Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype

PEX7 encodes the cytosolic receptor for the set of peroxisomal matrix enzymes targeted to the organelle by the peroxisome targeting signal 2 (PTS2). Mutations in PEX7 cause rhizomelic chondrodysplasia punctata (RCDP), a distinct peroxisome biogenesis disorder. In previous work we described three novel PEX7 mutant alleles, including one, L292X, with a high frequency due to a founder effect. We have now extended our analysis to 60 RCDP probands and identified a total of 24 PEX7 alleles, accounting for 95% of the mutant PEX7 genes in our sample. Of these, 50% are L292X, 13% are IVS9+1G>C, and the remainder are mostly private. IVS9+1G>C occurs on at least three different haplotypes and thus appears to result from recurrent mutation. The phenotypic spectrum of RCDP is broader than commonly recognized and includes minimally affected individuals at the mild end of the spectrum. To relate PEX7 genotype and phenotype, we evaluated the consequence of the disease mutation on PEX7 RNA by Northern analysis and RT/PCR. We evaluated the function of the encoded Pex7 protein (Pex7p) by expressing selected alleles in fibroblasts from RCDP patients and assaying their ability to restore import of a PTS2 marker protein. We find that residual activity of mutant Pex7p and reduced amounts of normal Pex7p are associated with milder and variant phenotypes.     

Angiotensin-(1-7)对血管平滑肌细胞钙化的影响及信号转导通路

目的:在钙化大鼠主动脉血管平滑肌细胞上观察血管紧张素-(1-7)[Angiotensin-(1-7)]对钙化的影响及其信号通道。方法:用β-磷酸甘油制备钙化的大鼠血管平滑肌细胞,再以血管紧张素-(1-7)、血管紧张素Ⅱ、血管紧张素Ⅱ 血管紧张素-(1-7)、选择性蛋白激酶A(PKA)或蛋白激酶C(PKC)抑制剂等干预,通过Von Kossa染色及检测钙含量、碱性磷酸酶活性、骨钙素浓度和Cbfa1 mRNA表达来探讨血管紧张素Ⅱ对钙化的影响及其信号通道。结果:血管紧张素-(1-7)抑制钙化大鼠血管平滑肌细胞的钙含量、碱性磷酸酶活性(P>0.05)、骨钙素浓度和Cbfa1 mRNA表达(P<0.05),也抑制血管紧张素Ⅱ对血管平滑肌细胞的钙含量、碱性磷酸酶活性、骨钙素浓度和Cbfa1 mRNA表达的促进作用(P<0.05);血管紧张素-(1-7)提高血管平滑肌细胞内cAMP浓度(P<0.05),PKA抑制剂可阻断血管紧张素-(1-7)对钙化血管平滑肌细胞的钙含量、碱性磷酸酶活性、骨钙素浓度和Cbfa1 mRNA表达的抑制作用(P<0.05)。结论:血管紧张素-(1-7)可抑制β-磷酸甘油诱导的血管平滑肌细胞钙化,并拮抗血管紧张素Ⅱ促进的血管平滑肌细胞钙化;这些效应与cAMP-PKA-Cbfa1信号途径有关。     

Renal function in relation to three candidate genes in a Chinese population

We recently found in a white population that the genes encoding angiotensin-converting enzyme (ACE, I/D polymorphism), -adducin (Gly460Trp), and aldosterone synthase (–344C/T) jointly influence renal function. We therefore investigated in a Chinese population the associations between the serum concentrations of creatinine and uric acid and these three genetic polymorphisms. We genotyped 471 ethnic Han Chinese subjects from 125 nuclear families recruited in northern China via random population sampling (75%) and at specialized hypertension clinics (25%). We performed population-based and family-based association analyses using generalized estimating equations (GEE) and quantitative transmission disequilibrium test (QTDT), respectively, while controlling for covariables. The participants were 39.7 years old and included 235 women (49.9%). The blood pressure measured at the subjects homes averaged 126/80 mmHg. Mean values were 71 µmol/l for serum creatinine, 111 ml min^–1 1.73 m^–2 for calculated creatinine clearance, and 236 µmol/l for serum uric acid. With adjustment for covariables, GEE analyses of single genes demonstrated that serum uric acid, but not serum creatinine, was positively associated with the ACE D allele. Serum uric acid concentrations were 15.8 µmol/l (95% confidence interval 3.3–28.2) and 25.7 µmol/l (11.1–40.2) higher in DD homozygotes than in ID and II subjects, respectively. Further GEE analyses of the three genes combined showed that the association between serum uric acid and the ACE polymorphism was confined to carriers of the -adducin Gly and/or aldosterone synthase C alleles. Sensitivity analyses in parents and offspring separately as well as QTDT analyses were confirmatory. Among 114 informative offspring carrying the -adducin Gly allele serum uric acid was significantly and positively associated with the transmission of the ACE D allele (=20.7 µmol/l). In conclusion, the present study extends our previous findings on the combined effects of the three candidate genes and supports the concept that these genetic polymorphisms jointly influence renal function.     

树突状细胞负载肝癌抗原肽疫苗体外诱导特异性免疫学反应的研究

目的：研究树突状细胞(dendritic cell，DC)负载肝癌抗原肽EPVTKAEML体外诱导特异性CTL的能力及其抑癌效果。方法：用顺序特异引物聚合酶链反应技术(PCR—SSP)选择HLA—B7表型供者，从脾组织中分离、培养DC-EPVTKAEML特异性CTL。用^51Cr释放法检测CTL的杀伤活性，并用抗HLA-1分子单抗(mAb)进行杀伤抑制实验。结果：找到4例HLA-B7杂合子供者，用DC负载HLA-B7限制的抗原肽EPVTKAEML可诱导特异性CTL反应，对肝癌细胞HHCC有较强的杀伤作用。结论：DC负载抗原肽EPVTKAEML在体外可诱发较强的特异性免疫反应。     

Afterpotentials following penicillin-induced paroxysmal depolarizations in rat hippocampal CA1 pyramidal cells in vitro

Epileptic discharges were induced by superfusion of rat hippocampal slices with penicillin. Under these conditions the neurons generated paroxysmal depolarization shifts (PDS) after electrical stimulation of Schaffer collaterals. The PDS were followed by large afterhyperpolarizations lasting about 2 s. The mechanisms causing these afterhyperpolarizations were studied in CA1 pyramidal cells. A late component of the after hyperpolarizations, which determined their overall duration, was blocked by intracellular application of EGTA and reduced by superfusion with 8-Br-cAMP. In the same neurons these drugs had a comparable effect on after hyperpolarizations following depolarizing current injections; it was therefore concluded that the late component of the PDS afterhyperpolarizations was caused by a slow Ca²⁺-activated K⁺ current. An initial fast component of PDS afterhyperpolarizations, which peaked about 60 ms after PDS onset, was reduced by EGTA but not affected by 8-Br-cAMP suggesting that the fast Ca²⁺-activated K⁺ current also contributed to the PDS afterhyperpolarizations. Superfusion of the slice with the -aminobutyric acid B receptor (GABA_B) antagonists phaclofen or 5-aminovalerate reduced the amplitude of the afterhyperpolarizations during the first 1000 ms but did not affect the late Ca²⁺-dependent component, indicating that a GABA_B-mediated K⁺ inhibitory postsynaptic potential (IPSP) contributed to the PDS afterhyperpolarization. Intracellular injection of Cl^– revealed that an early part of the afterhyperpolarizations lasting about 500 ms was Cl^–-dependent. This component was blocked by superfusion of the slices with bicuculline, suggesting that a GABA_A-mediated Cl^– IPSP contributed to the PDS afterhyperpolarization. The experiments show that different synaptic and intrinsic components with different time courses participate in the generation of PDS afterpotentials.     

Induction of B cell costimulatory function by recombinant murine CD40 ligand

T cell-dependent regulation of B cell growth and differentiation involves an interaction between CD40, a B cell surface molecule, and the CD40 ligand (CD40L) which is expressed on activated CD4⁺ T cells. In the current study, we show that recombinant membrane-bound murine CD40L induces B cells to express costimulatory function for the proliferation of CD4⁺ Tcells. CD40L- or lipopolysaccharide (LPS)-activated, but not control-cultured B cells were strong costimulators of anti-CD3 or alloantigen-dependent T cell responses. The molecular interactions responsible for the increased costimulatory functions were examined by analyzing the activated B cells for changes in the expression of two costimulatory molecules, B7 and heat-stable antigen (HSA), as well as by the use of antagonists of B7 and HSA (CTLA4.Fc and 20C9, respectively). The expression of both B7 and HSA was enhanced on B cells activated with LPS. As observed in previous studies, the costimulatory activity of the LPS-activated B cells was dependent on both B7 and HSA and was completely inhibited in the presence of a combination of CTLA4.Fc and 20C9. In contrast, activation of B cells with CD40L induced the expression of B7 but did not enhance the expression of HSA. In addition the costimulatory activity of the CD40L-activated B cells was partially, but not completely, inhibited by the combination of CTLA4.Fc and 20C9. These results demonstrate that CD40L regulates costimulatory function of B cells in part by inducing the expression of B7 and suggest that CD40L-activated B cells express an additional costimulatory activity that is not associated with LPS-activated B cells.     

Functionally active complement proteins C6 and C7 detected in C6- and C7-deficient individuals

Two sensitive sandwich ELISAs based on monoclonal antibodies directed to native C6 and C7 allowed the detection and quantitation of these complement proteins in 20 out of 37 serum samples from individuals who had previously been classified as deficient in these proteins as assessed by immunochemical and/or functional assays. Furthermore, serum from four C6-deficient and one combined C6-/C7-deficient individual showed an increase in the terminal complement complex (TCC) and a decrease in native C6 and C7 after complement activation as assayed by specific ELISAs. Despite their (incomplete) deficiencies, these individuals therefore possess functionally active terminal complement proteins with respect to their ability to generate the TCC. As these individuals have no history of a susceptibility to neisserial infections, even low concentrations of functionally active C6 and C7 may provide sufficient protection against those micro-organisms whose destruction requires TCC formation.     

ABC proteins: key molecules for lipid homeostasis

Forty-nine ABC protein genes exist on human chromosomes. Eukaryotic ABC proteins were originally recognized as drug efflux pumps involved in the multidrug resistance of cancer cells. However, it is now realized that one of their major physiological roles is cellular lipid transport and homeostasis, and their dysfunction is often associated with human diseases. ABCA1 and ABCA7 mediate the apolipoprotein-dependent formation of a high-density lipoprotein–cholesterol complex. ABCA3 is indispensable for pulmonary surfactant secretion. ABCG5 and ABCG8 are involved in the secretion of plant sterols and cholesterol into bile. However, the primary substrates and mechanism of action of these ABC proteins have not been precisely defined. In this review article, we first describe the general structure and functions of eukaryotic ABC proteins. The current model of ABCA1 functionality is then explained based on studies on a topological model, subcellular localization, apoA-I dependence of HDL formation, functional defects of Tangier disease mutants, and ATP hydrolysis of purified ABCA1. ABCA1 is supposed to function as a transporter of lipids as well as a receptor for apoA-I. ABCA3 is likely involved in accumulating phospholipids and cholesterol in lamellar bodies and in generating multivesicular structures.     

骨形成蛋白-7对人肾小管上皮细胞株HK-2凋亡的影响

研究不同浓度的骨成形蛋白 7(BMP 7)对人近曲小管上皮细胞株HK 2凋亡的影响并初步探讨其可能的机制。应用Hoechst 332 5 8荧光染色、DNA电泳检测凋亡细胞的形态学和生化改变 ,流式细胞仪定量检测细胞的凋亡率 ,并观察凋亡特异性蛋白酶Caspase 3活性变化的情况。结果显示 ,HK 2细胞加入 1 0ng/mlTGF β处理 2 4h后 ,可明显抑制细胞生长并诱导细胞凋亡 ,呈现明显的凋亡形态学改变 ,胞核或核质内可见浓度致密的颗粒状荧光 ,DNA电泳呈现典型的“梯形条带” ,而预先加入 5 0ng/ml、 1 0 0ng/mlBMP 7共处理 2 4h后 ,可减轻TGF β对细胞的抑制作用 ,凋亡细胞亦减少 ,Caspase 3蛋白酶活性明显下降 ,细胞凋亡率下降。提示BMP 7对人肾小管上皮细胞凋亡的具有抑制作用 ,可能是通过抑制Caspase蛋白酶活性而实现的