江苏徐州地区肝豆状核变性患者ATP7B基因8、13号外显子突变的检测和分析

目的研究徐州地区肝豆状核变性(Hepatolenticular degeneration,HLD)患者ATP7B基因8、13外显子突变情况,为本病的早期和产前诊断提供理论依据。方法采集33例HLD患者和30例对照组正常人外周血、提取DNA、PCR扩增ATP7B基因8和13外显子;对扩增产物分别进行限制性内切酶MspI及BtgI酶切分析,反应异常者行DNA测序,最后将突变结果与临床表型作相关性分析。结果 33例HLD患者中,15例存在MspI酶切异常,测序为Arg778Leu杂合或纯合突变,占45.45%(15/33);9例存在BtgI酶切异常,测序为Pro992Leu杂合突变,占27.27%(9/33)。30例正常对照组未检测出突变。结论 ATP7B基因第8、13外显子是徐州地区HLD患者的基因突变热区,筛选本地区HLD可疑患者时应优先检测。     

用荧光PCR对中国人肝豆状核变性进行早期诊断及携带者检测

目的 应用荧光聚合酶链反应技术对中国人肝豆状核变性（Wilson's disease,WD)进行早期诊断和携带者检测。方法 在66例WD患者,55名健康的家系成员和30例其他疾病患者（作为非WD对照组）中,检测了中国人WD基因的突变热点Arg778Leu;随机抽取3例样本（2例来自WD患者组,1例来自对照组）进行DNA测序以验证结果。结果 在66例WD患者中检出5例Arg778Leu突变纯合子和21例突变杂合子,总检出率为39．4％,在55名WD家系成员中检出了12例杂合子,并确定其中11例是WD致病基因携带者而不是症状前患者;DNA测序结果与荧光PCR结果一致。结论 8号外显子Arg778Leu突变是中国人WD的高频突变点,荧光PCR具有简便快速,准确,检出率高等优点。     

应用高分辨熔解曲线技术检测中国人Wilson病的常见基因突变

目的 应用PCR-高分辨熔解曲线分析技术检测中国人Wilson病(Wilson disease,WD)患者中ATP7B基因内高频突变区第8和13外显子.方法 酚-氯仿法提取外周全血基因组DNA;PCR扩增患者ATP7B基因第8和13外显子及两个外显子中的突变热点区域,PCR产物经HR-1进行高分辨熔解曲线分析;进一步以限制性内切酶或(和)DNA测序法对高分辨熔解曲线检测结果进行验证.结果 在30例WD患者中,检测到R778L纯合子3例,R778L杂合子6例,P992L杂合子6例,P992D/S975Y的复合杂合子1例,R778L/P992L复合杂合子2例和R778L/752.33delG复合杂合子1例.本组样本中,R778L、P992L和S975Y的基因频率分别为25%、15%和1.67%.测序及限制性内切酶分析结果完全与高分辨熔解曲线分析结果一致.结论 高分辨熔解曲线分析检测ATP7B基因突变具有简便、快速、特异和灵敏等优点,可作为Wilson病患者及携带者突变筛查的优选方法.     

肝豆状核变性基因表达产物及基因突变的研究

目的 探讨肝豆状核变性（Wilson's disease,WD)的发病机理。方法 将活检获得的WD患者肝标本体外分离,培养肝细胞,应用Western印迹法对WD患者肝细胞WD蛋白进行检测,同时扩增其基因组DNA并直接测序。结果 3例WD患者中有2例出现肝细胞WD蛋白特异条带密度降低。DNA测序发现其中一例患者存在ATP7B778位点CGG→CTG（Arg778Leu)杂合突变及770位点CTC→CTG改变。结论 WD基因在WD患者肝细胞的蛋白表达存在异常。可能与ATP7B基因突变有关。     

肝豆状核变性患者ATP7B基因突变分析及中医分型的相关性研究

目的用PCR-酶切和DNA直接双向测序法分析中国汉人ATP7B基因exon8,12突变特点及与WD中医分型的相关性。方法分子生物学方法及中医辨证分型方法。结果在102例WD患者中ATP7B基因exon8突变率27.5%,ex-on12突变率14.7%。中医辨证可将WD分为6型,各型在临床上有一定特点。结论exon8,12突变是WD患者的ATP7B基因最常见的突变,中医各型均与此突变密切相关;研究中还可发现一些国内罕见报道的点突变。     

中国人Wilson病ATP7B基因Thr935Met突变与临床表型的关系研究

目的　探讨Wilson病 (Wilsondisease,WD)ATP7B基因突变的高频位点之一Thr 935Met与临床表现的关系。方法　应用PCR技术扩增 90例WD患者和 30例正常人ATP7B基因第 12外显子 ,其PCR产物行限制性内切酶TaiI酶切分析。结果　正常人组酶切未见异常。 90例WD组第 12外显子有 10例Thr 935Met杂合突变 ,未见纯合突变 ,检出率为 11 1%。突变组的平均发病年龄为 19 10岁± 8 5 2岁 ,无突变组的平均发病年龄为 14 0 0岁± 7 4 2岁 ,差异有显著意义 (P =0 0 4 7)。结论　Thr 935Met突变组患者的发病年龄迟于未见该点突变组的患者 ,而性别、首发症状及铜生化水平与该点突变无明显相关性。     

应用变性高效液相色谱技术进行肝豆状核变性的基因突变筛查及产前诊断

目的 探讨变性高效液相色谱(denature high performance liquid chromatography,DHPLC)技术在肝豆状核变性(Wilson's disease,WD)的突变筛查及产前诊断中的临床应用.方法 以6个WD家系中的患者及其父母的DNA为模板,采用PCR技术扩增ATP7B基因的21个外显子及5'非翻译区,PCR产物经DHPLC技术进行突变筛查,对峰型有改变者进行测序验证.在确定了先证者突变类型的基础上,采用相同方法对其中4个家系(1个双胎和3个单胎)进行产前诊断.结果 6例患者中检测出5种已知的致病突变及8种多态类型.患者的父母均为相应突变类型的携带者.产前诊断结果显示,两例妊娠为异常胎儿,其中1例双胎为Arg778Leu/IVS4-1G>C双重杂合子,1例单胎为Ser975Tyr/Pro992Leu双重杂合子,这两对妊娠夫妇选择了终止妊娠.另两例妊娠中,1例为Ser975Tyr杂合子,1例完全正常,他们选择了继续妊娠,出生了表型正常儿.结论 DHPLC在Wilson病的突变检测和产前诊断中有良好的应用前景.     

安徽汉族人肝豆状核变性基因突变的检测与分析

目的建立PCR-LIS-SSCP银染法,筛选WD基因外显子突变,并经DNA测序确定之。方法应用PCR-LIS-SSCP银染法,对34例WD患者及10例正常人的ATP7B基因第8、12外显子突变进行检测,并对部分突变样品DNA测序。结果安徽WD患者的WD基因第8外显子突变率为38.2%,第12外显子突变率为14.7%,是安徽汉人WD基因突变的相对热区。结论PCR-LIS-SSCP加DNA自动测序,是一种操作较便捷的基因突变检测方法,可用于临床检出WD先证者、杂合子及进行症状前诊断和产前诊断。     

高分辨熔解曲线技术在Wilson病致病基因突变鉴定中的应用

目的在一个肝豆状核变性(WD)家系中进行致病突变鉴定和产前基因诊断。方法酚-氯仿法提取外周全血或妊娠13周胎儿绒毛组织基因组DNA;利用PCR-Sanger测序技术对先证者ATP7B基因外显子及外显子/内含子衔接区序列进行突变分析;针对先证者携带的突变位点,应用聚合酶链反应(PCR)-高分辨熔解曲线(HRM)方法对先证者家系4名成员进行突变鉴定,并在此基础上为患儿母亲提供产前基因诊断。结果先证者ATP7B基因第8外显子存在纯合致病突变c.2333GT(p.R778L);该家系5名成员和4名群体正常样本分属于3种不同熔解曲线。HRM分析结果与测序结果一致,即:患儿本人为R778L的纯合子,其父母、姐姐和母亲产前诊断的胎儿均为突变杂合子,4名群体正常对照为纯合野生型。结论在一个WD家系中检测到ATP7B基因热点突变c.2333GT(p.R778L),并针对该突变建立了一种基于PCR-HRM技术的快速突变鉴定方法,成功为家系提供产前基因诊断。     

儿童肝豆状核变性临床表型及ATP7B基因突变关联性分析

目的了解汉族儿童肝豆状核变性（WD）患儿的临床表型与ATP7B基因突变的相关性。方法以2005年7月至2012年12月就诊于复旦大学附属儿科医院确诊WD患儿为研究对象,按起病部位分为肝病型和神经型,以临床表现分为临床型和亚临床型。采用PCR技术扩增ATPTB基因全部外显子并直接测序。提取临床表型和基因型的信息;分析两者之间的相关性。结果52个无亲缘关系家庭的53例WD患儿进行分析。肝脏损害52例（98．5％）。肝病型41例,神经型12例;临床型19例,亚临床型34例。①肝病型A¨升高明显;神经型24h尿铜水平、胆汁酸水平和K—F环阳性率均显著高于肝病型;亚临床型起病年龄、ALT或AST异常率显著高于临床型;24h尿铜水平、胆汁酸升高比例和K·F环阳性率显著低于临床型。②53例WD患儿ATP7B基因外显子序列分析发现致病性突变等位基因97个,突变频率为91．5％。纯合突变8例,复合杂合36例,杂合突变9例。错义突变23种,插入／缺失突变8种,无义突变2种,剪接突变3种。③错义突变与非错义突变,纯合突变与杂合突变患儿在起病年龄、K—F环阳性率、铜蓝蛋白水平、24h尿铜水平和ALT、AST异常率等方面差异无统计学意义。④3种高发突变P．Arg778Leu（35．O％,34／97）、P．Pr0992Leu（15／97,15．5％）和P．Ala874Val／Pro（5／97,5．2％）在临床型和亚临床型、肝病型和神经型问的分布差异无统计学意义。⑤纯合、错义和错义＋剪接突变在临床型和亚临床型、肝病型和神经型间的分布差异无统计学意义。结论WD患儿几乎均有肝脏受累,多以肝病表现起病。ATPTB常见突变为P．Arg778Leu、P．Pr0992Leu和P．Ala874Val,常见基因型和临床表型间未发现显著相关性。P．Ala874Val／Pro突变患儿起病年龄较低,剪接突变对血清铜蓝蛋白影响较小。     

Asymmetry of the internal connections of the striate cortex of the cat in the projection zone of the center of the field of vision

Studies were carried out on the organization of the internal connections of the striate cortex in cats in the projection zone of the center (0–5°) of the field of vision by microintophoretic application of horseradish peroxidase to electrophysiologically identified orientational columns. The area containing neurons showing retrograde labeling in most cases extended in the mediolateral direction. Labeled cells were located in the upper (II, III) and lower (V, VI) layers of the cortex, and the shapes and orientations of the areas containing labeled neurons in these layers coincided. Spatial asymmetry was detected in the distribution of labeled neurons relative to the orientational column studied. Labeled cells were located predominantly medial to the columns, regardless of the distance from the projection of the area centralis. Considering the visuotopical map of field 17, the asymmetry detected here provides evidence that neurons in orientational columns have more extensive connections with neurons of the peripheral part of the cortex. An asymmetrical distribution of “silent” zones around the receptive fields of neurons in orientational columns is suggested, and that these appear to receive influences from the periphery of the visual field. Laboratory of Visual Physiology and Laboratory of Central Nervous System Morphology, I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, 6 Makarov Bank, 199034 St. Petersburg, Russia. Translated from Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 82, No. 12, pp. 23–29, December, 1996.     

Effects of the lesion of the postcommissural part of the septum on the behavior of the rat.

The effects of the lesion of the postcommissural part of the septum on behavior of the rat has been studied. Results may be summarized as follows. An increase in the exploratory behavior in the open field which decreases rapidly; a decrease in the number of defecations in this test and a decrease in time leaving a dark environment for exploration. In the shuttle box test, no facilitation of the acquisition, but a permanent and quite significant increase in the intertrial activity has been found. We conclude that the lesions tend to decrease the emotivity of the subjects. An interpretation on the basis of the species -- specific defensive reactions explains the transitory and permanent effects of the lesions on the spontaneous activity.     

The development of the ligament of the head of the femur

The hip joints of 30 human male and female fetuses and stillborns between 20 mm and 350 mm crown-rump length were studied by light microscopy. The ligament of the head of the femur developed in situ as a condensation of mesenchyme at the end of the second month of intra-uterine life (IUL), and was vascularized by branches of acetabular vessels early in the fourth month. In the majority of fetuses older than 5.5 months IUL, vessels in the ligament passed a short way into the femoral head within cartilage canals, to supply a small region around the fovea capitis. The remainder of the head was supplied by vessels in canals from around the upper part of the neck. The ligament changed from predominantly cellular to fibrous during the last 4 months of IUL. This increase in strength suggested significant mechanical functions in utero: limitation of adduction-flexion and opposition to postero-superior dislocation were the most likely.     

Counterpointing the functional role of the forebrain and of the brainstem in the control of the sleep-waking system

This paper reviews the lifetime contributions of the author to the field of sleep-wakefulness (S-W), reinterprets results of the early studies, and suggests new conclusions and perspectives. Long-term cats with mesencephalic transection show behavioral/polygraphic rapid eye movement sleep (REMS), including the typical oculo-pupillary behavior, even when the section is performed in kittens prior to S-W maturation. REMS can be induced as a reflex. Typical non-rapid eye movement S (NREMS) is absent and full W/arousal is present only after a precollicular section. The isolated forebrain (IF) rostral to the transection exhibits all features of W/arousal and NREMS [with electroencephalographic (EEG) spindles and delta waves], arousal to olfactory stimuli, and including the appropriate oculo-pupillary behaviors. These features also mature normally after neonatal transection. REMS is absent from the IF. After deprivation there is NREMS pressure and rebound in the IF, but the decerebrate cat only shows pressure for REMS. Most IF reactions to pharmacologic agents are within expectations, except for the tolerance/withdrawal effects of long-term morphine use which are absent. In contrast, these effects are supported by the brainstem (i.e. seen in the decerebrate cat). In cats with ablation of the telencephalon, or diencephalic cats, delta waves are absent in the thalamus. EEG thalamic spindle waves are seen triggering S for only 4-5 days after ablation. Therefore, true NREMS is absent in chronic diencephalic cats although pre- and postsomniac behaviors persist. These animals are hyperactive and show a pronounced, permanent insomnia; however, a low dose of barbiturate triggers a dramatic REMS/atypical NREMS rebound. Cats without the thalamus (athalamic cats), initially show a dissociation between behavioral hyperactivity/insomnia and the neocortical EEG, which for 15-20 days exhibits only delta and slower oscillations. Fast, low-voltage W rhythms appear later on, first during REMS, but spindle waves and S postures are absent from the start, such that these cats also display only atypical NREMS. Athalamic cats also show barbiturate-sensitive insomnia. Cats with ablation of the frontal cortices or the caudate nuclei remain permanently hyperactive. They also show a mild, but significant hyposomnia, which is permanent in afrontal cats, but lasts for about a month in acaudates. The polygraphic/behavioral features of their S-W states remain normal. We conclude and propose that: (a) the control of the S-W system is highly complex and distributed, but is organized hierarchically in a well-defined rostro-caudal manner; the rostral-most or highest level (telencephalon), is the most functionally complex/adaptative and regulates the lower levels; the diencephalic/basal forebrain, or middle level, has a pivotal role in inducing switching between S and W and in coordinating the lowest (brainstem) and highest levels; (b) W can occur independently in both the forebrain and brainstem, but true NREMS- and REMS-generating mechanisms exist exclusively in the forebrain and brainstem, respectively; (c) forebrain and brainstem S-W processes can operate independently from each other and are preprogrammed at birth; this helps understanding normal and abnormal polygraphic/behavioral dissociations in humans and normal dissociations/splitting in aquatic mammals; (d) NREMS homeostasis is present in the IF, but only REMS pressure after deprivation persists in the decerebrate cat; (e) the thalamus engages in both NREMS and W; (f) insomnia in diencephalic cats is the result of an imbalance between antagonistic W- and S-promoting cellular groups in the ventral brain (normally modulated by the telencephalon); (g) the EEG waves, which are signature for each S-W state, appear to truly drive the concomitant behaviors, e.g. a hypothetical human IF could alternate between behavioral NREMS and W/arousal/awareness; (h) a role for REMS is to keep the individual sleeping at the end of the self-limiting NREMS periods. The need for accelerating research on telencephaling NREMS periods. The need for accelerating research on telencephalic S-W processes and downstream control of the lower S-W system levels is emphasized.     

Topical organization of the projections of neurons of the substantia nigra and the ventral field of the tegmentum of the midbrain to the head of the caudate nucleus of the cat

Neuroscience and Behavioral Physiology -     

Involvement of the striatum in the central regulation of the hormonal functions of the gonads

Studies reported here show that intrastriatal administration of corticoliberin to rats decreases the blood testosterone level. However, in conditions of chemical deficiency of dopaminergic transmission in the dorsal striatum induced by injection of 6-hydroxydopamine, the effect of this neurohormone did not appear. It is concluded that extrahypothalamic corticoliberin is involved in regulating the hormonal reproductive system acting via dopaminergic mechanisms. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 4, pp. 594–597, April, 1999.     

Ultrastructure of the endothelium of the drainage system of the eye

The endothelium of the ocular drainage system (Schlemm’s canal, collector tubules, and aqueous veins) in primary juvenile glaucoma undergoes degenerative dystrophic changes with compensatory hypertrophy and proliferation at the initial stages of the glaucomatous process and atrophy and desquamation at advanced and terminal stages. Progressive decrease in the pinocytous function of endotheliocytes, reduction of the protein-synthesizing and mitochondrial compartments of the cytoplasm, and formation of autophagosomes reflect the process of endotheliocyte degeneration in general. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 574–577, May, 2008