Ventral specification and perturbed boundary formation in the mouse midbrain in the absence of Hedgehog signaling.

Although Hedgehog (HH) signaling plays a critical role in patterning the ventral midbrain, its role in early midbrain specification is not known. We examined the midbrains of sonic hedgehog (Shh) and smoothened (Smo) mutant mice where HH signaling is respectively attenuated and eliminated. We show that some ventral (Evx1+) cell fates are specified in the Shh-/- mouse in a Ptc1- and Gli1-independent manner. HH-independent ventral midbrain induction was further confirmed by the presence of a Pax7-negative ventral midbrain territory in both Shh-/- and Smo-/- mice at and before embryonic day (E) 8.5. Midbrain signaling centers are severely disrupted in the Shh-/- mutant. Interestingly, dorsal markers are up-regulated (Wnt1, Gdf7, Pax7), down-regulated (Lfng), or otherwise altered (Zic1) in the Shh-/- midbrain. Together with the increased cell death seen specifically in Shh-/- dorsal midbrains (E8.5-E9), our results suggest specific regulation of dorsal patterning by SHH, rather than a simple deregulation due to its absence.     

Shh/Gli1信号参与小鼠脊髓损伤后血-脊髓屏障渗透性改变及运动功能恢复

目的:探讨sonic hedgehog/Gli1(Shh/Gli1)信号在小鼠脊髓损伤后病理变化及运动功能恢复中的作用。方法:成年雄性C57野生型、Gli1lz和Gli1lz/lz小鼠行T8节段脊髓夹伤及假手术。Gli1lz小鼠脊髓损伤及假手术后3 d行X-gal染色;7 d行Shh/PDGFr-α、Shh/GFAP、LacZ/GFAP染色,观察Shh/Gli1信号在小鼠脊髓损伤后的激活。C57野生型和Gli1lz/lz小鼠脊髓夹伤后7 d行GFAP染色和实时定量RT-PCR,观察星形胶质细胞反应;术后14 d尾静脉注射伊文氏兰观察血-脊髓屏障改变;术后1、3、5 d和7 d行BMS评分评价小鼠后肢运动功能。结果:脊髓损伤7 d后,损伤区Shh表达升高;Shh/Gli1信号报告基因LacZ表达升高,主要表达于反应性星形胶质细胞;免疫组织化学及实时定量RT-PCR结果显示Gli1基因敲除不影响脊髓损伤后GFAP表达;伊文氏兰染色及其定量分析显示Gli1lz/lz小鼠脊髓损伤后脊髓组织伊文氏兰外渗增加。BMS评分显示Gli1lz/lz小鼠运动功能恢复显著差于野生型小鼠。结论:Shh/Gli1信号在小鼠脊髓损伤后激活,可能参与脊髓损伤后血-脊髓屏障渗透性的改变,并影响脊髓损伤后的运动功能恢复。     

Frizzled10 mediates WNT1 and WNT3A signaling in the dorsal spinal cord of the developing chick embryo

Background: WNT1 and WNT3A drive a dorsal to ventral gradient of β‐catenin‐dependent Wnt signaling in the developing spinal cord. However, the identity of the receptors mediating downstream functions remains poorly understood. Results: In this report, we show that the spatiotemporal expression patterns of FZD10 and WNT1/WNT3A are highly correlated. We further show that in the presence of LRP6, FZD10 promotes WNT1 and WNT3A signaling using an 8xSuperTopFlash reporter assay. Consistent with a functional role for FZD10, we demonstrate that FZD10 is required for proliferation in the spinal cord. Finally, by using an in situ proximity ligation assay, we observe an interaction between FZD10 and WNT1 and WNT3A proteins. Conclusions: Together, our results identify FZD10 as a receptor for WNT1 and WNT3A in the developing chick spinal cord. Developmental Dynamics 243:833–843, 2014. © 2014 Wiley Periodicals, Inc.     

Reorganization of the ependyma during axolotl spinal cord regeneration: changes in intermediate filament and fibronectin expression.

Changes in intermediate filament content and extracellular matrix material showed that the injury response of ependymal cells in lesioned axolotl spinal cord involves an epithelial-to-mesenchymal transformation, and that fibrous astrocytes are excluded from the remodeling lesion site. Antibody localization was used to visualize cytokeratin-, vimentin-, and glial fibrillary acidic protein- (GFAP-) containing intermediate filaments, as well as the adhesive glycoprotein fibronectin. In normal axolotl spinal cord cytokeratins were found near the apical surface of the ependymal cells. Transmission electron microscopic examination suggested that these cytokeratins were in tonofilaments. Cytokeratin expression was lost and vimentin production was initiated in ependymal cells 2-3 weeks following spinal cord injury. There was a period of approximately 1-2 weeks when cytokeratins and vimentin were co-expressed in vivo. This co-expression was maintained in vitro by culture on a fibronectin-coated substratum. As the central canal reformed, vimentin expression was lost. Ependymal cells lacked GFAP intermediate filaments, but GFAP was present in fibrous astrocytes of the neuropil and white matter. Following injury, GFAP localization showed that fibrous astrocytes disappeared from the remodeling lesion site and reappeared only after the ependymal epithelium reformed and newly myelinated axons were found. Fibronectin expression closely followed the expression of vimentin during mesenchymal ependymal cell outgrowth. These results suggest that the ependymal cell outgrowth requires changes in cell shape followed by changes in production of extracellular matrix.     

Curcumin improves the recovery of motor function and reduces spinal cord edema in a rat acute spinal cord injury model by inhibiting the JAK/STAT signaling pathway

Curcumin, a yellow pigment extracted from Carcuma longa, has been demonstrated to have extensive pharmacological activity in various studies, and it exhibits protective effects on injuries involving a number of human organs. The present study was designed to evaluate the potential effect and underlying mechanism of curcumin on the motor function and spinal cord edema in a rat acute spinal cord injury (SCI) model. The SCI model was induced by a heavy object falling. At 30 min after the SCI was successfully induced, the animals were intraperitoneally given 40 mg/kg curcumin. The Basso, Beattie and Bresnahan scores showed that curcumin moderately improved the recovery of the motor function in the injured rats, and hematoxylin–eosin staining demonstrated the role of this compound in reducing the hemorrhage, edema and neutrophil infiltration of the traumatic spinal cord. Furthermore, curcumin also inhibited the SCI-associated aquaporin – 4 (AQP4) overexpression and glial fibrillary acidic protein (GFAP) and repressed the unusual activation of the JAK/STAT signaling pathway. In conclusion, our data demonstrate that curcumin exhibits a moderately protective effect on spinal cord injury, and this effect might be related to the inhibition of overexpressed AQP4 and GFAP and the activated JAK/STAT signaling pathway. Curcumin may have potential for use as a therapeutic option for spinal cord injuries.     

Signaling pathways regulating the expression of Prx1 and Prx2 in the chick mandibular mesenchyme

Prx1 and Prx2 are members of the aristaless‐related homeobox genes shown to play redundant but essential roles in morphogenesis of the mandibular processes. To gain insight into the signaling pathways that regulate expression of Prx genes in the mandibular mesenchyme, we used the chick as a model system. We examined the patterns of gene expression in the face and the roles of signals derived from the epithelium on the expression of Prx genes in the mandibular mesenchyme. Our results demonstrated stage‐dependent roles of mandibular epithelium on the expression of Prx in the mandibular mesenchyme and provide evidence for positive roles of members of the fibroblast and hedgehog families derived from mandibular epithelium on the expression of Prx genes in the mandibular mesenchyme. Our studies suggest that endothelin‐1 signaling derived from the mesenchyme is involved in restricting the expression of Prx2 to the medial mandibular mesenchyme. Developmental Dynamics 237:3115–3127, 2008. © 2008 Wiley‐Liss, Inc.     

RhoA和ROCK2在SOD1-G93A转基因小鼠脊髓中的表达

目的:通过研究Rho A和ROCK2在SOD1-G93A转基因小鼠脊髓内的表达变化以阐明Rho/ROCK信号通路在肌萎缩侧索硬化症(ALS)病程中的作用。方法:饲养SOD1-G93A转基因小鼠和同窝野生型小鼠至发病早期、中期和晚期,部分小鼠冰上剥离新鲜脊髓组织,利用RT-PCR方法检测Rho A和ROCK2 mRNA的表达,利用Western Blot方法检测Rho A和ROCK2蛋白的表达;部分小鼠行心脏灌注并剥离其脊髓组织制成冰冻切片,利用免疫组织化学染色方法检测Rho A和ROCK2蛋白的表达。结果:在SOD1-G93A鼠发病的早期、中期和晚期,转基因小鼠脊髓中Rho A和ROCK2的mRNA及蛋白表达均上调。免疫组织化学染色实验结果显示,野生型小鼠脊髓中Rho A和ROCK2弥散分布于胞质和突起中,阳性染色浅,SOD1-G93A转基因小鼠脊髓中Rho A和ROCK2阳性染色深,大量聚集在细胞膜及细胞质。结论:Rho A和ROCK2在SOD1-G93A转基因小鼠脊髓中异常高水平表达与ALS脊髓区病变密切相关,可能参与ALS疾病进程。     

Comparative transcriptome analysis of the calcium signaling and expression analysis of sodium/calcium exchanger in Aspergillus cristatus

Aspergillus cristatus develops into various stages under different Na concentrations: the sexual stage in 0.5 M NaCl and asexual development stage in 3 M NaCl. In order to explore whether the Ca²⁺ signaling pathway in A. cristatus responded to the changes in the salt stress, we analyzed the gene expression levels in A. cristatus respectively cultured in 0.5 M NaCl and 3 M NaCl. According to the BLAST analysis results, we identified 25 Ca²⁺‐signaling proteins in A. cristatus. The expression levels of most genes involved in the Ca²⁺‐signaling pathway in A. cristatus cultured in different salt concentrations showed significant differences, indicating that the Ca²⁺ signaling pathway was involved in the response to the changes in the salt stress. In yeasts, only calcium ion influx proteins were reported to be involved in the response to the changes in the salt stress. So far, the protein for the exchanger of calcium/sodium ions has not been reported. Therefore, we obtained the sodium/calcium exchanger (termed NCX) proteins from the KEGG Database. The ncx gene of A. cristatus was cloned and characterized. The full length of ncx gene is 3055 bp, including a 2994‐bp open reading frame encoding 994 amino acids. The expression levels of ncx in the sexual development stage and asexual development stage were respectively ～8.94 times and ～2.57 times of that in the hyphal formation stage. Therefore, we suggested that ncx gene was up‐regulated to resist the sodium stress. The study results provide the basis for further exploring the Ca²⁺‐signaling mechanism and ion exchanger mechanism.     

葛根素对臂丛神经根性撕脱伤脊髓前角iNOS、CGRP蛋白表达及PI3K/Akt信号通路的影响

目的　探讨葛根素对臂丛神经根性撕脱伤（brachial plexus root avulsion injury,BPRAI）脊髓前角iNOS、CGRP蛋白表达及PI3K/Akt信号通路的影响。 方法　将50只雄性SD大鼠随机分为正常组、模型组、葛根素低、中、高剂量治疗组,每组10只。模型组,葛根素低、中、高剂量治疗组进行BPRAI造模,撕脱大鼠右侧C5~7脊神经前根,后根剪断,术后3个治疗组予腹腔注射葛根素,剂量分别为50、100、200 mg·kg-1·d-1,正常组、模型组腹腔注射等体积生理盐水,持续4周。采用尼氏染色、免疫荧光化学、Western blot方法,观察损伤侧脊髓前角α运动神经元（alpha motorneurons,α-MNs）的存活率,iNOS、CGRP、PI3K/Akt通路相关蛋白的表达。 结果　第4周时,低、中、高剂量的葛根素治疗可抑制α-MNs丢失（P<0.05或P<0.01）;中、高剂量的葛根素治疗可抑制iNOS表达（P<0.05）;高剂量的葛根素治疗可促进CGRP蛋白表达（P<0.05或P<0.01）;低、中、高剂量葛根素均可显著抑制p-Akt1/2/3表达（P<0.01）。 结论　葛根素可改善BPRAI造模引起的α-MNs死亡,其机制可能与葛根素能抑制iNOS蛋白的表达、促进CGRP蛋白的表达有关,并且PI3K/Akt信号通路参与其调控。     

葛根素对臂丛神经根性撕脱伤脊髓前角iNOS、CGRP蛋白表达及PI3K/Akt信号通路的影响

目的　探讨葛根素对臂丛神经根性撕脱伤（brachial plexus root avulsion injury,BPRAI）脊髓前角iNOS、CGRP蛋白表达及PI3K/Akt信号通路的影响。 方法　将50只雄性SD大鼠随机分为正常组、模型组、葛根素低、中、高剂量治疗组,每组10只。模型组,葛根素低、中、高剂量治疗组进行BPRAI造模,撕脱大鼠右侧C5~7脊神经前根,后根剪断,术后3个治疗组予腹腔注射葛根素,剂量分别为50、100、200 mg·kg-1·d-1,正常组、模型组腹腔注射等体积生理盐水,持续4周。采用尼氏染色、免疫荧光化学、Western blot方法,观察损伤侧脊髓前角α运动神经元（alpha motorneurons,α-MNs）的存活率,iNOS、CGRP、PI3K/Akt通路相关蛋白的表达。 结果　第4周时,低、中、高剂量的葛根素治疗可抑制α-MNs丢失（P<0.05或P<0.01）;中、高剂量的葛根素治疗可抑制iNOS表达（P<0.05）;高剂量的葛根素治疗可促进CGRP蛋白表达（P<0.05或P<0.01）;低、中、高剂量葛根素均可显著抑制p-Akt1/2/3表达（P<0.01）。 结论　葛根素可改善BPRAI造模引起的α-MNs死亡,其机制可能与葛根素能抑制iNOS蛋白的表达、促进CGRP蛋白的表达有关,并且PI3K/Akt信号通路参与其调控。     

Gene expression and association analysis of the epithelial membrane protein 1 gene in major depressive disorder in the Japanese population

The epithelial membrane protein 1 (EMP1) plays a role in neuronal differentiation and neurite outgrowth, which are involved in the pathogenesis of major depressive disorder (MDD). We sought to determine whether the EMP1 gene is implicated in MDD. We determined the mRNA expression levels of the EMP1 gene in peripheral-blood leukocytes of patients and control subjects (n=27 each). Next, we performed case-control association analyses (MDD, n=182; controls, n=350) in the Japanese population. The level of expression of the EMP1 mRNA was significantly lower in medication-free patients compared with control subjects (P<0.001). The association analysis revealed an absence of association between the polymorphisms studied and MDD, whereas a gender-specific association was observed between male controls and male patients for marker rs7315725 (permutation P=0.039). Our results suggest that the EMP1 gene may be implicated in the pathophysiology of MDD in the Japanese population.     

The development of the dendritic organization of primary and secondary motoneurons in the spinal cord of Xenopus laevis. An HRP study

Summary During embryonic and larval development of the clawed toad, Xenopus laevis, two different populations of motoneurons appear in the spinal cord. In this study the development of primary motoneurons which innervate the axial musculature (used during embryonic locomotion) and of secondary motoneurons which innervate the extremity musculature (used for locomotion during metamorphosis and thereafter) was analyzed with horseradish peroxidase (HRP) as a neuronal marker. After application of HRP to the axial musculature (rostral five postotic myotomes) the first labeled primary motoneurons were found at stage 24/25. During development gradually more labeled neurons were observed. These primary motoneurons send their dendrites into the marginal zone (white matter). At first only dorsal and lateral dendrites develop (stages 25–33), followed by ventral dendrites (stage 37/38). Up till stage 48 the developing dendrites extend throughout the marginal zone. Hereafter the marginal zone increases particularly at the dorsolateral edge, a development which is not followed by the dendrites of the primary motoneurons. The dendrites of mature primary motoneurons (stages 58–62) occupy the ventral and ventrolateral parts of the marginal zone.At stage 48, shortly after the hindlimb bud arises (stage 46, early metamorphosis), the first neurons related to this developing extremity could be labeled in the ventrolateral part of the lumbar spinal cord. At first these secondary motoneurons bear only a few dorsal dendrites of which only the tips reache out in the adjacent white matter. Already at stage 50 these dorsal dendrites have invaded the whole dorsolateral part of the marginal zone. Also the first ventral dendrites were observed at this stage. Later, at stage 53/54 also some ventral dendrites have reached the white matter together with a few lateral dendrites. At these early metamorphic stages already some primary afferent fibers were found making contact with the dorsomedial dendrites. At stage 58 for the first time recurrent axon collaterals were found, which extend into the ventromedial part of the marginal zone. The development of motoneurons in the spinal cord seems to be characterized by two phases: (1) establishment of contacts between motoneurons and target muscles, and (2) subsequent formation of connections of these motoneurons with other nerve cells within the central nervous system. The dendrites of primary motoneurons follow the development of the marginal zone, while dendrites of secondary motoneurons develop into an already well developed marginal zone. Generally, the dendrites of mature motoneurons of the axial musculature were observed in the ventromedial and ventrolateral parts of the marginal zone. The dendrites of themotoneurons which innervate the musculature of the hindlimbs were observed predominantly in the dorsolateral and ventromedial parts of the marginal zone.     

缺失半胱氨酸富集区的HIV-1 HXB2株Tat蛋白在大肠杆菌中的高效表达及免疫原性分析

目的 删除人免疫缺陷病毒Ⅰ型(HIV-1) HXB2株Tat蛋白(长度101个氨基酸)的半胱氨酸富集区(22～37位氨基酸)以提高其在大肠杆菌中的表达量和分子稳定性,并分析缺失半胱氨酸富集区后的Tat蛋白[Tat(△C)蛋白]免疫原性的改变.方法 应用PCR方法对Tat基因的半胱氨酸富集区(64～111位核苷酸)进行缺失突变,获得其突变体序列[Tat(△C)DNA] ,并构建其重组表达质粒pET-32a-Tat(△C).相同的条件下将pET-32a-Tat(△C)和pET-32a-Tat质粒转入大肠杆菌BL21(DE3)中进行诱导表达及纯化.用pET-32a-Tat(△C)融合蛋白免疫家兔制备抗血清,ELISA和Western blot方法对抗血清进行免疫原性分析.结果 pET-32a-Tat(△C)蛋白在大肠杆菌中的表达水平(7.12 mg/ml)明显高于pET-32a-Tat蛋白(1.50 mg/ml);pET-32a-Tat蛋白在表达纯化后及25℃和4℃放置7 d后均有明显二聚体的产生,而pET-32a-Tat(△C)蛋白则未形成二聚体;pET-32a-Tat(△C)蛋白免疫家兔可诱导产生高滴度的抗体(1∶320 000),该抗体与Tat(△C)蛋白、Tat蛋白(1～101 AA)及化学合成Tat(1～86 AA)蛋白均呈特异性反应.结论 缺失HIV-1 Tat蛋白的半胱氨酸富集区可明显提高其原核表达水平和蛋白的稳定性并较好地保留其免疫原性,为HIV-1 Tat疫苗研究提供了一种新型免疫原.     

HDM2 overexpression and focal loss of p14/ARF expression may deregulate the p53 tumour suppressor pathway in meningeal haemangiopericytomas. Study by double immunofluorescence and laser scanning confocal microscopy

AIMS: To investigate the p53 pathway in meningeal haemangiopericytomas (MHPCs), p14/ARF, p53 protein expression and two wild-type (wt) p53-induced proteins (HDM2 and p21/WAF1) were studied in 18 MHPCs, 11 primary, four of them recurrent on one, one, two and four occasions. METHODS: Immunohistochemical detection of p14/ARF, p53, p21/WAF1, HDM2 and Ki67 proliferative index (PI) protein expression. RESULTS: Ki67 index was > 5% in eight out 18 cases (44.4%). The PI in recurrent cases increased with neoplastic progression. Simultaneous p53 and wt p53 transactivated gene (p21/WAF, HDM2) expression occurred in all cases. This argues against p53 mutation. HDM2 overexpression was observed in 10 cases (55.5%). Double-immunofluorescence staining and laser scanning confocal microscopy (LSCM) displayed HDM2 and p53 colocalization. This strongly suggests that HDM2 binds and inactivates p53 that could be pathogenic for MHPCs, by a different mechanism than point mutation. p14/ARF expression > 5% was observed in 12 cases (66.6%). A normal (diffuse) pattern of expression was seen in 13 cases (72.2%). Focal loss of expression was observed in five patients (27.7%): three primary cases and two recurrences. Therefore, p14/ARF down-regulation may also contribute to the development of MHPC. CONCLUSION: HDM2 overexpression, sometimes combined with focal loss of p14/ARF expression, may play a pathogenic role in MHPCs.