The construction of siRNA plasmid targeting mouse HIF-1α and in vitro study of its inhibition effect

Objective

To construct effective RNA-interference plasmids targeting mouse HIF-1α gene and testify their effects and specificities in interfering HIF-1α expression.

Methods

Three RNA-interference plasmids targeting mouse HIF-1α gene, pBS/U6/HIF-1α-siRNAI～III, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1α in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1αi-II in SH-SY5Y cell line was further investigated.

Results

All the three RNA-interference plasmids, especially pBS/U6/HIF1αi-II, showed significant inhibition in HIF-1α expression in 293T cell line. pBS/U6/HIF1αi-II could also inhibit HIF-1α expression in SH-SY5Y cell line, in a dose-dependent way.

Conclusion

Plasmid pBS/U6/HIF1αi-II constructed in our study can effectively and specifically inhibit HIF-1α expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/HIF1αi-II plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and ischemia development.     

Axl和Tyro-3受体表达受神经生长因子调控并参与PC12细胞的分化

目的 Axl和Tyro-3受体在脑内有区域性的分布,但两者在中枢神经系统中的生物学功能尚不明确.本研究旨在探讨Axl和Tyro-3受体在神经元分化中的作用.方法 PC12细胞分别转染CMV-Axl-eGFP、CMV-Tyro-3-eCFP和CMV-eGFP质粒后,给予神经生长因子(nerve growth factor...     

HLB-1 functions as a new regulator for the organization and function of neuromuscular junctions in nematode Caenorhabditis elegans

Objective To study the role of HLB-1 in regulating the organization and function of neuromuscular junctions in nematode Caenorhabditis elegans

Methods

To evaluate the functions of HLB-1 in regulating the organization and function of neuromuscular junctions, effects of hlb-1 mutation on the synaptic structures were revealed by uncovering the expression patterns of SNB-1::GFP and UNC-49::GFP, and pharmacologic assays with aldicarb and levamisole were also used to test the synaptic functions. Further rescue and mosaic analysis confirmed HLB-1’s role in regulating the organization and function of neuromuscular junctions.

Results

Loss of HLB-1 function did not result in defects in neuronal outgrowth or neuronal loss, but caused obvious defects of SNB-1::GFP and UNC-49::GFP puncta localization, suggesting the altered presynaptic and postsynaptic structures. The mutant animals exhibited severe defects in locomotion behaviors and altered responses to an inhibitor of acetylcholinesterase and a cholinergic agonist, indicating the altered presynaptic and postsynaptic functions. Rescue and mosaic analysis experiments suggested that HLB-1 regulated synaptic functions in a cell nonautonomously way. Moreover, HLB-1 expression was not required for the presynaptic active zone morphology. Genetic evidence further demonstrated that hlb-1 acted in a parallel pathway with syd-2 to regulate the synaptic functions.

Conclusion

HLB-1 appeared as a new regulator for the organization and function of neuromuscular junctions in C. elegans.     

携带人内皮抑素基因的重组腺病毒构建及表达*☆

目的：内皮抑素是目前发现的作用最强的内皮细胞抑制因子，但内皮抑素蛋白极不稳定，难以制备及应用，故需要借助基因治疗发挥其抑制血管生成的作用。实验利用AdEasy-1系统构建并鉴定人内皮抑素重组腺病毒，并在人血脐静脉内皮细胞中表达出内皮抑素蛋白。 方法：实验于2006-03/10在中山大学生命科学院完成。以Pshuttle-Endostatin质粒为模板，应用特异引物，通过聚合酶链反应扩增纯化得到Endostatin基因片断，经测序鉴定后，酶切插入穿梭质粒pAdTrack-CMV中，再与骨架质粒AdEasy-1在大肠杆菌BJ5183中进行同源重组，重组质粒经筛选、提取、纯化后，经酶切及测序鉴定正确，再大量扩增为腺病毒载体，线性化后利用脂质体2000转染AAV293细胞包装并扩增，得到的病毒液纯化扩增后，感染人脐静脉内皮细胞，反转录-聚合酶链反应检测感染细胞中目的基因的表达，蛋白免疫印迹检测感染细胞中蛋白的表达。 结果：获得的Endostatin基因片段经酶切及测序鉴定正确，重组腺病毒质粒经酶切及测序鉴定正确，成功转染AAV293细胞，包装并扩增出病毒液，纯化后测滴度为2.06×1010 pfu/mL，进一步感染人脐静脉内皮细胞，在其中检测到目的基因及蛋白表达。 结论：人内皮抑素重组腺病毒pAd-Endo构建成功，且可在人脐静脉内皮细胞中表达出相应的目的基因及蛋白。     

Endophilin isoforms have distinct characteristics in interactions with N-type Ca2+ channels and dynamin I

Objective Formation of the endophilin II-Ca 2+ channel complex is Ca 2+ -dependent in clathrin-mediated endocytosis. However, little is known about whether the other two endophilin isoforms have the same features. The present study aimed to investigate the characteristics of the interactions of all three isoforms with Ca 2+ channels and dynamin I. Methods N-type Ca 2+ channel C-terminal fragments (NCFs) synthesized with a 3 H-leucine-labeled kit, were incubated with endophilin-GST fusion proteins, followed by pull-down assay. Results were counted on a scintillation counter. In addition, the different endophilin isoforms were each co-transfected with dynamin I into 293T cells, followed by flow cytometry and co-immunoprecipitation assay. Immunostaining was performed and an image analysis program was used to evaluate the overlap coefficient of cells expressing endophilin and dynamin I. Results All three isoforms interacted with NCF. Endophilins I and II demonstrated clear Ca 2+ -dependent interactions with NCF, whereas endophilin III did not. Co-immunoprecipitation showed that, compared to endophilin I/II, the interaction between endophilin III and dynamin I was significantly increased. Similar results were obtained from flow cytometry. Furthermore, endophilin III had a higher overlap coefficient with dynamin I in co-transfected 293T cells. Conclusion Endophilin isoforms have distinct characteristics in interactions with NCF and dynamin I. Endophilin III binding to NCF is Ca 2+ -independent, implying that it plays a different role in clathrin-mediated endocytosis.     

RETRACTED ARTICLE: Pro-protein convertase-2/carboxypeptidase-E mediated neuropeptide processing of RGC-5 cell after in vitro ischemia

Objective

To observe the change of the neuropeptide pro-protein processing system in the ischemic retina ganglion cell-5 (RGC-5) cells, pro-protein convertase-2 (PC2), carboxypeptidase-E (CPE) and preproneuropeptide Y (preproNPY) protein levels in the ischemic RGC-5 cells and conditioned medium were analyzed.

Methods

The RGC-5 cell was differentiated in 0.1 μmol/L staurosporine for 24 h and then stressed by different doses of oxygen and glucose deprivation (OGD). The acute or chronic OGD-induced cell death rates were obtained by using PI or TUNEL staining. The protein expression levels were determined by using the Western blot method and PC2 activity analysis.

Results

The ischemia caused substantial cell death in an OGD dose-dependent manner. In the cells, proPC2 and preproNPY protein levels gradually increased whereas proCPE gradually decreased. After OGD, PC2 activity was decreased. In the conditioned medium, proPC2 and PC2 proteins gradually decreased whereas proCPE, CPE, and preproNPY proteins gradually increased.

Conclusion

These results demonstrated that OGD inhibited the neuropeptide pro-protein processing system by reducing PC2 activity and the maturation of proPC2. The aggregation of the pro-proteins and the increase of the active CPE excision adversely exacerbated the cell injury. The pro-protein processing system might play a critical role in the ischemic stress of RGC-5 cells.     

Differential distributions and trafficking properties of dopamine D1 and D5 receptors in nerve cells

Objective

To explore the possible differential trafficking properties of the dopamine D1-like receptor subtypes, D1 receptor and D5 receptor.

Methods

To visualize distributions of dopamine D1-like receptor subtypes at subcellular level, the yellow and cyan variants of green fluorescent protein (GFP) were used to tag D1 and D5 receptors. After transfection with the tagged dopamine receptors, the neuroblastoma cells NG108-15 were treated with D1 agonist SKF38393 or acetylcholine (ACh). Then we observed the subcellular distributions of the tagged receptors under the confocal microscopy and tried to determine trafficking properties by comparing their distribution patterns before and after the drug treatment.

Results

In resting conditions, D1 receptors located in the plasma membrane of NG108-15 cells, while D5 receptors located in both plasma membrane and cytosol. With the pre-treatment of SKF38393, the subcellular distribution of D1 receptors was changed. The yellow particle-like fluorescence of tagged D1 receptors appeared in the cytosol, indicating that D1 receptors were internalized into cytosol from the cell surface. Same situation also occurred in ACh pre-treatment. In contrast, the subcellular distribution of D5 receptors was not changed after SKF38393 or ACh treatment, indicating that D5R was not translocated to cell surface. Interestingly, when D1 and D5 receptors were co-expressed in the same cell, both kept their distinct subcellular distribution patterns and the trafficking properties.

Conclusion

Our present study reveals that in NG108-15 nerve cells, dopamine D1 and D5 receptors exhibit differential subcellular distribution patterns, and only D1 receptor has a marked trafficking response to the drug stimulation. We further discuss the potential role of the differential trafficking properties of D1-like receptors in complex modulation of DA signaling.     

Characterization of neuroblastoma bone invasion/metastasis in established bone metastatic model of SY5Y and KCNR cell lines

Objects

To determine the mechanism of neuroblastoma (NB) bone invasion/metastasis, it is necessary to investigate the bone invasion/metastasis-related factors in the bone invasion/metastasis process. Some evidence has suggested that various proteins were involved in bone osteolytic response. The invasion/metastasis property and gene expression of NB, however, are still unknown.

Methods

Single-cell suspensions of SY5Y and KCNR cells were injected directly into the femur of nude mice. Radiological and histological analyses, immunohistochemistry analyses, and western blot assay were performed to characterize bone metastasis mechanism in these bone metastasis models.

Results

SY5Y and KCNR NB cells result in osteolytic responses in bone metastasis model. Osteoprotegerin (OPG), receptor activator of NF-kappaB ligand (RANKL), parathyroid hormone-related peptide (PTHrP), endothelin 1 (ET-1), and CXCR4 were examined and compared among in vitro, in vivo, and normal bone, respectively. PTHrP, OPG, RANKL, and ET-1 except CXCR4 in SY5Y and KCNR NB cells xenografts were strikingly upregulated compared with normal bone and NB cells. However, significantly stronger expression of PTHrP and RANKL was presented than ET-1 and OPG; furthermore, the ratios of expression of PTHrP, RANKL to OPG, and ET-1 were also markedly increased in vivo versus in vitro.

Conclusions

Our study provided evidence that NB cell may enhance bone invasion through PTHrP, OPG, RANKL, and ET-1, especially PTHrP and RANKL which may display stronger effects. CXCR4 appeared not participating in bone invasion, but in tumor growth, and homing to bone. Targeting PTHrP, OPG, ET-1, and RANKL may provide a new insight and method for patient therapy by inhibiting NB bone metastasis and invasiveness.     

Expression of the apoptosis-related proteins caspase-3 and NF-κB in the hippocampus of Tg2576 mice

Objective

To investigate the relations between neuroapoptosis and the onset and development of Alzheimer’s disease (AD), especially the role of NF-κB in the regulation of neuroapoptosis.

Methods

Caspase-3 and NF-κB (p50) expressions in the CA3 region of the hippocampus in APPswe Tg2576 transgenic mice were studied from postnatal day 0–180, using Nissl staining, immunohistochemistry and RT-PCR methods.

Results

Both neuronal apoptosis and NF-κB activity decreased gradually with the increase of age in wild type and Tg2576 mice. However, the number of caspase-3-positive or NF-κB-positive pyramidal cells in Tg2576 mice was greater than that in age-matched wild type mice, with significant differences after postnatal day 14 (P < 0.01 or P < 0.05). Linear regression analyses of caspase-3 and NF-κB expression demonstrated a correlation between neuroapoptosis and activity of NF-κB.

Conclusion

The process of neuroapoptosis is consistent with the onset and development of AD. Furthermore, the observed correlation between neuroapoptosis and NF-κB activity suggests a role of NF-κB in hippocampal neuroapoptosis.     

铜离子对MES23.5细胞的毒性作用机制与线粒体功能障碍有关

目的探讨铜离子对MES23．5多巴胺能细胞的毒性作用及可能机制。方法用MTF法检测铜离子对细胞存活率的影响;用半定量逆转录聚合酶链反应（RT-PCR）,Western blotting以及高效液相色谱电化学检测法（HPLC-ECD）检测细胞内酪氨酸羟化酶（tyrosine hydroxlase,TH）mRNA、蛋白的表达以及多巴胺含量的改变：用流式细胞仪检测线粒体跨膜电位的改变。结果100μmol／L和200／2mol／L铜离子对细胞存活率没有影响,400μmol／L和800μmol／L铜离子可以造成细胞存活率降低（P〈0．01）。200μmol／L铜离子孵育细胞24h,THmRNA、蛋白表达量以及多巴胺含量较正常对照组降低（P〈0．01,P〈0．01,P〈0．05）。200μmol／L铜离子孵育细胞24h,线粒体跨膜电位较正常对照组明显降低（P〈0．01）。结论铜离子对MES23．5多巴胺能细胞具有毒性作用,降低细胞功能的表达,其机制可能与线粒体功能障碍有关。     

Genetic analysis of the UPB1 gene in two new Chinese families with β-ureidopropionase deficiency and the carrier frequency of the mutation c.977G>A in Northern China

Purpose

The purpose of the study was to investigate mutations of the UPB1 gene in two Chinese families with β-ureidopropionase deficiency and the heterozygous carrier frequency in Chinese.

Methods

Genomic DNA was extracted from peripheral blood leukocytes from all available family members and 500 unrelated healthy controls. Then, all exons and flanking intron regions of the UPB1 gene were amplified by PCR and analyzed by direct sequencing in two patient-families. Finally, the carrier frequency of the c.977G>A (p.R326Q) mutation was identified by PCR restriction fragment length polymorphism in 500 healthy controls.

Results

The two patients had the same homozygous missense mutation in exon 9 (c.977G>A; p.R326Q), and the carrier frequency of this mutation was 2.8 % in the Northern Chinese population, which suggests that about 1:5,102 Chinese are expected to suffer from UPB1 deficiency.

Conclusions

The c.977G>A (p.R326Q) is the most common mutation of the UPB1 gene in Chinese. The predicted incidence indicates that β-ureidopropionase deficiency is significantly underdiagnosed in the Chinese population. It should be necessary to add β-ureidopropionase deficiency to high-risk screening for the symptomatic patients group.     

Pharmacological modulation of brain Nav1.2 and cardiac Nav1.5 subtypes by the local anesthetic ropivacaine

Objective

The present study was aimed to investigate the pharmacological modulatory effects of ropivacaine, an amide-type local anesthetic, on rat Nav1.2 (rNav1.2) and rNav1.5, the two Na⁺ channel isoforms heterologously expressed in Xenopus oocytes and in HEK293t cell line, respectively.

Methods

Two-electrode voltage-clamp (TEVC) and whole-cell patchclamp recordings were employed to record the whole-cell currents.

Results

Ropivacaine induced tonic inhibition of peak Na⁺ currents of both subtypes in a dose- and frequency-dependent manner. rNav1.5 appeared to be more sensitive to ropivacaine. In addition, for both Na⁺ channel subtypes, the steady-state inactivation curves, but not the activation curves, were significantly shifted to the hyperpolarizing direction by ropivacaine. Use-dependent blockade of both rNav1.2 and rNav1.5 channels was induced by ropivacaine through a high frequency of depolarization, suggesting that ropivacaine could preferentially bind to the 2 inactivated Na⁺ channel isoforms.

Conclusion

The results will be helpful in understanding the pharmacological modulation by ropivacaine on Nav1.2 subtype in the central nervous system, and on Nav1.5 subtype abundantly expressed in the heart.     

Initial bradykinin triggers calcium-induced calcium release in C6 glioma cells and its significance

Objective

To investigate the underlying mechanism for the selective modulation of the permeability of blood-tumor barrier (BTB) by small dose of bradykinin (BK).

Methods

C6 glioma cells were treated with BK, and changes of intracellular nitric oxide (NO) and intracellular calcium level were measured with fluorescent spectrophotometer.

Results

The initial application of BK easily triggered extracellular calcium influx, which resulted in intracellular calcium store release in C6 glioma cells. The above mechanism was also named ryanodine mediated calcium induced calcium release (CICR). We also detected a long-lasting intracellular NO elevation in C6 glioma cells upon BK treatment. Further study showed that ryanodine mediated CICR contributed greatly to the secondary NO elevation induced by BK treatment.

Conclusion

These results suggested that BK triggered CICR in C6 glioma cells and the associated NO generation might be the underlying mechanism for the selective modulation of BTB permeability by BK.     

Dose- and time-dependent α-synuclein aggregation induced by ferric iron in SK-N-SH cells

Objective

Intracellular formation of Lewy body (LB) is one of the hallmarks of Parkinson’s disease. The main component of LB is aggregated α-synuclein, present in the substantia nigra where iron accumulation also occurs. The present study was aimed to study the relationship between iron and α-synuclein aggregation.

Methods

SK-N-SH cells were treated with different concentrations of ferric iron for 24 h or 48 h. MTT assay was conducted to determine the cell viability. Thioflavine S staining was used to detect α-synuclein aggregation.

Results

With the increase of iron concentration, the cell viability decreased significantly. At the concentrations of 5 mmol/L and 10 mmol/L, iron induced α-synuclein aggregation more severely than at the concentration of 1 mmol/L. Besides, 48-h treatment-induced aggregation was more severe than that induced by 24-h treatment, at the corresponding iron concentrations.

Conclusion

Ferric iron can induce α-synuclein aggregation, which is toxic to the cells, in a dose- and time-dependent way.     

Functional analysis of calcium channel-mediated exocytosis in synaptic terminals by FM imaging technique

Objective

Presynaptic voltage-gated Ca²⁺ channels mediate rapid Ca²⁺ influx into the synaptic terminal which triggers synaptic vesicle exocytosis and neurotransmitter release. The FM 1-43 dye was firstly introduced as a fluorescence probe by Betz and his colleagues in 1992, and has been used to monitor exocytosis, endocytosis and endosomal traffic in a variety of cell types. The present study aims to investigate the feasibility of applying the FM 1-43 dye in the functional analysis of calcium channel-mediated exocytosis in synaptic terminals.

Methods

The hippocampi were isolated from embryos of pregnant rats, and hippocampal neurons were then transfected with Ds-Red conjugated plasmid. The neurons were then loaded with 8 μmol/L FM 1-43 and 47 mmol/L KCl for 90 s after transfection. After that, 90 mmol/L KCl was employed to induce FM dye destaining, which was recorded by FM imaging system.

Results

The neuron synaptic terminals of rat hippocampus could be effectively stained by the FM 1-43 dye. Besides, the destaining of the labeled neuron terminals was in accordance with the transmitter release, which could be blocked by the application of nifedipine (inhibitor for L-type calcium channel).

Conclusion

The FM imaging technique is an advanced and effective method for analyzing synaptic vesicle exocytosis and neurotransmitter release, and can be applied in various synaptic functional studies.     

Medulloblastoma-derived tumor stem-like cells acquired resistance to TRAIL-induced apoptosis and radiosensitivity

Objects

Medulloblastoma (MB) is the most malignant primary brain tumor in early childhood that contains cellular and functional heterogeneity. Recent evidence has demonstrated that the tumor stem cells (TSC) may explain the radiochemoresistance of brain tumors, including MB. The aim of the present study is to investigate the possible role of TNF-related apoptosis-inducing ligand (TRAIL) in viability and tumorigenicity of MB cells and MB-derived TSC.

Methods

MB-associated TSC were isolated and cultured by serum-free medium with bFGF and EGF. The parental MB cells and MB-TSC cells were treated with TRAIL in different concentrations and assessed for cell viability, invasion ability, colony forming ability, and radiotherapy effect.

Results

We enrich a subpopulation of MB-TSC cells using tumor spheroid formation approach. MB-TSC display enhanced self-renewal and highly expressed “stemness” genes (CD133, Sox-2, Bmi1, Nestin). Additionally, MB-TSC showed significant resistance to TRAIL-induced apoptosis and radiosensitivity compared to the parental MB cells due antiapoptotic gene (c-FLIP, Caspase 8, Bcl-2, and Bax) upregulation.

Conclusions

Our data suggest that MB-TSC are resistant to TRAIL-induced apoptosis and tumorigenic properties. Understanding the molecular mechanisms by which to operate the physiological characteristics in MB-TSC cells offers attractive approach for MB treatment.     

Combining Self-Affirmation and Implementation Intentions: Evidence of Detrimental Effects on Behavioral Outcomes

Background

There is limited evidence that self-affirmation manipulations can promote health behavior change.

Purpose

The purpose of this study was to explore whether the efficacy of a self-affirmation manipulation at promoting exercise could be enhanced by an implementation intention intervention.

Methods

Participants (Study 1?N?=?120, Study 2?N?=?116) were allocated to one of four conditions resulting from the two (self-affirmation manipulation: no affirmation, affirmation) by two (implementation intention manipulation: no implementation intention, implementation intention) experimental design. Exercise behavior was assessed 1 week post-intervention.

Results

Contrary to prediction, those participants receiving both manipulations were significantly less likely to increase the amount they exercised compared to those receiving only the self-affirmation manipulation.

Conclusion

Incorporating an implementation intention manipulation alongside a self-affirmation manipulation had a detrimental effect on exercise behavior; participants receiving both manipulations exercised significantly less in the week following the intervention.     

Possible association between Interleukin-1beta gene and schizophrenia in a Japanese population

Background

Disrupted-in-Schizophrenia 1 (DISC1) gene is one of the most promising candidate genes for major mental disorders. In a previous study, a Finnish group demonstrated that DISC1 polymorphisms were associated with autism and Asperger syndrome. However, the results were not replicated in Korean population. To determine whether DISC1 is associated with autism in Chinese Han population, we performed a family-based association study between DISC1 polymorphisms and autism.

Methods

We genotyped seven tag single nucleotide polymorphisms (SNPs) in DISC1, spanning 338 kb, in 367 autism trios (singleton and their biological parents) including 1,101 individuals. Single SNP association and haplotype association analysis were performed using the family-based association test (FBAT) and Haploview software.

Results

We found three SNPs showed significant associations with autism (rs4366301: G > C, Z = 2.872, p = 0.004; rs11585959: T > C, Z = 2.199, p = 0.028; rs6668845: A > G, Z = 2.326, p = 0.02). After the Bonferroni correction, SNP rs4366301, which located in the first intron of DISC1, remained significant. When haplotype were constructed with two-markers, three haplotypes displayed significant association with autism. These results were still significant after using the permutation method to obtain empirical p values.

Conclusions

Our study provided evidence that the DISC1 may be the susceptibility gene of autism. It suggested DISC1 might play a role in the pathogenesis of autism.