Molecular detection of genetic defects in congenital adrenal hyperplasia due to 21-hydroxylase deficiency: A study of 27 families

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase (21-OHase) deficiency is inherited as an autosomal recessive trait. Patients can present with the salt wasting, simple virilizing or a non-classical form of the disease. The gene for P450C21, the enzyme carrying 21-OHase activity, has been mapped to the major histocompatibility complex on chromosome 6p. Using molecular hybridisation techniques we have studied the genetic defect in 27 families with one or more affected off-spring diagnosed and treated at the University Hospital of Essen. DNA samples were digested with restriction endonucleaseTaqI,PvuII,BglII, andEcoRI and analysed by Southern blot hybridisation with the cDNA probe pC21/3c. Eleven of 40 haplotypes associated with the salt wasting form were found to have a large deletion of 30 kb affecting the 5 end of the active 21-OHase gene and the 3 end of the closely linked pseudogene. Results in another 11 cases are compatible with gene conversion; 18 cases were not informative. The 30 kb deletion was associated with a combination of the HLA antigens Bw47 and DR7 in 7 of 11 cases. In the haplotypes with gene conversion, no linkage disequilibrium to HLA antigens was found. No apparent gene alterations were detected in simple virilizing and non-classical haplotypes. The direct detection of the genetic defect in 55% of the salt wasting haplotypes may help to improve predictive testing in families with CAH.     

No effect of a Taql polymorphism in DNA at the endothelin I (EDN1) locus on normal blood pressure level or variability

Endothelin is a peptide reported to be one of the most potent vasoconstrictors known. Presumably, endothelin could play a role in the physiological regulation of blood pressure in healthy or hypertensive people. We have studied a normal restriction fragment length polymorphism (RFLP) at the endothelin-I (EDN1) locus detected with the restriction enzyme TaqI. In three different series comprising 166, 120 and 207 unrelated individuals, we found no evidence for association between genotype in this polymorphism and level of systolic or diastolic blood pressure. In two series of 156 and 117 monozygotic (MZ) twin pairs, respectively, there was no difference between genotypes in within-pair variation in systolic or diastolic blood pressure. Thus neither "level gene" nor "variability gene" effects of normal genes at the EDN1 locus could be detected with the polymorphism analyzed, in healthy population samples.     

Tonotopic Order in the Adult and Developing Auditory System of the Rat as Shown by c-fos Immunocytochemistry

Immediate éarly genes such as the proto-oncogene c- fos can be expressed in neurons following synaptic excitation by sensory stimulation. C- fos immunocytochemistry has subsequently been shown to be a very sensitive marking technique for neuronal activity. Here, antibodies against the c- fos protein product Fos were used to map the tonotopic organization in the auditory system of adult and developing rats. After stimulating adult rats with pure-tone pulses, bands of Fos-immunoreactive neurons revealed the frequency representation in seven brainstem nuclei: all three subdivisions of the cochlear nucleus, the lateral superior olive, the medial nucleus of the trapezoid body, the ventral nucleus of the trapezoid body, the rostral periolivary nucleus, the dorsal nucleus of the lateral lemniscus and the inferior colliculus. With the exception of the dorsal cochlear nucleus and the inferior colliculus, tonotopicity has not been previously demonstrated in the brainstem nuclei of the rat. During development two striking results were obtained. First, beginning at postnatal day 14 (i.e. ∼2 days after physiological hearing begins in rats), not only low but also high frequencies were able to induce strong Fos immunoreactivity, indicating that gradual recruitment of formerly unresponsive high-frequency sites does not occur in the rat. Second, a gradual age-related shift of the position of isofrequency bands was not seen in any of the nuclei, suggesting that changes in frequency-place code do not occur after 2 weeks postnatally. These results indicate that the rat's auditory brainstem nuclei achieve their adult-like tonotopic organization early on, implying a somewhat different developmental time course than is found in other mammalian species.     

Basal expression of c-Fos and Zif268 in the rat basal ganglia: immunohistochemical characterization of striatal Zif268-positive neurons

Basal expression of the protein products of the inducible immediate early genes (IEGs), c-Fos and Zif268, was investigated in five regions of the rat basal ganglia using immunohistochemistry. In particular, high basal levels of Zif268 but very low levels of c-Fos were seen in the caudate-putamen (CPu). Double immunostaining revealed that many of the constitutively expressed Zif268-positive neurons were GABAergic but very few were cholinergic or neuronal nitric oxide synthase (nNOS)-positive, and some of the Zif268-positive neurons were also immunopositive for a glutamate NMDA receptor subunit NR1 or NR2A. No regional difference between the medial and lateral parts of the CPu was observed in the cellular phenotypes of Zif268-positive neurons. Almost no basal levels of Zif268 were seen in the other four regions: the globus pallidus, entopeduncular nucleus, subthalamic nucleus and substantia nigra pars reticulata. As in the CPu, negligible levels of c-Fos were seen in these four regions. Differential expression of these two IEGs may suggest gene-specific and region-specific functions of c-Fos and Zif268 in the basal ganglia. Constitutive expression of Zif268 existing mainly in the GABAergic neurons in the CPu may at least in part be maintained by glutamatergic afferents.     

高同型半胱氨酸血症及MTHFR基因与中青年脑梗死的研究

目的 :探讨高同型半胱氨酸血症 (HHC)及N5,N10-亚甲四氢叶酸还原酶(MTHFR)基因型与中青年脑梗死的关系。方法 :采用高压液相色谱法及PCR -RFLP技术测定80例脑梗死患者和40例正常对照者的血浆总同型半胱氨酸水平 (tHcy)和MTHFR基因多态性。结果 :患者组中MTHFR基因T/T纯合型频率为33.75 % ,T等位基因频率为58.75 % ,显著高于对照组的20 %和36.25 % (P<0.05) ;患者组血浆tHcy均值为22.70μmol/L ,对照组仅为13.19μmol/L ,患者组HHC31例 ,对照组仅为2例 ,2组差别有统计学意义,且2组T/T型者血浆tHcy水平高于T/C型和C/C型者。结论 :MTHFR基因突变可能是脑梗死发生的一个遗传易感因素 ,tHcy可能与血脂血糖一样是脑卒中发病的一个独立危险因素     

经内镜活检对消化道肿瘤MDR1和MRP表达的研究

目的 :探讨消化道不同部位实体肿瘤多药耐药基因 (MDR1)和多药耐药基因相关蛋白 (MRP)的表达情况。方法 :应用RT_PCR反应技术 ,测定71例未进行任何化疗的初次内镜检查患者消化道肿瘤的MDR1和MRP。结果 :MDR1阳性表达率为87 3 % (62/71) ,其中食管癌100 % (12/12)表达 ,胃癌82 5 % (33/40)表达 ,结肠癌89 5 %(17/19)表达。3者比较无显著差异 (χ2=2.662,P>0.05)。MRP阳性表达率为43 7 % (31/71) ,其中食管癌25 0 % (3/12)表达 ,胃癌43 2 % (16/37 ,3例RNA降解 )表达 ,结肠癌66 7 % (12/18 ,1例RNA降解 )表达。3处比较无显著差异 (χ2=5.332,P>0.05)。胃内肿瘤在贲门胃底、胃体、胃角和胃窦随着发生部位的下移 ,MDR1阳性表达率逐渐升高 (50 %～100 %) ,但尚无显著性差异 (χ2=7.754,P>0.05) ,而胃内MRP不同部位的阳性表达不同 ,分别为5/6,1/9,2/10和8/15 ,组间比较有显著性差异 (χ2=9.902,P<0.05) ,其中贲门胃底具有高表达。结论 :(1)未经放化疗的消化道肿瘤MDR1具有高度表达。(2)MRP与MDR1表达并不一致。(3)胃内不同部位肿瘤MDR1表达与肿瘤发生的部位无关。MRP的表达则与肿瘤发生的部位有关。     

肥胖基因蛋白的研究进展

肥胖基因蛋白 (Leptin简称Lep)是由肥胖基因 (OB基因 )编码并由脂肪细胞分泌的一种蛋白质 ,它是脂肪细胞传递大脑有关脂肪组织储存能量情况的信号 ,能减少能量摄取 ,增加能量消耗 ,抑制脂肪合成 ,从而达到减肥目的。另外 ,该药耐受性好 ,无系统或局部反应 ,无明显不良反应     

大肠杆菌胞嘧啶脱氨酶基因的克隆及其真核表达载体的构建

目的 克隆大肠杆菌胞嘧啶脱氨酶(CD)基因，构建真核表达载体，本研究拟探索该基因在肿瘤基因治疗中的应用基础。方法 根据GenBank数据库提供的CD基因核苷酸序列，设计并合成一对引物，采用PCR方法，从大肠杆菌基因组DNA中扩增出CD基因，并与pcDNA3．1定向连接，构建受控于人巨细胞病毒启动子的重组真核载体pcDNA3．1-CD，并用限制性内切酶、PCR和DNA测序进行鉴定。结果克隆了大肠杆菌CD基因，并构建了真核表达载体，经限制性内切酶酶切、PCR扩增和DNA测序证实了其正确性。结论 pcDNA3．1-CD真核表达载体构建成功。     

大鼠脑损伤时神经细胞c-fos 、c-jun和c-myc基因表达变化

目的观察大鼠脑损伤后不同时间皮质神经细胞早期快反应基因c-fos 、c-jun和c-myc表达变化.方法应用Northern杂交、原位杂交和免疫组织化学方法检测皮质神经细胞中c-fos、c-jun和c-myc的表达.结果脑损伤后15 min,伤侧皮质神经细胞出现上述3种基因mRNA的表达,随时间的延长,表达逐渐增加,于伤后3 h达高峰并持续至伤后24 h,伤后72 h恢复至对照水平.c-fos和c-jun蛋白表达亦于伤后15 min出现,c-myc蛋白表达于伤后30 min出现,并均于伤后3 h达高峰,持续至伤后6 h,伤后24 h表达消失.结论脑损伤后上述3种基因表达特点与其它脑损害不同,可能与损伤机制和继发性神经细胞损害有关.