Distribution of growth hormone-like immunoreactive cells and somatostatin receptors in the nervous system and Hatschek''s pit of amphioxus, Branchiostoma belcheri

Using immunohistochemical method and double staining technique, the localization of growth hormone (GH) and somatostatin receptors in the nervous system and Hatschek's pit of amphioxus has been investigated. The results showed that the growth hormone-like nerve cells and endocrine cells as well as three subtypes of somatostatin receptors exist in the nervous system and Hatschek's pit, and GH-like nerve cells and endocrine cells co-exist with three subtypes of somatostatin receptors in the brain vesicle and Hatschek's pit. It is suggested that a primitive control system of inhibitory growth hormone secretion in Hatschek's pit could have been developed in amphioxus, as in vertebrates. The present study provides new evidence for the endocrinology and the evolution of Hatschek's pit.     

Distribution of growth hormone-like immunoreactive cells and somatostatin receptors in the nervous system and Hatschek''''s pit of amphioxus, Branchiostoma belcheri

Using immunohistochemical method and double staining technique, the localization of growth hormone (GH) and somatostatin receptors in the nervous system and Hatschek's pit of amphioxus has been investigated. The results showed that the growth hormone-like nerve cells and endocrine cells as well as three subtypes of somatostatin receptors exist in the nervous system and Hatschek s pit, and GH-like nerve cells and endocrine cells co-exist with three subtypes of somatostatin receptors in the brain vesicle and Hatschek s pit. It is suggested that a primitive control system of inhibitory growth hormone secretion in Hatschek s pit could have been developed in amphioxus, as in vertebrates. The present study provides new evidence for the endocrinology and the evolution of Hatschek's pit.     

mmunohistochemical localization of gonadotropin-releasing hormone receptors (GnRHR) in the nervous system, Hatschek's pit and gonads of amphioxus, Branchiostoma belcheri

Using gonadotropin-releasing hormone (GnRH) anti-idiotypic antibodies and APA im-munohistochemical method, the immunoreactivity of GnRHR in the nervous system, Hatschek's pit and gonads of amphioxus has been located. It is found for the first time that the immunoreactivity of GnRHR exists in the nerve cells and fibers in the amphioxus's brain and nerve tube and the epithelial cells of Hatschek's pit at the different stages of gonadal development. At the same time, it is also found that GnRHR also exists in the ovary and testis of different developed stages. These findings provide morphological new proof for the informative transfer and regulation between brain and Hatschek's pit mediation by GnRHR, and for the understanding of the mechanism of action on the reproductive endocrine control axis among brain-Hatschek's pit-go-nads.     

Distribution of α-MSH-like immunoreactive cells in the nervous system, Hatschek''''s pit and other tissues of amphioxus, Branchiostoma belcheri

Immunohistochemical localization of a-melanocyte-stimulating hormone (α-MSH) in the nervous system, Hatschek's pit and other tissues of amphioxus (Branchiostoma belcheri) was performed using the antibody against synthetic α-MSH. The results revealed that α-MSH-like immunoreactive cells were distributed at the dorsal side and ventral side of brain vesicle, the dorsal side and the surrounding of nerve tube, and in the epithelial cells of Hatschek's pit, the zone 1, 3, and 6 of endostyle and gut. The immunoreactive substance was also found in the primary oocytes of the small and large growth stage of ovary and early stage spermatogenic cells in testis. These findings indicate that α-MSH is an ancient and highly conserved hormone and it is extensively distributed in amphioxus. Although Hatschek's pit in amphioxus does not have a structure of the intermediate lobe of vertebrate adenohypophysis, it has already hosted α-MSH-like endocrine cells, implying that the functional differentiation of α-MSH-like cells occurred earlier than the differentiation of the tissue structure. The results of the present study provided a new evidence for the endocrinology of Hatschek's pit and for the origin and evolution of vertebrate adenohypophysis.     

Localization and expression of LHβmRNA in the brain and Hatschek's pit of amphioxus, Branchiostoma belcheri

To determine whether gonadotropin-like substance in the brain and Hatschek' s pit of amphioxus issynthesized by the tissue in situ or transported from other tissue, a histochemical study was carried out by in situ hy- bridization using digoxigenin (DIG)-labelled LHβRNAprobes. The results showed that LHβmRNA expressed in the nerve cells of brain and the epithelial cells of Hatschek' s pit, thus providing new evidence for the homology of pituitary of ver- tebrates with Hatschek' s pit and the functional evolution of gonadotropin.     

Immunohistochemical localization of gonadotropin-releasing hormone receptors (GnRHR) in the nervous system, Hatschek’s pit and gonads of amphioxus,Branchiostoma belcheri

Using gonadotropin-releasing hormone (GnRH) anti-idiotypic antibodies and APA immunohistochemical method, the immunoreactivity of GnRHR in the nervous system, Hatschek’s pit and gonads of amphioxus has been located. It is found for the first time that the immunoreactivity of GnRHR exists in the nerve cells and fibers in the amphioxus’s brain and nerve tube and the epithelial cells of Hatschek’s pit at the different stages of gonadal development. At the same time, it is also found that GnRHR also exists in the ovary and testis of different developed stages. These findings provide morphological new proof for the informative transfer and regulation between brain and Hatschek’s pit mediation by GnRHR, and for the understanding of the mechanism of action on the reproductive endocrine control axis among brain-Hatschek’s pit-gonads.     

Distribution of ghrelin-like immunoreactive cells in amphioxus, Branchiostoma belcheri – A study of immunohistochemistry

The distribution of ghrelin-like immunoreactive cells in amphioxus （Branchiostoma belcheri） was investigated by using immunohistochemical staining with rabbit antiserum against synthetical mammalian ghrelin. The results showed that ghrelin-like immunoreactive cells were distributed widely in the nervous system, Hatschek＇s pit, wheel organ, digestive tract and gonads （ovary and testis）. In nervous system, ghrelin-like immunoreactive neurons and their protrusions were distributed specifically on the dorsal side, ventral side and funnel part of brain vesicle, with a few dispersive immunoreactive nerve cells and their fibers in nerve tube. Ghrelin-like immunoreactivities were also detected in Hatschek＇s pit epithelial cells and wheel organ cells, with positive substance located along cell membrane. In digestive tract, ghrelin-like immunoreactive cells existed in hepatic diverticulum, anterior and posterior region of midgut, and could be classified into two types, closed- and opened-type endocrine cells. The number of positive cells was most in hepatic diverticulum, secondary in posterior region of midgut and least in anterior region of midgut. In gonads, ghrelin-like immunoreactive substance was detected in oogonia, oocytes and follicle cells in ovary at the small and large growth stages and in early spermatogenic cells and Sertoli cells in testis. The extensive distribution of ghrelin-like cells in amphioxus suggested that these kinds of cells are conservative in evolution and diversified in function. At the same time, we found for the first time that ghrelin-like immunoreactive cells existed in brain vesicle and Hatschek＇s pit, which provided new morphological evidence for the existence of an activation pathway between brain vesicle and Hatschek＇s pit for the regulation of growth hormone excretion.     

Distribution of CRH-and ACTH-like immunoreactive cells in amphioxus,Branchiostoma belcheri

Immunocytochemical studies on the nervous system,Hatschek's pit,digestive tract and gonads tissues of an amphioxus(Branchiostoma belcheri)were performed using polyclonal antibodies against human corticotrophin-releasing hormone(CRH)and human adrenocorticotropin(ACTH).The results showed that many CRH-like immunoreactive neurons were distributed specifically on the dorsal side and ventral side of brain vesicle,while a few CRH-like neurons and their fibers in spinal cord.At the same time,the epithelial cells in the basic region of Hatschek's pit were shown immunopositive to CRH antibody.In gonads(ovary and testis),CRH-immunopositive substance was localized in the cytoplasm near oocyte nucleus and in early spermatogenic cells.ACTH-like immunoreactivities were observed specially in the neurons and their protrusions localized on the ventral side of the brain vesicle and in spinal cord,and also in epithelial cells of Hatschek's pit,enteric neurons of digestive tract,oocytes in ovary and in early spermatogenic cells as well.It was found for the first time that CRH-like neurons existed in the middle region of brain vesicle(corresponding to the hypothalamus of vertebrates)and ACTH-like immunopositive cells existed in Hatschek's pit,implying that a control mechanism between brain vesicle and Hatschek's pit maybe had been already built in amphioxus as that in vertebrates.The present study will provide new morphological evidence for the origin and evolution of ACTH.In addition,the immunoreactivities of CRH and ACTH in the digestive tract and gonads suggested other physiological function of CRH and ACTH in amphioxus.     

Distribution of a-MSH-like immunoreactive cells in the nervous system,Hatschek's pit and other tissues of amphioxus,Branchiostoma belcheri

Immunohistochemical localization of α-melanocyte-stimulating hormone （α-MSH） in the nervous system, Hatschek＇s pit and other tissues of amphioxus （Branchiostorna belcheri） was performed using the antibody against synthetic α-MSH. The results revealed that α-MSH-like immunoreactive cells were distributed at the dorsal side and ventral side of brain vesicle, the dorsal side and the surrounding of nerve tube, and in the epithelial cells of Hatschek＇s pit, the zone 1, 3, and 6 of endostyle and gut. The immunoreactive substance was also found in the primary oocytes of the small and large growth stage of ovary and early stage spermatogenic cells in testis. These findings indicate that α-MSH is an ancient and highly conserved hormone and it is extensively distributed in amphioxus. Although Hatschek＇s pit in amphioxus does not have a structure of the intermediate lobe of vertebrate adenohypophysis, it has already hosted α-MSH-like endocrine cells, implying that the functional differentiation of α-MSH-like cells occurred earlier than the differentiation of the tissue structure. The results of the present study provided a new evidence for the endocrinology of Hatschek＇s pit and for the origin and evolution of vertebrate adenohypophysis.     

Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system of the human tumor cell growth

Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the censitivity of chemotherapy and radiotherapy.E1A have the ability to integrate into the host genome,resulting in long-time expres-sion that induces Rb gene inactivation and animal cells im-mortalization.This prompted us to select the E1A protein for treatment of cancer in order to overcome the limitations of E1A gene therapy.Thus,we firstly comstructed E1A eu-caryotic expression vector (pPIC9/E1A),transformated the pichia pastoris yeast cells(GS115) and screened the high-expressing recombinant strains.The positive yeast strains were cultured in the shake flask,and induced for 3d.The crude E1A protein was purified using two steps of col-umu chromatography on HiTrap Q and HiTrap SP.The pu-rified E1A protein was identified by SDS-PAGE and Western blot.E1A protein was mostly located at cellular unclear when Cheriot delivered E1A protein into cells.The analysis in vitro indicated that the E1A protein arrested LN686 cell cycle at G2/M phase,and significantly inhibited the growth of LN686 tumor cells.The current studies firstly provided an experimental basis to further develop E1A protein for tumor treatment.     

Immunorecognition of estrogen and androgen receptors in the brain and thoracic ganglion mass of mud crab, Scylla paramamosain

The brain and the thoracic ganglion of crustacean can synthesize and secrete gonad-stimulating hormone (GSH) which stimulates the maturation of gonad. In the previous experiments, sex steroid hormones (estradiol, testosterone, progesterone, etc.) have been detected from crustacean. However, the feedback regulation of sex steroid hormones on the brain and thoracic ganglion of crustacean has not been reported so far. In the present experiment, monoclonal antibodies were applied to investigate the immunorecognition of estrogen receptor (ER) and androgen receptor (AR) in the brain and thoracic ganglion mass of Scylla paramamosain. The results showed that the distribution of the immunopositive substances of ER and AR was extremely similar. They distributed in the protocerebrum, deutocerebrum and tritocerebrum of the brain, and mainly in protocerebrum. In the thoracic ganglion mass, immunopositive substances distributed in the subesophageal ganglion, thoracic ganglion and abdomen ganglion, and mostly in subesophageal ganglion. Immunopositive substances of ER and AR mostly existed in the cytoplasm of neurons. The present study will provide morphological evidence for the origin and evolution of ER and AR. In addition, the immunoreactivities of ER and AR suggested that the estrogen and androgen may be involved in the feedback regulation of crustacean neuroendocrine.     

Immunorecognition of estrogen and androgen receptors in the brain and thoracic ganglion mass of mud crab, Scylla paramamosain

The brain and the thoracic ganglion of crustacean can synthesize and secrete gonad-stimulating hormone (GSH) which stimulates the maturation of gonad. In the previous experiments, sex steroid hormones (estradiol, testosterone, progesterone, etc.) have been detected from crustacean. However, the feedback regulation of sex steroid hormones on the brain and thoracic ganglion of crustacean has not been reported so far. In the present experiment, monoclonal antibodies were applied to investigate the immunorecognition of estrogen receptor (ER) and androgen receptor (AR) in the brain and thoracic ganglion mass of Scylla paramamosain. The results showed that the distribution of the immunopositive substances of ER and AR was extremely similar. They distributed in the protocerebrum, deutocerebrum and tritocerebrum of the brain, and mainly in protocerebrum. In the thoracic ganglion mass, immunopositive substances distributed in the subesophageal ganglion, thoracic ganglion and abdomen ganglion, and mostly in subesophageal ganglion. Immunopositive substances of ER and AR mostly existed in the cytoplasm of neurons. The present study will provide morphological evidence for the origin and evolution of ER and AR. In addition, the immunoreactivities of ER and AR suggested that the estrogen and androgen may be involved in the feedback regulation of crustacean neuroendocrine.     

CTCF通过抑制p53信号通路促进胆管癌细胞的生长、迁移和侵袭

为了探索CCCTC结合因子(CCCTC-binding factor, CTCF)对人胆管癌细胞的生长、迁移和侵袭能力的影响及其潜在的分子作用机制,利用慢病毒感染法获得稳定过表达或敲低CTCF的胆管癌细胞系,采用蛋白质免疫印迹法(Western blotting, Wb)检测CTCF蛋白表达水平,采用Cell Counting Kit-8(CCK-8)法和克隆形成实验检测细胞生长情况,采用Transwell小室实验检测细胞迁移和侵袭能力,采用GraphPad软件(v6)的Mann-Whitney U检验分析CTCF基因在癌和癌旁组织间的mRNA差异表达.结果显示:过表达CTCF可显著促进HCCC-9810和RBE胆管癌细胞的生长、迁移和侵袭能力;敲低CTCF呈现相反的表型.通过通路富集分析发现:CTCF的表达水平在胆管癌中与p53信号通路活性显著负相关,过表达CTCF可显著下调p53及其靶基因p21的mRNA和蛋白表达水平,而敲低CTCF则显著促进p53和p21的mRNA和蛋白表达水平.综上,本研究揭示CTCF可能通过抑制p53信号通路而促进胆管癌细胞生长、迁移和侵袭而发挥促癌基因的功能.     

Leptomeningeal metastasis from central nervous system tumors: A study of classification and stage in the spinal canal of 58 patients

Leptomeningeal metastasis (LM) is caused by the spread of malignant tumor cells into the subarachnoid space.However,classification and staging of LM in the spinal canal is rare in the literature.The authors reviewed the records of 58 Chinese patients with LM for clinical features,neuroimaging,and treatments.Gadolinium-enhanced magnetic resonance imaging (MRI) of brain and spinal cord were performed in all patients.Removal of intracranial tumors was performed in all patients and diagnoses were confirmed by histology.The study group consisted of 58 patients,with 29 cases presenting with intraspinal symptoms.Of the 58,8 patients underwent intraspinal tumor removal,8 received radiotherapy alone,9 received chemotherapy alone,and 34 patients received combined radiochemotherapy.We classified LM into 3 types:type L or leptomeninges LM,is subdivided into 2 subtypes (subtype LI and LII (a,b)),type N or nerve root LM is subdivided into 2 subtypes (subtype NI and NII (a,b)),and type M or mixed-type LM.We also divided LM into stages of I-IV according to the symptoms and the volume of the tumor based on spinal axial MRI.Type LI LM often occurs in patients with intracranial and intraspinal tumors found simultaneously.Patients who receive surgery for intracranial tumors may present with type N LM.Surgery is suitable for patients with NI LM and LIIb LM in stages III-IV,presenting with severe spinal symptoms.The prognosis is better for type N LM than type L LM.     

Cloning and characterization of 37 kD laminin receptor precursor in pearl oyster, Pinctada fucata

A 1063 bp cDNA clone encoding a putative 37 kDa laminin receptor precursor （37 kDa LRP） is isolated from the mantle tissue of pearl oyster, Pinctadafucata. The amino acid sequence predicted from the cDNA sequence is 301 residues long, with a calculated molecular mass of 33.5 kDa. RT-PCR analysis shows that 37 kDa LRP mRNA is especially highly expressed in the mantle while widely expressed in several tissues. In situ hybridization analysis reveals that 37 kDa LRP is expressed in the outer epithelial ceils of the mantle edge, suggesting its involvement in cell proliferation and secretion in P. fucata. The identification and characterization of 37 kDa LRP in the pearl oyster will help us to further understand the signal transduction in the processes of mantle epithelial cell proliferation and tissue formation.     

Cloning and characterization of 37 kD laminin receptor precursor in pearl oyster, Pinctada fucata

A 1063bp cDNA clone encoding a putative 37 kD laminin receptor precursor (37 kD LRP) is isolated from the mantle tissue of pearl oyster, Pinctada fucata. The amino acid sequence predicted from the cDNA sequence is 301 residues long, with a calculated molecular mass of 33.5 kD. RT-PCR analysis shows that 37 kD LRP mRNA is especially highly expressed in the mantle while widely expressed in several tissues. In situ hybridization analysis reveales that 37 kD LRP is expressed in the outer epithelial cells of the mantle edge, suggesting its involvement in cell proliferation and secretion in P. fucata. The identification and characterization of 37 kD LRP in the pearl oyster will help us to further understand the signal transduction in the processes of mantle epithelial cell proliferation and tissue formation.     

广西北海大冠沙白骨壤树上大型固着动物的数量及其分布

本文研究了广西北海大冠沙白骨壤树上大型固着动物的种类组成、数量分布特点及其对植物体的危害。结果表明,大冠沙白骨壤群落中的大型固着生物有10种,分属3门4纲6科,其中在白骨壤植株上的主要种类为白条小藤壶、潮间藤壶、褶牡蛎和黑荞麦蛤4种。危謇程度最大的是白条小藤壶,其湿重生物量占总湿重生物量的百分比高达72.3%～100%。受害程度最大的是向海林带的枝和叶,其动物生物量干重与植物生物量干重的比值(a/p)可达30.49×10~(-2)和30.43×10~(-2)。就整个红树林区而言,危害程度为向海林带(21.68×10~(-2))>中间林带(7.88×10~(-2))>向陆林带(0.01×10~(-2)).同一林带的植株受害程度为较低树层的大于较高树层的.密度和生物量的分布也与此相类似.     

Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system on the human tumor cell growth

Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the sensitivity of chemotherapy and radiotherapy. E1A have the ability to integrate into the host genome, resulting in long-time expression that induces Rb gene inactivation and animal cells immortalization. This prompted us to select the E1A protein for treatment of cancer in order to overcome the limitations of E1A gene therapy. Thus, we firstly constructed E1A eucaryotic expression vector (pPIC9/E1A), transformated the pichia pastoris yeast cells (GS115) and screened the high-expressing recombinant strains. The positive yeast strains were cultured in the shake flask, and induced for 3 d. The crude E1A protein was purified using two steps of column chromatography on HiTrap Q and HiTrap SP. The purified E1A protein was identified by SDS-PAGE and Western blot. E1A protein was mostly located at cellular nuclear when Chariot delivered E1A protein into cells. The analysis in vitro indicated that the E1A protein arrested LN686 cell cycle at G2/M phase, and significantly inhibited the growth of LN686 tumor cells. The current studies firstly provided an experimental basis to further develop E1A protein for tumor treatment.     

Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system

The human adenovirus type 5 E1A, a tumor- suppressor gene[1], codes for two major related proteins of 243 amino acids (12S) and 289 amino acids (13S) by al-ternative splicing in two exons[2]. Studies have been shown that E1A can regulate expression of many genes and cell cycle[3]. Both in vitro and in vivo experiments indicated that E1A could induce tumor cells differentia-tion, convert tumor cells into an epithelial phenotype, in-hibit tumor cell growth and metastasis and strongly en-ha…     

大气CO2浓度和温度变化对石莼生长及其叶绿素荧光特性的影响

【目的】为了研究大型海藻在全球气候变化背景下的生态功能,探讨大气 CO2浓度升高和温度变化对石莼（Ulva lactuca）生长及其叶绿素荧光特性的影响。【方法】在4种条件（390μL／L CO2＋15℃;700μL／L CO2＋15℃;390μL／L CO2＋25℃;700μL／L CO2＋25℃）下培养石莼,10 d后测定藻体生长、叶绿素荧光参数以及生化组分。【结果】经10℃低温6 h处理,石莼受到光抑制;经35℃高温6 h处理,25℃正常空气条件下生长的石莼表现出较高的最大光量子产量（Fv／Fm）,光能利用效率（α）,非光化学淬灭（NPQ）和光化学淬灭（qP ）,25℃高CO2浓度条件下生长的石莼最大相对电子传递速率（rETRmax）和饱和电子传递速率（Ek ）大于其他生长条件下的石莼;经40℃高温处理6 h,15℃生长的石莼光合机构受损;相对于25℃与正常空气条件下生长的石莼,25℃与高CO2浓度条件下生长的石莼Fv／Fm、a、NPQ和qP 的下降程度较小,而且具有较高的rETRmax 和Ek。【结论】高CO2浓度促进石莼的生长,但其对石莼可溶性蛋白（SP ）、可溶性碳水化合物（SC ）、叶绿素 a （Chl a）和类胡萝卜素（Car ）等生化组分含量影响具有温度依赖性。另外,CO2浓度升高使得石莼抗高温的能力增强。