Noncoordinate expression of M band proteins in slow and fast embryonic chick muscles

The expression of the myosin-associated M band proteins myomesin and M protein in differentiating muscle fibers in the anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) muscles during embryonic chicken development was examined by immunocytochemistry using monoclonal antibodies. Early in the embryonic development of both muscles, both myomesin and M protein are expressed in primary and secondary myotubes. However, beginning at 10 days in ovo, M protein is gradually suppressed first in primary, then in secondary, presumptive slow-tonic type 3 fibers. M protein is transiently suppressed in presumptive fast-twitch type 2 fibers derived from primary myotubes but continuously expressed in those derived from secondary myotubes. Thus, initially all myotubes have a common intrinsic M band composition with respect to myomesin and M protein, whereas at later stages the expression of M protein is fiber-type specific. Intrafusal spindle fibers, which are segregated from extrafusal fibers around 14 days in ovo, have a heterogeneous M band composition atypical of extrafusal fibers.     

Developmental changes in KCC1, KCC2, and NKCC1 mRNA expressions in the rat brain

We investigated the expressions of KCC1, KCC2 and NKCC1 mRNAs in the developing rat brain. The neuroepithelium showed abundant KCC1 and NKCC1 mRNA expressions, while KCC2 mRNA was not detected there. In contrast, KCC2 mRNA was preferentially expressed in postmitotic mature neurons. These results suggest that the appearance of KCC2 expression mainly depends on the maturation of individual neurons.     

Nurr1在MN9D细胞中的过表达及其对酪氨酸羟化酶的影响

目的 :观察MN9D细胞中Nurr1的表达和分布及Nurr1过表达对酪氨酸羟化酶的影响。 方法 :应用高压液相色谱、免疫细胞化学、转基因、Westernblot和Northernblot等方法检测了MN9D细胞中多巴胺及其代谢物的水平和Nurr1蛋白在细胞中的分布 ,建立了过表达Nurr1的细胞株并观察了Nurr1过表达对TH基因的影响。结果 :MN9D细胞裂解液中含有大量多巴胺及其代谢物 ;Nurr1蛋白主要分布在MN9D细胞的胞质中 ,尤以核周最多 ;MN9D细胞中有Nurr1的mRNA存在和蛋白质的表达 ;过表达Nurr1的MN9D细胞A1株酪氨酸羟化酶表达增高。结论 :Nurr1与多巴胺能神经元发育有关 ,有可能用于帕金森病的基因治疗。     

Id1, Id2, and Id3 gene expression in neural cells during development

Id1, Id2, and Id3 mRNA are expressed mainly in the proliferating ependymal cell zone of the mouse brain during embryogenesis. In this study, the expression pattern and cell phenotypes of the Id family mRNA were examined in postnatal and adult rat brain. The expression of Id1 and Id3 mRNA in rat brain was observed in the cortex layer 1, corpus callosum, ventricular/ subventricular zone (VZ/ SVZ), and the CA1-4 layers of the hippocampus at postnatal day 1 (P1) through P14, whereby it declined at 2 months. In general, the developmental pattern of Id1 mRNA coincided with the pattern observed for Id3 mRNA. Similar to Id1 and Id3, Id2 mRNA was highly expressed in the corpus callosum, VZ/ SVZ, and the hippocampus. Examination of Id2 mRNA revealed high levels in the cortex and caudate putamen at P1 through P14, whereas a decline was observed in its expression in the adult cortex. In P5 rat cerebellum, all Id mRNA examined were found in the internal granular cell layers; however, at this time point, only Id2 mRNA expression was detected in the differentiating zone of the external granular cell layers, preferentially localizing to adult Purkinje cells. Furthermore, only Id2 mRNA expression in brain was observed in NF+ neurons at P5. Examination of S100α+ and GFAP+ astrocytes, revealed the presence of all three mRNAs, whereas the expression of Id2 and Id3 mRNA was absent in O4+ immature oligodendrocytes. These data suggest that the spatial and temporal kinetic patterns during development, as well as cellular specificity, of the Id gene family may play a critical role in neural precursor cell proliferation and cell divergence. GLIA 24:372–381, 1998. © 1998 Wiley-Liss, Inc.     

Inflammation of peripheral tissues and injury to peripheral nerves induce differing effects in the expression of the calcium‐sensitive N‐arachydonoylethanolamine‐synthesizing enzyme and related molecules in rat primary sensory neurons

Elevation of intracellular Ca²⁺ concentration induces the synthesis of N‐arachydonoylethanolamine (anandamide) in a subpopulation of primary sensory neurons. N‐acylphosphatidylethanolamine phospholipase D (NAPE‐PLD) is the only known enzyme that synthesizes anandamide in a Ca²⁺‐dependent manner. NAPE‐PLD mRNA as well as anandamide's main targets, the excitatory transient receptor potential vanilloid type 1 ion channel (TRPV1), the inhibitory cannabinoid type 1 (CB1) receptor, and the main anandamide‐hydrolyzing enzyme fatty acid amide hydrolase (FAAH), are all expressed by subpopulations of nociceptive primary sensory neurons. Thus, NAPE‐PLD, TRPV1, the CB1 receptor, and FAAH could form an autocrine signaling system that could shape the activity of a major subpopulation of nociceptive primary sensory neurons, contributing to the development of pain. Although the expression patterns of TRPV1, the CB1 receptor, and FAAH have been comprehensively elucidated, little is known about NAPE‐PLD expression in primary sensory neurons under physiological and pathological conditions. This study shows that NAPE‐PLD is expressed by about one‐third of primary sensory neurons, the overwhelming majority of which also express nociceptive markers as well as the CB1 receptor, TRPV1, and FAAH. Inflammation of peripheral tissues and injury to peripheral nerves induce differing but concerted changes in the expression pattern of NAPE‐PLD, the CB1 receptor, TRPV1, and FAAH. Together these data indicate the existence of the anatomical basis for an autocrine signaling system in a major proportion of nociceptive primary sensory neurons and that alterations in that autocrine signaling by peripheral pathologies could contribute to the development of both inflammatory and neuropathic pain.     

Localization of mRNA for SHIP2, SH2 domain-containing inositol polyphosphate 5-phosphatase, in the brain of developing and mature rats

The localization of mRNA for SHIP2, SH2 domain containing inositol 5-phosphatase SHIP isozyme, was examined by in situ hybridization histochemistry in the brain of developing and mature rats. SHIP2 mRNA was first detected in the ventricular germinal zone at embryonic stages. As the postnatal development proceeded, the expression signal was evident in cell of the white matters, presumptive oligodendrocytes, and no significant expression was seen in neurons throughout the development.     

在急性局灶性脑缺血早期VEGF、FLT-1、FLK-1 mRNA的表达及作用研究

目的 检测血管内皮生长因子（VEGF）及其受体（FLT-1，FLK-1）mRNA在急性局灶性脑缺血中的表达，并探讨血管内皮生长因子在急性局灶性脑缺血中的作用。方法 建立SD大鼠永久大脑中动脉闭塞（MCAO）模型。应用原位杂交技术检测血管内皮生长因子及其受体基因在局灶性脑缺血后不同时间点的表达。结果 原位杂交显示VEGF及FLT-1，FLK-1mRNA在局灶性脑缺血后3h即开始表达增强。VEGFmRNA和FLT-1，FLK-1mRNA于48h达高峰，后VEGFmRNA逐渐下降。至14d恢复到对照水平。而FLT-1，FLK-1mRNA在达高峰后，下降更加缓慢，约在21d时达对照水平，VEGFmRNA主要在缺血半影区神经元表达，同时在胶质细胞和血管内皮细胞表达，而FLT-1，FLK-1mRNA主要在缺血周边血管内皮细胞表达，但也在神经元表达。在缺血后48h半影区周边出现血管增生。并逐渐向中心区延伸，于缺血后7d达高峰，整个半影区可见明显敌国管增生。结论 急性半影区周边出现血管增生，并逐渐向中心区延伸，于缺血后7d达高峰，整个半影区可见明显血管增生。结论 急性局灶性脑缺血早期VEGFmRNA及FLT-1，FLK-1mRNA在神经细胞。胶质细胞，血管内皮细胞均有表达，可促进缺血半影区的血管增生。对改善急性局灶性脑缺血早期缺血半影区血供有重要作用。     

Expression of CHL1 and L1 by neurons and glia following sciatic nerve and dorsal root injury

Cell adhesion molecules (CAMs), particularly L1, are important for axonal growth on Schwann cells in vitro. We have used in situ hybridization to study the expression of mRNAs for L1 and its close homologue CHL1, by neurons regenerating their axons in vivo, and have compared CAM expression with that of GAP-43. Adult rat sciatic nerves were crushed (allowing functional regeneration), or cut and ligated to maintain axonal sprouting but prevent reconnection with targets. In other animals lumbar dorsal roots were transected to produce slow regeneration of the central axons of sensory neurons. In unoperated animals L1 and CHL1 mRNAs were expressed at moderate levels by small- to medium-sized sensory neurons and L1 mRNA was expressed at moderate levels by motor neurons. Many large sensory neurons expressed neither L1 nor CHL1 mRNAs and motor neurons expressed little or no CHL1 mRNA. Neither motor nor sensory neurons showed any obvious upregulation of L1 mRNA after axotomy. Increased CHL1 mRNA was found in motor neurons and small- to medium-sized sensory neurons 3 days to 2 weeks following sciatic nerve crush, declining toward control levels by 5 weeks when regeneration was complete. Cut and ligation injuries caused a prolonged upregulation of CHL1 mRNA (and GAP-43 mRNA), indicating that reconnection with target tissues may be required to signal the return to control levels. Large sensory neurons did not upregulate CHL1 mRNA after axotomy and thus regenerated within the sciatic nerve without producing CHL1 or L1. Dorsal root injuries caused a modest, slow upregulation of CHL1 mRNA by some sensory neurons. CHL1 mRNA was also upregulated by many presumptive Schwann cells in injured nerves and by some satellite cells around large sensory neurons after sciatic nerve injuries and was transiently upregulated by some astrocytes in the degenerating dorsal columns after dorsal rhizotomy.     

神经干细胞生存因子在大鼠脊髓损伤前后的表达变化

目的观察大鼠脊髓损伤后干细胞来源的神经干细胞生存因子（SDNSF）mRNA在大鼠正常和损伤脊髓的农达变化,以及SDNSF的表达与Ⅵ类中间丝蛋白的表达之间的关系。方法按改良的Allen重物打击法制备大鼠脊髓损伤模型,采用RT—PCR、原位杂交方法,观察SDNSF mRNA在大鼠脊髓中的表达位置及在损伤脊髓中的表达变化。应用免疫组化的方法,显示脊髓中nestin的表达。结果RT-PCR检测SDNSF mRNA在正常大鼠脊髓中的表达,损伤后4天SDNSF的mRNA表达上升,损伤8天剑达高峰,此后SDNSF的mRNA表达逐渐减少,到16天恢复到正常水平;脊髓切片原位杂交结果发现SDNSF的mRNA阳性细胞主要分布十脊髓灰质细胞中,可能足神经元细胞,结果表明正常脊髓可表达SDNSF;脊髓损伤后8犬,原位杂交硅示SDNSF阳性细胞明显增多。同时与此切片相邻层面的切片免疫组化证实nestin阳性细胞增殖、变大、向周围发出突起,但这些阳性细胞在分布上与SDNSF无关。结论（1）SDNSF在脊髓中表达于灰质,脊髓损伤后SDNSF的mRNA表达随时间发生变化。（2）随着脊髓损伤的修复,nestin阳性细胞增殖,但是这些细胞并不表达SDNSF。     

Differential expression of phospholipase D1 in the developing retina

Expression patterns of phospholipase D1 (PLD1) in the developing rat retina were investigated using immunocytochemistry and Western blot analysis and compared with the expression patterns of glutamine synthetase. PLD1 immunoreactivity appeared first in a few neuroblasts in the middle of the mantle zone of the primitive retina by embryonic (E) day 13. PLD1-immunoreactive primitive ganglion cells were characterized in the ganglion cell layer by E17. Faint immunoreactivity at E17 profiled radially orientated cells and this pattern appeared up to postnatal (P) day 7. In the ganglion cell layer at P3, displaced amacrine cells and ganglion cells were classified. At P5, presumptive horizontal cells and amacrine cells were identified. By P7, a thin outermost layer of newly formed segments of the photoreceptor cells was also PLD1 immunoreactive. PLD1 immunoreactivity at P8 was limited to radial Müller cells and the outer segment layer of the photoreceptor cells, and the expression pattern was conserved to adulthood. Western blot analysis showed relatively high amounts of PLD1 protein at E17 and P3, a decrease at P7, and moderate amounts from P8 onward. Co-expression of PLD1 with glutamine synthetase in the retina appeared first after birth in differentiating neurons and in Müller cells by P8; thereafter the pattern was maintained. The expression pattern of the PLD1 during development of the retina suggests that PLD1 plays important roles in glutamate-associated differentiation of both specific neurons and radial glial cells, and in glutamate-mediated cellular signalling in Müller cells.     

A novel putative M9.2 isoform of V-ATPase expressed in the nervous system

We have identified a cDNA encoding a novel putative neuron-specific isoform of vacuolar proton-translocating ATPase (V-ATPase), NM9.2, from rat and mouse. Sequence analysis revealed that NM9.2 conserved similar characteristic amino acid sequences with 60-70% identity to M9.2 previously isolated from V-ATPase in chromaffin granules. Using Northern blot analysis, NM9.2 mRNA was specifically detected in the brain, whereas M9.2 mRNA was widely expressed in various tissues. In situ hybridization showed that NM9.2 gene expression was restricted mainly to neuronal cells and consistent with that of the a1/Ac116 subunit of V-ATPase. NM9.2 is a putative neuronal isoform of the 9.2 kDa subunit in V-ATPase.     

人脑胶质瘤CXCL12基因的甲基化分析研究

目的 检测胶质瘤CXCL12基因启动子区的甲基化状态及其mRNA表达水平,以及受体CXCR4、DNA甲基转移酶等基因的mRNA表达情况,分析甲基化在CXCL12/CXCR4生物轴参与胶质瘤恶性进展中的调控机制.方法 半定量RT-PCR和实时定量PCR检测CXCL12、CXCR4、DNMT1、DNMT3A和DNMT3B基闪在76例胶质瘤及10例正常脑组织中的表达情况;甲基化PCR检测CXCL12基因启动子区的甲基化状态.结果 (1)CXCR4 mRNA随胶质瘤恶性程度的增高而表达增加;(2)CXCL12基因在胶质瘤中的甲基化率为34.2%,甲基化率随胶质瘤恶性程度的增高而降低;(3)CXCL12基因的甲基化主要发生在低度恶性胶质瘤中,其甲基化状态与mRNA表达密切相关;(4)DNMT1、DNMT3A和DNMT3B在CXCL12基因甲基化胶质瘤中的表达明显高于未发生甲基化的胶质瘤.结论 CXCR4基因有望成为胶质瘤恶性程度的生物学标志;CXCL12基因启动子区的甲基化主要发生在低度恶性胶质瘤中,其CXCL12基因甲基化下调mRNA的表达;DNMT1、DNMT3A和DNMT3B的过表达可能参与CXCL12基因甲基化的调控.     

PEX基因对C6胶质瘤干细胞MMP-2和VEGF表达的影响

目的 探讨PEX基因对C6胶质瘤干细胞(GSCs)中基质金属蛋白酶2(MMP-2)和血管内皮生长因子(VEGF)表达的影响.方法 采用血清培养基培养C6胶质瘤细胞系,再取其单细胞悬液悬浮培养于无血清培养基中传代培养.采用单细胞克隆和免疫荧光染色分离鉴定GSCs,运用本实验室成功构建的重组子pDsRed2-C1-PEX,以脂质体为介导转染体外培养的GSCs,逆转录酶-聚合酶链反应(RT-PCR)检验真核表达载体在GSCs中的表达及PEX基因对GSCs表达MMP-2mRNA、VEGFmNA的影响.结果 成功培养出GSCs并成功转染,PEX在GSCs中高表达并且体外对MMP-2、VEGF的表达有明显抑制作用,差异有统计学意义(P＜0.05).结论 PEX基因可以在GSCs中表达,并且体外可以抑制MMP-2和VEGFmRNA在GSCs中的表达,为胶质瘤的治疗奠定理论基础.     

GDNF, RET and GFRalpha-1-3 mRNA expression in the developing human spinal cord and ganglia.

The identification of endogenous neurotrophic factors and their receptors in human spinal cord is important not only to understand development, but also in the consideration of possible future therapies for neurodegenerative disorders and trauma. Using in situ hybridization, the expression of glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), persephin (PSP), GFRalpha-1, GFRalpha-2, GFRalpha-3 and RET mRNA in human fetal spinal cord was studied. Strong GDNF mRNA hybridization signal, presumably restricted to Clarke's nucleus, was detected in the thoracic spinal cord. mRNA encoding GFRalpha-1 was expressed in the entire spinal cord gray matter with particularly high expression in the ventral horn. GFRbeta-1 was also expressed more weakly in dorsal root ganglia. NTN and persephin mRNA were not detected in either the fetal spinal cord or the dorsal root ganglia. mRNA coding for GFRalpha-2, however, was found in most cells of the spinal cord gray matter. A strong expression of GFRalpha-3 mRNA was detected in dorsal root ganglia cells and Schwann cells. The transducing receptor RET was expressed strongly in motorneurons and dorsal root ganglion neurons. We conclude that basic features concerning the role of the GDNF family of ligands and their receptors revealed in rodents applies to humans.     

The association of NF2 (neurofibromin 2) gene polymorphism and the risk of medulloblastomas

To explore the relationship between NF2 promoter gene mutation and the risk of medulloblastomas (MBs). We collected tissues from 16 MB patients and 7 age-matched non-MB controls. Gene sequencing, qPCR (real-time quantitative polymerase chain reaction), IHC (immunohistochemistry), and WB (Western blot) were used to analyze the changes in the NF2 gene sequence and expression between patients and controls. We found that NF2 promoter gene mutations occurred in MB patients. The NF2 mRNA expression was higher in the controls than in patients (p?=?0.03?<?0.05); however, the results of IHC and WB demonstrated that the NF2 protein expression was significantly higher in patients than in the controls (IHC: p?=?0.0001; WB: p?=?0.01). There was no significant difference in the CRL4 mRNA and protein levels. In addition, NF2 protein was mainly expressed in the nucleus in MB patients, while the NF2 protein was mainly expressed in the cytoplasm in the controls. NF2 promoter mutations exist in MB patients. NF2 mRNA expression was higher in controls than patients; whereas NF2 protein level was higher in patients than in controls.