The Expression of Tenascin-X in Developing and Adult Rat and Human Eye

Tenascin-X has been studied in developing and adult rat eye and in foetal and adult human eyes, using immunohistochemistry and frozen sections. The data were compared with the distribution of tenascin-C. The immunoreactivity for tenascin-X was seen in a basement membrane-like feature in different structures of embryonic (E) day 16–17 rat eyes. Postnatal (P) day 2 and older rat eyes showed immunoreactivity for tenascin-X in different connective tissues. In the epithelial basement membrane zone of the cornea, immunostaining was positive in P5 eyes, negative in P10 and P15 eyes and again positive in P30 and adult eyes. In the 20-week-old human foetus, immunoreactivity for the tenascin was seen in the posterior parts of the conjunctival stroma adjacent to the sclera and in a basement membrane-like fashion in anterior conjunctiva. In the adult human eye, immunoreactivity for tenascin-X was seen in the anterior one-third stroma of cornea as thin fibrils, in the stroma of the limbus and conjunctiva, and in blood vessels. Immunostaining for tenascin-C was seen in the posterior aspect of the further cornea, and in mesenchyme adjacent to cornea in E16–17 rat eyes. Corneal keratocytes and Descemet's membrane showed immunoreactivity for tenascin-C in P2–P15 rat eyes. Sclera and the junction of the cornea, and sclera expressed tenascin-C in P2 and older rat eyes. In human foetal eyes, immunostaining for tenascin-C was seen in the anterior parts of the corneal stroma, in the basement membrane zone and Bowman's membrane of the corneal epithelium, in the posterior one-fifth of the corneal stroma and the sclera starting from the junction of the cornea and sclera. In normal human adult eyes, immunostaining for tenascin-X was seen in the anterior one-third stroma of cornea, in the stroma of limbus and conjunctiva, and in blood vessels. The association of tenascin-X and basement membranes in early development evokes a question of its potential function in the development of the basement membrane. The results also suggest the association of tenascin-X with connective tissue development as well as the association of tenascin-C with the migration of keratocytes during the development of the corneal stroma.     

人小肠三叶因子在大肠杆菌中的表达

利用PCR技术将人小肠三叶因子(hITF)基因重组入表达载体pGEX-4T-1,构建了融合蛋白GST-hITF的重组表达质粒pGTF,在大肠杆菌中诱导表达。表达的融合蛋白经亲和层析、凝血酶切和凝胶过滤层析得到纯化的hITF蛋白。测定了重组蛋白的氨基酸组成、分子量及其对酸和蛋白酶的抗性。Western印迹表明重组蛋白具有hITF的抗原性,并对大鼠胃溃疡具有明显的预防和保护作用。     

人三叶因子3在毕赤酵母中表达条件的研究

为提高人三叶因子 3 (HumanTrefoilfactor 3 ,hTFF3 )在毕赤酵母中的表达量 ,研究了转化子生长的培养条件 ,包括不同碳源对转化子生长的影响和接种量、甲醇浓度、pH值、摇瓶转速及不同诱导时间对人三叶因子 3表达的影响。结果表明转化子在生长阶段加入葡萄糖生长旺盛 ,培养 14h后OD₆₀₀ 就可达到 50。在 100mL生长培养基上的菌液以 1∶1接入诱导培养基时蛋白表达量最高 ;转化子在 1%的甲醇、pH60、摇瓶转速240r/min的条件下诱导4 8h ,菌体密度OD₆₀₀为 15 ,目的蛋白表达量达到 20mg L。用 5L发酵罐进行了高密度发酵 ,经2%甲醇32h诱导 ,最终菌体密度OD₆₀₀ 达到 120 ,每升发酵液中含目的蛋白100mg。     

Ventral Tegmental Area Inactivation Suppresses the Expression of CA1 Long Term Potentiation in Anesthetized Rat

The hippocampus receives dopaminergic projections from the ventral tegmental area (VTA). Modulatory effect of dopamine on hippocampal long term potentiation (LTP) has been studied before, but there are conflicting results and some limitations in previous reports. Most of these studies show a significant effect of dopamine on the late phase of LTP in CA1 area of the hippocampus, while few reports show an effect on the early phase. Moreover, they generally manipulated dopamine receptors in the hippocampus and there are few studies investigating influence of the VTA neural activity on hippocampal LTP in the intact brain. Besides, VTA neurons contain other neurotransmitters such as glutamate and GABA that may modify the net effect of dopamine. In this study we examined the effect of VTA reversible inactivation on the induction and maintenance of early LTP in the CA1 area of anesthetized rats, and also on different phases of learning of a passive avoidance (PA) task. We found that inactivation of the VTA by lidocaine had no effect on CA1 LTP induction and paired-pulse facilitation, but its inactivation immediately after tetanic stimulation transiently suppressed the expression of LTP. Blockade of the VTA 20 min after tetanic stimulation had no effect on the magnitude of LTP. Moreover, VTA inactivation immediately after training impaired memory in the passive avoidance task, while its blockade before or 20 min after training produced no memory deficit. It can be concluded that VTA activity has no effect on CA1 LTP induction and acquisition of PA task, but involves in the expression of LTP and PA memory consolidation.     

人小肠三叶因子（hITF）基因在生菜中的整合与表达

用根癌土壤杆菌（Agrobacterium tumefaciens(Smith et Townsend)conn)介导的叶盘法,将人小肠三叶因子（hITF)导入生菜（Lactuca sativa L.)中,在含有除草上培养基上筛选,获得抗性植株,通过PCR和Southern印迹分析证明,hITF cDNA已整合到生菜基因组中,Western印迹分析证明hITF在生菜中的表达。ELISA检测表明,hITF在生菜新鲜叶片中的表达量为200－300ng/g,最高达700ng/g,约占总可溶性蛋白的0．1％。     

肠三叶因子表达载体构建及在酵母菌GS115中的表达

目的:构建肠三叶因子(ITF)的真核表达载体,并在酵母菌GS115中表达,为进一步研究ITF的生理和药理功能奠定基础。方法:通过RT-PCR获得ITF cDNA片断,将目的基因插入pPICZαA酵母表达载体,得到重组载体pPICZαA-ITF。经BspHI线性化后氯化锂转化进入GS115,Zeocin筛选转化酵母菌,PCR鉴定目的基因。阳性转化子经摇瓶表达,取上清TCA沉淀后做Tricine SDS-PAGE分析及Western blot检测表达蛋白。结果:经测序及PCR证实ITF cDNA准确插入酵母表达载体pPICZαA中。Tricille SDS-PAGE分析证明ITF的分子量约为14×10~3,表达蛋白具有良好的抗原性和特异性。结论:成功构建了真核表达载体pPICZαA-ITF,在酵母菌GS115中表达,获得重组ITF蛋白。     

Developmental Expression of Go in Neuronal Cultures from Rat Mesencephalon and Hypothalamus

The developmental expression of the alpha-subunit of Go was examined in neuronal cultures derived from rat mesencephalon (MES) and hypothalamus (HYP). These cultures were essentially free of contaminating glia and were maintained as a stable population for periods up to 3 weeks. Immunoblotting utilizing specific antisera against Go indicated that in neurons from both brain regions, membrane concentrations of Go increased dramatically during the first 2 weeks in vitro. Thereafter, increases in the amount of Go per neuron kept pace with increasing process (axons and dendrites) formation. Multiple forms of immunoreactive Go were detected in MES and HYP neurons, and the proportions of these forms changed between 4 and 14 days in culture. Finally, increasing neuron density significantly increased membrane levels of Go in MES but not HYP cultures.     

Expression of the Low-Affinity p75 Nerve Growth Factor Receptor in the Developing Rat Pituitary Gland

We investigated, by means of in situ hybridization with a digoxigenin-labelled RNA probe, the expression of the low-affinity p75 nerve growth factor receptor (NGFR) in the developing pituitary primordium of the rat. In 13-day pc embryos, intense staining of p75 NGFR mRNA was present in the cytoplasm of all cells of Rathke's pouch. In day-17 pc embryos p75 NGFR expression was present primarily in the cells of the intermediate lobe. In the newborn rat pituitary only very weak staining was observed, predominantly in the intermediate lobe. In neural structures the staining at day 13 pc was comparable to that of day 17 pc. Since p75 expression is seen very early during pituitary development and declines during the time the expression of pituitary hormonal phenotypes are steadily increasing, we suggest that the p75 NGFR expression in Rathke's pouch may play a temporally defined role in the commitment rather than in the differentiation of the various pituitary cell types.     

Increased Expression of Protease-Activated Receptor 4 and Trefoil Factor 2 in Human Colorectal Cancer

Protease-activated receptor 4 (PAR4), a member of G-protein coupled receptors family, was recently reported to exhibit decreased expression in gastric cancer and esophageal squamous cancer, yet increased expression during the progression of prostate cancer. Trefoil factor 2 (TFF2), a small peptide constitutively expressed in the gastric mucosa, plays a protective role in restitution of gastric mucosa. Altered TFF2 expression was also related to the development of gastrointestinal cancer. TFF2 has been verified to promote cell migration via PAR4, but the roles of PAR4 and TFF2 in the progress of colorectal cancer are still unknown. In this study, the expression level of PAR4 and TFF2 in colorectal cancer tissues was measured using real-time PCR (n = 38), western blotting (n=38) and tissue microarrays (n = 66). The mRNA and protein expression levels of PAR4 and TFF2 were remarkably increased in colorectal cancer compared with matched noncancerous tissues, especially in positive lymph node and poorly differentiated cancers. The colorectal carcinoma cell LoVo showed an increased response to TFF2 as assessed by cell invasion upon PAR4 expression. However, after intervention of PAR4 expression, PAR4 positive colorectal carcinoma cell HT-29 was less responsive to TFF2 in cell invasion. Genomic bisulfite sequencing showed the hypomethylation of PAR4 promoter in colorectal cancer tissues and the hypermethylation in the normal mucosa that suggested the low methylation of promoter was correlated to the increased PAR4 expression. Taken together, the results demonstrated that the up-regulated expression of PAR4 and TFF2 frequently occurs in colorectal cancer tissues, and that overexpression of PAR4 may be resulted from promoter hypomethylation. While TFF2 promotes invasion activity of LoVo cells overexpressing PAR4, and this effect was significantly decreased when PAR4 was knockdowned in HT-29 cells. Our findings will be helpful in further investigations into the functions and molecular mechanisms of Proteinase-activated receptors (PARs) and Trefoil factor factors (TFFs) during the progression of colorectal cancer.     

人三叶因子3在毕氏酵母中的表达及生物活性分析

利用PCR方法在人胎儿胎盘cDNA中扩增了人三叶因子3基因（hTFF3）,插入到含有AOX1启动子和α－因子信号肽序列的表达载体pPIC9K中,采用毕氏酵母表达系统对其进行了高效的分泌表达,并用G418筛选高拷贝整合转化子。2％甲醇诱导酵母表达48h后,上清经SDS－PAGE和Western印迹证明重组蛋白质以双体的形式分泌表达,占总蛋白质的45％,能被抗hTFF3抗体所识别。表达产物经S－Sepharose、Q-Sepharose离子交换及Sephacryl S-100纯化后纯度达到95％以上。体内生物学活性研究证实hTFF3可有效的防止盐酸诱导的大鼠胃溃疡。     

Expression of the Cytoskeletal-Associated Protein Filamin in Adult Rat Organs

Filamin is a well-characterized actin-associated protein first isolated from chicken smooth muscle. Subsequently, this polypeptide and its nonmuscle homolog actin-binding protein have been shown to be expressed in avian muscle tissue, mammalian smooth muscle, mammalian macrophages and other blood cell types, as well as several cultured cell lines. In this report, the occurrence of this polypeptide in adult mammalian organs has been investigated. Immunoblot analysis using three anti-filamin monoclonal antibodies showed that this protein was largely detected in adult rat organs that possess a substantial smooth muscle component. Furthermore, the limited expression of filamin in smooth muscle tissue was corroborated by immunohistochemical analysis. In contrast to avian systems, filamin was never found in detectable quantities in either mammalian cardiac or skeletal muscle. Quantitative immunoblot analysis demonstrated that filamin amounts roughly correlated with the abundance of the smooth muscle component of a given organ, comprising as much as 16.5% of the total SDS-extractable protein in bovine aorta. Work in avian systems and cells in culture has suggested that filamin is a rather ubiquitous cytoskeletal element. By contrast, this work demonstrates that filamin is highly restricted in its expression in mammalian organ systems, in situ.     

Changes in Glial Cell Line-derived Neurotrophic Factor Expression in the Rostral and Caudal Stumps of the Transected Adult Rat Spinal Cord

Limited information is available regarding the role of endogenous Glial cell line-derived neurotrophic factor (GDNF) in the spinal cord following transection injury. The present study investigated the possible role of GDNF in injured spinal cords following transection injury (T₉–T₁₀) in adult rats. The locomotor function recovery of animals by the BBB (Basso, Beattie, Bresnahan) scale score showed that hindlimb support and stepping function increased gradually from 7 days post operation (dpo) to 21 dpo. However, the locomotion function in the hindlimbs decreased effectively in GDNF-antibody treated rats. GDNF immunoreactivty in neurons in the ventral horn of the rostral stump was stained strongly at 3 and 7 dpo, and in the caudal stump at 14 dpo, while immunostaining in astrocytes was also seen at all time-points after transection injury. Western blot showed that the level of GDNF protein underwent a rapid decrease at 7 dpo in both stumps, and was followed by a partial recovery at a later time-point, when compared with the sham-operated group. GDNF mRNA-positive signals were detected in neurons of the ventral horn, especially in lamina IX. No regenerative fibers from corticospinal tract can be seen in the caudal segment near the injury site using BDA tracing technique. No somatosensory evoked potentials (SEP) could be recorded throughout the experimental period as well. These findings suggested that intrinsic GDNF in the spinal cord could play an essential role in neuroplasticity. The mechanism may be that GDNF is involved in the regulation of local circuitry in transected spinal cords of adult rats.     

Glutamate Stimulation of [3H]Dopamine Release from Dissociated Cell Cultures of Rat Ventral Mesencephalon

In dissociated cell cultures of fetal rat ventral mesencephalon preloaded with [3H]dopamine, glutamate (10(-5)-10(-3) M) stimulated the release of [3H]dopamine. Glutamate stimulation of [3H]dopamine release was Ca2+ dependent and was blocked by the glutamate antagonist, cis-2,3-piperidine dicarboxylic acid. Glutamate stimulation of [3H]dopamine release was not due to glutamate neurotoxicity because (1) glutamate did not cause release of a cytosolic marker, lactate dehydrogenase, and (2) preincubation of cultures with glutamate did not impair subsequent ability of the cells to take up or release [3H]dopamine. Thus, these dissociated cell cultures appear to provide a good model system to characterize glutamate stimulation of dopamine release. Release of [3H]dopamine from these cultures was stimulated by veratridine, an activator of voltage-sensitive Na+ channels, and this stimulation was blocked by tetrodotoxin. However, glutamate-stimulated [3H]dopamine release was not blocked by tetrodotoxin or Zn2+. Substitution of NaCl in the extracellular medium by sucrose, LiCl, or Na2SO4 had no effect on glutamate stimulation of [3H]dopamine release; however, release was inhibited when NaCl was replaced by choline chloride or N-methyl-D-glucamine HCl. Glutamate-stimulated [3H]-dopamine release was well maintained (60-82% of control) in the presence of Co2+, which blocks Ca2+ action potentials, and was unaffected by the local anesthetic, lidocaine. These results are discussed in terms of the receptor and ionic mechanisms involved in the stimulation of dopamine release by excitatory amino acids.     

Expression of en in the Prosencephalon Heterotopically Transplanted into the Mesencephalon

The alar plate of the prosencephalon differentiates into a tectum-like structure when transplanted and integrated into the mesencephalon around 10-somite stage. Homeobox containing gene, en , is expressed in the mesencephalon with caudorostral gradient from 7-somite stage. Here expression of en in the prosencephalic transplant in the mesencephalon was studied immunohistochemically with anti- en antibody. Transplantation was carried out between quail and chick. It was shown that the transplant was stained with anti- en antibody at the caudal part of the mesencephalon. At the rostral part, the transplant did not express en . When the transplant was not contiguous to the caudal part of the host mesencephalon, it did not express en . The present study suggests that the prosencephalic transplant differentiates into the tectum-like structure when it expresses en at the caudal part of the mesencephalon.     

肠三叶因子在大肠杆菌中的表达、纯化及其抗体制备

目的:获得肠三叶因子(ITF)的原核表达产物及抗rITF抗体,为深入研究ITF的作用机制及其受体研究奠定基础。方法:常规提取人小肠组织总RNA,用RT-PCR获得ITF编码基因片段,克隆至质粒pET32a获得原核表达栽体,双酶切和测序后转化至Origami B(DE3)用IPTG诱导表达,优化条件获得最大表达产量;用SDS-PAGE、Western blot鉴定表达产物,亲和层析纯化获得的重组蛋白rITF皮下多点注射家兔,制备多克隆抗体,并用此抗体进行大鼠肠组织免疫组化研究。结果:测序证实PCR扩增获得ITF全长基因序列与基因文库中的完全一致,将该基因片段正确插入表达载体pET32a中、优化表达条件后,重组蛋白的表达量达到50mg/L;Western blot证明重组蛋白具有良好的抗原性和特异性;通过Ni-NTA亲和层析、超滤离心后,得到90%纯度的蛋白;收集兔血清,纯化后获得特异性良好的ITF抗体,免疫组化染色肠组织显示ITF表达的部位定位于杯状细胞。结论:成功构建了表达载体pET32a-ITF,在大肠杆菌中表达并纯化获得纯度较高的rITF,并获得了生物活性较高的ITF抗体,ITF主要在肠道杯状细胞分泌表达。     

大鼠大脑皮层中钙调神经磷酸酶活力的时空变化

以ＰＮＰＰ为底物测定了超离心制备的大鼠出生后早期和成年大脑皮层亚细胞各组分中钙调神经磷酸酶的活力。实验结果表明：（ｌ）钙调神经磷酸酶活力广泛地存在于胞液和突触部分,并且各亚细胞组分有明显差异。成年大鼠大脑皮层中ＣａＮ活力相对最高水平是在突触体,突触质,胞液,重的和轻的突触膜部分。（２）大鼠大脑皮层突触体中ＣａＮ活力在出生后第２周和第３周出现高峰的平台期,这与突触发生的高峰期是一致的。在胞液和重的突触膜中ＣａＮ活力最高水平是在出生后的第７ｄ,而在突触质和轻的突触膜中是在第２０ｄ。总之,这些发现证实,在脑发育期间,ＣａＮ活力是依照区域和时间性控制的,提示ＣａＮ可能参与了突触功能作用。