signaling pathways, which may accumulate in mitogenic responses. With respect to the initial steps, the activation of BCR-associated non-receptor tyrosine kinases appears to be indispensible. In this report we show that Shc proteins become tyrosine phosphorylated after BCR ligation on both transformed and normal human B cells. This is accompanied by the association of Shc with Grb2 proteins and a yet unidentified 145-kDa tyrosine phosphorylated protein. Subcellular fractionation revealed that this activation-induced multimeric Shc complex rapidly translocates towards the plasma membrane. Co-ligation of the BCR with the CD19 molecule results in a marked increase of these events, whereas CD19 cross-linking alone does not induce Shc tyrosine phosphorylation or translocation. Thus, in B cells the Shc complex may represent a molecular junction between the BCR and the mitogenic p21^ras cascade.     

抗HER-2抗体chA21对人乳腺癌SKBR3细胞凋亡的诱导作用及其机制

目的探讨抗HER-2工程抗体chA21在体外对高表达HER-2的人乳腺癌SKBR3细胞凋亡的诱导作用及其分子机制。方法采用透射电镜和原位末端标记技术(TUNEL)观察和检测chA21对SKBR3细胞凋亡的诱导,采用免疫细胞化学技术检测凋亡相关基因bc l-2、bax、Fas及caspase-3表达的改变。结果chA21作用72 h,可见SKBR3细胞凋亡,chA21高浓度组(5.4mg/L)凋亡指数显著高于低浓度组(0.2 mg/L)(P<0.01);chA21处理组SKBR3细胞的bax、Fas及caspase-3表达增加,而bc l-2表达及bc l-2/bax比值降低,上述改变在chA21高、低两个浓度组间有显著差异(P<0.01)。结论chA21在体外可诱导SK-BR3细胞凋亡,其分子机制与调节凋亡相关基因bax、bc l-2、Fas及caspase-3的表达有关。     

大鼠急性肺栓塞后cytokeratin-19表达下降

目的研究急性肺血栓栓塞大鼠的肺组织中细胞角蛋白19(cytokeratin-19,CK-19)及其分解产物CYFRA21-1的表达变化。方法建立大鼠急性肺血栓栓塞模型并做肺泡灌洗。用半定量RT-PCR法和Western blot法检测CK-19的mRNA和蛋白表达;免疫组化方法检测CK-19在肺组织内的分布;放免法检测血清和肺泡灌洗液中CYFRA21-1的水平;检测动脉血氧饱和度。结果急性肺栓塞后CK-19mRNA和蛋白质水平均逐渐降低;肺泡上皮细胞CK-19的表达明显下降;血清和肺泡灌洗液中CYFRA21-1水平升高;动脉血氧饱和度呈明显下降趋势。结论急性肺栓塞不仅导致CK-19表达降低,而且分解代谢增强,因此CYFRA21-1的检测对于判断急性肺栓塞的严重程度及预后具有重要意义。     

Subtle translocation (18;21) confirmed by FISH in a patient with Down syndrome

The patient presented with the typical features of Down syndrome: hypotonia, brachycephaly, flattened occiput, bilateral prominent medical epican-thic folds, flat nasal bridge, protruding tongue, low-set dysplastic ears, short broad hands, bilateral clinodactyly and simian crease. The karyotype of this child was originally reported as normal. High-resolution chromosomes revealed extra material on the long arm of chromosome 18. The mother's karyotype showed a reciprocal translocation between the long arm of 18 and the long arm of 21 at band q23 and q22.1, respectively. FISH performed separately with two different 21q cosmid probes gave two signals on the mother's metaphases and three signals on the prob-and. These findings confirmed that the proband is trisomic for the long arm of chromosome 21 at loci D21S65 and D21S19.     

Mutations in Steroid 21-Hydroxylase (CYP21)

The inherited inability to synthesize cortisol is termed congenital adrenal hyperplasia. More than 90% of cases are caused by 21-hydroxylase deficiency. This syndrome is characterized by signs of androgen excess and often mineralocorticoid deficiency. Steroid 21-hydroxylase (P450c2l) is a microsomal enzyme expressed in the adrenal gland that catalyzes conversion of 17-hydroxyprogesterone and progesterone to 11-deoxycortisol and deoxycorticosterone respectively. In man, this enzyme is encoded by the CYP21 (CYP21B) gene which is located in the HLA major histocompatibility complex along with a pseudogene, CYP21P (CYP21A). Mutations in CYP21 causing congenital adrenal hyperplasia are almost all generated by recombinations between CYP21 and CYP21P. These recombinations either delete CYP21 or transfer deleterious mutations from CYP21P to CYP21, a process termed apparent gene conversion. The degree of enzymatic compromise caused by each mutation is correlated with the clinical severity of the deficiency observed in patients carrying that mutation. © 1994 Wiley-Liss, Inc.     

Fibroblast growth factor 17 is over-expressed in human prostate cancer

Over-expression of fibroblast growth factor 8 (FGF8) in human prostate cancer is associated with clinically aggressive disease. Among different members of the FGF family, FGF17 and FGF8 share high sequence homology and have similar patterns of expression during embryogenesis. In this study, the clinical significance of FGF17 expression and its in vitro function in prostate cancer cells were tested. Forty resected prostate specimens from patients with benign prostatic hyperplasia (BPH, n = 12) and prostate cancer (CaP, n = 28; Gleason sum scores 3-10) were studied using semi-quantitative RT-PCR. In addition, 85 cases of CaP (Gleason sum scores 5-9) and 20 cases of BPH were examined using immunohistochemistry and findings were correlated with clinical parameters. In vitro experiments using prostate cancer cell lines examined the functional significance of FGF17 in prostate cancer. These studies revealed a significant linear correlation between increasing Gleason sum scores and FGF17 expression using both immunohistochemistry (p < 0.0001, rho = 0.99) and RT-PCR (p = 0.008, rho = 0.99). Immunohistochemistry demonstrated upregulation of FGF17 in CaP compared with BPH (p < 0.0001) and, when comparing high-grade CaP (Gleason sum score 7-10) with BPH, RT-PCR showed a fourfold upregulation of FGF17 mRNA expression (p < 0.0001). Men with tumours displaying high levels of FGF17 expression had a worse outcome on survival analysis (p = 0.044) and a higher risk of progression with metastases (p < 0.0001). Proliferation assays showed low-dose recombinant (r) FGF17 (1 ng/ml) to be a more potent mitogen than rFGF1 and rFGF8 in prostate cancer cell lines (LNCaP, DU145, and PC3M) (p < 0.001). Furthermore, FGF8 was shown to induce expression of FGF17 in these cell lines. These data support a role for FGF17, and a model of co-expression of multiple FGFs, with FGF17 as a potential mediator of FGF8 function, in human prostate carcinogenesis.     

siRNA干扰跨膜转运蛋白21的表达对KO小鼠胚胎成纤维细胞γ分泌酶活性的影响

目的探讨跨膜转运蛋白21（TMP21）对γ分泌酶活性的影响。 方法将淀粉样前体蛋白基因缺陷型KO小鼠胚胎成纤维细胞[MEF（KO）]分为转染组（T）和对照组（C），转染组转染载有TMP21小分子干扰RNA（siRNA）的质粒，对照组转染空质粒。每组均制备包含细胞全膜蛋白的样品（T1、C1），经CHAPSO进一步溶解后超速离心制备所得纯化全膜蛋白样品（T2、C2），用γ分泌酶组分早老蛋白1（PS-1）特异性抗体免疫沉降C2或T2后获得的γ分泌酶样品（IPT2、IPC2）。蛋白质印迹法检测各类样品中γ分泌酶重要组分TMP21、PS-1和Nicastrin（NCT）蛋白表达量；应用ELISA法检测β淀粉样肽40和β＆#61472;淀粉样肽42的生成总量。 结果蛋白质印迹法检测显示，TMP21蛋白表达量在转染组样品IPT2中为（5294±247）ng/ml，在对照组样品IPC2中为（19110±579）ng/ml，组间差异有统计学意义（P〈0.05）；各类样品中PS-1与NCT蛋白表达量无明显变化；TMP21可与PS-1共沉降。ELISA法检测显示，转染组样品IPT2与饱和浓度底物C99反应生成的β淀粉样肽40和β淀粉样肽42总量为（348±18）pg/ml，对照组样品IPC2为与饱和浓度底物C99反应生成的β淀粉样肽40和β淀粉样肽42总量为（342±18）pg/ml，组间差异有统计学意义（P〈0.01）。 结论TMP21可能为γ分泌酶的组分之一。在TMP21蛋白低表达状态下γ分泌酶活性升高，提示TMP21为γ分泌酶的负调控因子之一。     

Antisense oligonucleotides and short interfering RNAs silencing the cyclin-dependent kinase inhibitor p21 improve proliferation of Duchenne muscular dystrophy patients’ primary skeletal myoblasts

Increased levels of the cyclin-dependent kinase inhibitor p21 associated with decreased myoblast proliferation may be involved in the dystrophic process in Duchenne muscular dystrophy (DMD). Therefore we are interested to improve the proliferation of primary myoblasts of DMD patients by a reduction in p21 using either antisense oligonucleotides (ASO) or short interfering RNAs (siRNA). After transient transfection of myoblasts in cell culture proliferation was analyzed using a 5-bromo-2-deoxyuridine assay comparing specific transfected cells with untransfected cells and cells transfected with scrambled ASO and luciferase siRNA, respectively. Four of five Dystrophin-deficient (Dys^–) cell culture samples revealed an increase in proliferation between 7% and 18% compared to untransfected cells and between 8% and 36% compared to cells transfected with scrambled ASO. Transfection with siRNA was performed for selected samples to determine whether siRNA is more effective in gene silencing than ASO. The increase in proliferation using luciferase siRNA as reference was comparable to or less than ASO data using scrambled ASO as reference. Using untransfected cells as reference, the increase in proliferation was higher for siRNA than ASO (20–47% vs. 7–18%), but the data must be carefully interpreted with respect to nonspecific effects on gene expression by siRNA. Our findings of transient p21 gene silencing represent a basis for viral vector-mediated drug-inducible p21 shRNA expression in Dys^– myoblasts which might enhance, prolong and regulate the proliferation effect.S. Endesfelder and A. Kliche contributed equally to this work     

Allicin stimulates lymphocytes and elicits an antitumor effect: a possible role of p21ras

Allicin, the main organic allyl sulfur component in garlic, exhibits immune-stimulatory and antitumor properties. Allicin stimulated [(3)H]thymidine incorporation in mouse splenocytes and enhanced cell-mediated cytotoxicity in human peripheral mononuclear cells. Multiple administration (i.p.) of allicin elicited a marked antitumor effect in mice inoculated with B-16 melanoma and MCA-105 fibrosarcoma. The immune-stimulatory and antitumor effects of allicin are characterized by a bell-shaped curve, i.e. allicin at high, supra-optimal concentrations is less effective or inhibitory. Allicin induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in human peripheral mononuclear cells, and also in wild-type Jurkat T-cells. Allicin failed to activate ERK1/2 in Jurkat T cells that express p21(ras), in which Cys118 was replaced by Ser. These cells are not susceptible to redox-stress modification and activation. We postulate that the immune stimulatory effect of allicin is mediated by redox-sensitive signaling such as activation of p21(ras). It is suggested that the antitumor effect of allicin is related to its immune-stimulatory properties.