Gap junction proteins exhibit early and specific expression during intramembranous bone formation in the developing chick mandible

The spatial and temporal expression of three closely related members of the connexin family of gap junction proteins (connexin42, Cx42; connexin43, Cx43; and connexin45, Cx45) was evaluated during bone formation in the mandibular process of the chick embryo. Mandibles of chick embryos from Hamburger and Hamilton stage 25 (approximately 5 days) through 19 days of development were dissected, serially sectioned and processed for immunocytochemical localization, employing site-specific anti-connexin antibodies. Our data revealed that (1) Cx43 was present throughout mandibular bone formation; (2) although it appeared to be associated with all bone cell types, Cx43 was concentrated in mesenchymal cells during the earliest stages in the osteogenic lineage; (3) most importantly, the localization of Cx43 at sites of bone formation appeared to precede the overt expression of the osteogenic phenotype; (4) by contrast, Cx45 was more restricted, spatially and temporally, in its distribution; (5) Cx42 expression was not detected in osteogenic tissue during mandibular bone formation. From all of the data obtained, Cx45 appeared to be associated with stages of bone formation characterized by the elaboration of matrix and the progressive expression of the differentiated osteogenic phenotype. Cx43 appeared to be associated with condensation of mesenchyme and the earliest stages of osteogenesis. Because of these associations, we propose that connexin expression may be necessary for the initiation of bone formation and the full expression of the osteogenic phenotype.     

Irregular connexin43 expressed in a rare cardiac hamartoma containing adipose tissue in the crista terminalis

Cardiac hamartomas are very rare and are demarcated masses of enlarged, hypertrophied, mature myocytes and collagen tissue. Cardiac hamartomas are generally circumscribed in the right ventricle or atrium, but not reported in the crista terminalis (CRT). The CRT is crucial in electrophysiology, is related to arrhythmogenesis, and is targeted by radiofrequency catheter procedures. Previous works only described the benign natures of prominent CRT using non-invasive methods. This study describes an unusual cardiac hamartoma originating from the CRT and extending toward the tricuspid valve. Microscopically, this hamartoma comprised dense collagen and adipose tissue, mixed with hypertrophy, but with disarrayed cardiomyocytes. An irregular gap junction, connexin43, was demonstrated in this cardiac hamartoma.     

成人与新生儿心脏连接蛋白43表达差异

目的　探讨成人与新生儿心脏连接蛋白 4 3(Cx4 3)表达差异。　方法　应用SP免疫组织化学和图像分析方法 ,观测成人与新生儿心脏Cx4 3的蛋白表达。　结果　 1 新生儿心脏Cx4 3在心房和心室均呈斑点状遍布于心肌细胞侧面连接处和细胞质内 ,闰盘处极少。 2 成人Cx4 3表达在心房肌非均质分布于细胞侧面连接处和端闰盘处 ;心室肌典型地排列在闰盘处。 3 图像分析表明 ,心肌细胞Cx4 3分布密度 ,新生儿心房 <心室 ,成人心房 >心室。成人心房、心室均低于新生儿。　结论　新生儿Cx4 3主要分布于心肌细胞侧连接处 ,成人心房和心室存在差异。Cx4 3分布密度新生儿心房 <心室 ,成人心房 >心室 ;成人心脏低于新生儿。提示 ,Cx4 3有增龄变化。     

Cx43在造血调控及重建中的作用

目的:采用条件性基因敲除技术构建造血系统间隙连接蛋白43(Cx43)基因敲除(Cx43~(-/-))小鼠模型,并探讨Cx43在维持造血细胞自我更新及功能稳定中的作用。方法:将引进的2对转基因小鼠Cx43 loxP/loxP和Lyz-Cre/+杂交,选取F1雌性子代Cx43 loxP/-_Lyz-Cre/+与雄性Cx43 loxP/loxP合笼回配,提取所获得子代小鼠鼠尾组织基因组DNA,采用PCR方法鉴定小鼠基因型,RT-PCR方法筛选Cx43~(-/-)小鼠,同时分析小鼠不同器官中Cx43基因的表达差异;该类小鼠经5-氟尿嘧啶(5-FU;125 mg/kg)处理,在化疗前及化疗后第5、10和15天经眼球取血分析其血象变化。Cx43~(-/-)及Cx43~(+/+)小鼠予7.5 Gy(~(60)Co-γ)的致死量照射,剂量率1 Gy/min,照射后6 h分别给予事先准备就序的骨髓细胞,每只3×10~6细胞于尾静脉注入,2周后处死小鼠检测造血是否重建:分离股骨切片后,收集骨髓细胞进行细胞表型分析(选用的单抗为CD45R、Gr-1、CD4、 CD8a、TCRαβ、Mac-1、抗sIgM、TER119、Sca-1及CD117);同时进行体外造血细胞集落实验观察造血细胞的体外增殖能力。结果:本研究通过2种转基因小鼠间杂交和回交,成功获得造血系统选择性Cx43基因敲除小鼠;该类小鼠骨髓及外周血细胞无Cx43表达,参与造血的组织,如肝脏和脾脏中Cx43表达也显著下调(P0.01),而心脏和肾脏的Cx43表达则无影响,小鼠成年后外周血象分析并无明显异常,但应急代偿能力下降,经5-FU处理后,其造血功能恢复显著减缓,处理15 d后,Cx43~(+/+)小鼠造血功能已接近正常水平,而Cx43~(-/-)小鼠仍无明显的恢复迹象,血红蛋白、白细胞及血小板仍处低位,2者差别有统计学显著性(P0.01);体外集落试验也证实Cx43~(-/-)小鼠造血干/祖细胞的增殖能力下降,其CFU-GM或CFU-E集落数均明显少于Cx43~(+/+)小鼠(P0.01),但流式细胞术结果显示,Cx43~(-/-)小鼠骨髓中Lin~-/c-Kit~+/Sca-1~+细胞亚群数量与Cx43~(+/+)小鼠相比差异并无统计学显著性;Cx43~(-/-)小鼠在化疗或移植后其骨髓造血功能重建均延迟,且化疗15 d后骨髓切片及涂片均证实其骨髓中造血细胞增生程度明显降低,脂肪组织显著增多,而且T、B细胞发育也有异常。此外,其外周血中CD4~+CD8~+细胞比例比野生型小鼠增多(P0.05),但CD4~+T细胞显著减少(P0.01),尤其是TCRαβ亚群细胞减少最为明显(P0.01)。同样,Cx43~(-/-)小鼠外周血中CD45R~+sIgM~-细胞亚群比例与野生型小鼠相比显著减少(P0.01)。结论:骨髓中Cx43基因表达在造血干/祖细胞发育(尤其是应急状态时)具重要作用,敲除Cx43基因后造血干/祖细胞增殖减缓,造血及免疫重建功能受损。     

Cx40和Cx43调节大鼠肠系膜上动脉收缩反应性与钙敏感性的机制

目的：研究连接蛋白Cx40/Cx43对大鼠肠系膜上动脉内膜依赖的血管收缩反应性与钙敏感性的调节作用机制。方法:以SD大鼠肠系膜上动脉（SMA）为研究对象，用Cx40或Cx43反义寡脱氧核苷酸（Cx40/Cx43AODN）阻断SMA Cx40或Cx43表达，观察缺氧处理后SMA的收缩反应性、钙敏感性、肌球蛋白轻链磷酸酶/激酶（MLCP/MLCK)的活性、20 kD的肌球蛋白轻链（MLC₂₀）磷酸化程度的变化。结果:Cx40AODN可以降低正常组、1 h和3 h缺氧组SMA MLCP活性，增加MLC₂₀磷酸化水平，改善血管的钙敏感性和内膜依赖的收缩反应性；Cx43AODN可以增加各组血管的MLCP活性，减少MLC₂₀磷酸化水平，降低血管的钙敏感性和内膜依赖的收缩反应性。Cx40和Cx43AODN对SMA MLCK活性无明显作用。结论:Cx40和Cx43主要通过调节血管平滑肌细胞的MLCP活性和MLC₂₀磷酸化水平调节血管的钙敏感性，从而调节休克后内膜依赖的血管收缩反应性。     

GJB2: the spectrum of deafness-causing allele variants and their phenotype

Genetic testing was completed on 1,294 persons with deafness referred to the Molecular Otolaryngology Research Laboratories to establish a diagnosis of DFNB1. Exon 2 of GJB2 was screened for coding sequence allele variants by denaturing high-performance liquid chromatography (DHPLC) complemented by bidirectional sequencing. If two deafness-causing mutations of GJB2 (encoding Connexin 26) were identified, further screening was not performed. If only a single deafness-causing mutation was identified, we screened for the g.1777179_2085947del (hereafter called del(GJB6-D13S1830); GenBank NT_024524.13) and mutations in the noncoding region of GJB2. Phenotype-genotype correlations were evaluated by categorizing mutations as either protein truncating or nontruncating. A total of 205 persons carried two GJB2 exon 2 mutations and were diagnosed as having DFNB1; 100 persons carried only a single deafness-causing allele variant of exon 2. A total of 37 of these persons were c.35delG carriers, and 51 carried other allele variants of GJB2. Persons diagnosed with DFNB1 segregating two truncating/nonsense mutations had a more severe phenotype than persons carrying two missense mutations, with mean hearing impairments being 88 and 37%, respectively (P < 0.05). The number of deaf c.35delG carriers was greater than expected when compared to the c.35delG carrier frequency in normal-hearing controls (P < 0.05), suggesting the existence of at least one other mutation outside the GJB2 coding region that does not complement GJB2 deafness-causing allele variants.     

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3:5-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g_j) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas g_j was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g_j in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (_j) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the _j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [³²P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [³²P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g_j, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.     

三磷酸腺苷对人横纹肌肉瘤细胞系诱导分化作用的研究

为了探讨三磷酸腺苷（ＡＴＰ０对人横纹肌肉瘤细胞增殖和分化的影响，用ＡＴＰ作用于人横纹肌肉瘤细胞亚系（ＲＤＬ６）细胞，观察到ＡＴＰ可抑制ＲＤＬ６细胞的增殖，使其生长速度明显减慢，作用第５ｄ时增殖抑制率为８１％，流式光度术检测；观察到ＡＴＰ ＲＤＬ６细胞Ｓ期的细胞数明显增多，说明细胞停滞在Ｓ期，用罗氏黄荧光染料传法实，ＡＴ家恢复ＲＤＬ６细胞间隙加接通讯功能的作用。用 光细胞化学方法观察到经ＡＴＰ处理后     

中国东北汉族一个先天性白内障家系致病基因的鉴定

目的鉴定一个先天性白内障家系的致病基因。方法根据已知与先天性白内障有关的12个致病基因的染色体上的定位,分别选取3～4个的微卫星标记位点,对该家系进行连锁分析。通过测序鉴定致病基因。结果在1q21.1GJA8位点显示最大Lod值2.44。致病基因定位于1q21.1区的GJA8基因,构成缝隙连接的缝隙连接蛋白Connexin50。DNA序列分析鉴定显示其第2外显子的第191个碱基杂合突变T>G导致其蛋白产物第64位缬氨酸转变为甘氨酸。结论Connexin50的V64G新生突变是导致该家系的致病原因。     

大鼠骨髓间质干细胞体外诱导分化为心肌样细胞Cx43通讯连接功能检测

目的： 探讨大鼠骨髓间充质干细胞（MSCs）体外诱导分化为心肌样细胞Cx43的分布与通讯连接功能状态。方法： 取健康SD大鼠骨髓，用5-氮杂胞苷体外诱导培养。取诱导培养2、3、4周的MSCs为Ⅰ、Ⅱ、Ⅲ组，另取急性分离的心肌细胞为对照组，用激光共聚焦技术检测Cx43的分布及平均荧光漂白恢复率。结果： 对Cx43在细胞内分布检测发现，随诱导培养时间的延长，细胞内蛋白颗粒密度逐渐增加；诱导培养4周后MSCs内蛋白颗粒密度与对照组比较无显著差异（63.87±12.43，64.87±12.15，P>0.05）。各组细胞平均荧光漂白率的变化趋势与Cx43分布变化相似，即随诱导培养时间的延长，细胞平均荧光漂白率逐渐增加；Ⅰ、Ⅱ、Ⅲ组及对照组分别为19.59%±6.08%、37.17%±3.84%、46.82%±2.69%、49.71%±5.53%，Ⅲ组与对照组比较无显著差异（P>0.05）。结论： 大鼠MSCs在体外诱导培养4周后已分化为心肌样细胞，其细胞Cx43的分布与通讯连接功能与正常心肌细胞相似。