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.     

Distribution of α-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 (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 the brain vesicle, the dorsal side and the surrounding of central tube in the nerve tube, the epithelial cells of Hatschek’s pit, and zones 1, 3 and 6 in endostyle and hindgut. 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 do 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 present study provided new evidence for the endocrinology of Hatschek’s pit and the origin and evolution of vertebrate adenphypophysis.     

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 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.     

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.     

Neuroendocrine regulation of structure and function in the endostyle of amphioxus, Branchiostoma belcheri

Using histological and histochemical methods the structure of the endostyle in amphioxus at different gonadal developmental stages is studied, and the immunocytochemical localization of thyroglobulin (Tg) and thyrotropin (TSH) in the endostyle, Hatschek's pit and brain vesicle is investigated. The results not only confirm the previous report that the endostyle is composed of 6 zones and the cells of zone 5 are thyroid hormone synthesizing cells, but also find the thyroxine synthesis in and secretion from zone 3. In addition, the epithelial cells in Hatschek's pit and the neural cells in brain vesicle are immunopositive for TSH, and the immunoactivity is correlated with gonadal cycle. The present study may provide morphological proof for the hypothesis that the secretory activity of thyroid cells is regulated by TSH from Hatschek's pit and brain vesicle.     

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 speci?cally on the dorsal side, ventral side and funnel part of brain vesicle, with a few dispersive immunoreactive nerve cells and their ?bers 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 ?rst 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 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.     

Immunocytological evidence for the presence of vertebrate FSH- and LH-like substances in the brain and thoracic ganglion of the swimming crab，Portunus trituberculatus

Investigating the distribution of follicle stimulating hormone （FSH） and luteinizing hormone （LH） in the swimming crab, Portunus trituberculatus, helps in the understanding of its reproductive regulation mechanism. This study has detected the location of vertebrate FSH- and LH-like substances in the brain and the thoracic ganglion mass with immunocytochemical techniques. Their immunoreaction is very weak or negative during the immature period and the post-spawning period, while it is stronger in the mature period. Localization of FSH-like immunoreactive cells resembles that of LH-like cells. These immunoreactive cells were observed chiefly in the anterior median cell-duster of the protocerebrum with stronger immunostaining. There were only a few in the inboard cell-cluster of the olfactory lobe of the deutocerebrum and the posterior cell-cluster of the tritocerebrum. Immunoreactive cells were located in all the three ganglia in the thoracic ganglion mass. These results indicated that substances resembling the vertebrate FSH and LH exist in P. trituberculatus, and their expression has some correlation with the developmental stage of ovaries, thus they might engage in the ovary development and ovulation.     

Positional variation of antipodal cells in polyembryonic rice ApⅢ before and after fertilization

The variation in the position of antipodal cells before and after fertilization in polyembryonic rice strain ApⅢ was studied by using conventional sectioning technique. It was shown that initially the three antipodal cells lie at the chalazal end of the young embryo sacs. Along with the development of embryo sac, the antipodal cells proliferate into a multicellular structure containing up to about 20 cells. Most of the cells migrate to its dorsal side when the embryo sacs turn into mature. In a few embryo sacs the antipodal cells are situated in the positions adjacent to the egg apparatus at the micropylar end, while in other sacs, they spread from the midst of the embryo sac to the micropylar end clinging to the nucellar tissue. Furthermore, it is reported for the first time that cells of some egg apparatus or proembryos disorganize when antipodal cells lie at the micropylar end, indicating that the function of the antipodal cells may vary during the embryogenesis in rice ApⅢ.     

Corrosion behavior of high-strength spring steel for high-speed railway

The corrosion resistance and evolution of corrosion products in medium-carbon high-strength spring steels were investigated in a neutral salt spray (5wt% NaCl solution). A formation model of γ-FeOOH and a transformation model describing the conversion of γ-FeOOH to α-FeOOH were constructed. The results indicated that, at the initial corrosion stage, the corrosion resistance was gradually improved with the addition of Cr; however, with the addition of alloying element V, the corrosion resistance decreased. These results were attributed mainly to the initial corrosion stage being closely related to the matrix microstructure parameters such as grain-boundary character and dislocation density. After the rust layer was formed at a later corrosion stage, the corrosion resistance was reinforced with the addition of Cr and V because Cr strongly influenced the composition, structure, and morphology of the corrosion products. The results presented herein show that Cr was conducive to the transformation of γ-FeOOH into α-FeOOH. Moreover, V and Cr exhibited obvious synergy and were enriched in the inner layer of the corrosion products.     

Mechanisms of plant somatic embryogenesis

Based on our experimental results and the related research reports, this review addresses some important issues about plant somatic embryogenesis. Once plant somatic cells develop into embryonic cells, the organelles of the cells increase, and their function is active. In the early stage of embryogenic cells, the reaction products of AT-Pase are mainly deposited on the plasma and vacuole's membranes, then the activity of ATPase can be found in the cytoplasm, vacuole and nucleus in the late stage of embryogenic cells. ATPase-catalyzed reaction occurs actively in the thickened wall of embryogenic cells. Endogenous hormone is a critical factor affecting somatic embryogenesis. Superox-ide dismutase (SOD) activity, which is closely related to embryogenic cell differentiation, is much higher in differentiated embryogenic cells than in undifferentiated cells, indicating that embryogenic cells have stronger function of antioxida-tion. In addition, the lower level H2O2 can promote embryogenic cell differentiation. The programmed cell death (PCD) exists in the process of embryogenic cell differentiation and development, and the active oxygen species plays an important role in inducing of plant PCD.     

Regulation of BMP4 on the proliferation and differentiation in SVZa neural stem cells

The neural stem cells in the anterior subventricular zone (SVZa) mainly generate the progenitors that will differentiate into neurons, and along a highly circumscribed migratory access Rostral migratory stream (RMS), they migrate to the olfactory bulbs (OB). To understand the effects of BMPs on SVZa neural stem cells, in this study BMP4 at various concentrations was used to induce SVZa neural stem cells, and the living cell labeling using BMP4 promotor conjugated with red fluorescence protein showed the expression of BMP4 dynamically. The results demonstrated that low BMP4 doses (1-5 ng/mL) promoted while high doses (10-100 ng/mL) inhibited the proliferation of SVZa neural stem cells, and BMP4 promotedneuron differentiation in the early stage (1-3 d), howeverm, it inhibited the neuron commitment after 4 d. Noggin, the antagonist of BMP4, blocked the physiological effects of BMP4. In OB, BMP4 is mainly to accelerate the progenitors to withdraw from the cell cycle and trigger the differentiation, and in RMS, it promotes the proliferation of committed progenitors and not differentiation, further in SVZa, BMP4 enhances astrocyte commitment.     

Side Structure Sensitivity to Passenger Car Crashworthiness During Pole Side Impact

Side impact accidents of passenger cars with fixed poles may result in severe injuries to the vehi-cle occupants. In this paper, side structure intrusion was considered as the criterion for passenger cars crashworthiness during side impact with a pole. The relationship between side intrusions and the side struc-ture stiffness was analyzed. The acceleration of the unstruck side vehicle body was selected as the criterion for studying the influence of different side structure components on the side structure stiffness during pas-senger car pole side impacts. The behavior was analyzed using finite element simulations. The results show that the rocker and the lower part of the B-pillar are the key parts of the side structures in determining the passenger car side stiffness. Passenger car pole side impact crashworthiness is, therefore, most sensitive to these two components.     

Induction of embryonic stem cells to hematopoietic cellsin vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright’s staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34⁺ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34^－CD38⁺. Wright’s staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Comparative study on the effect of integrin αvβ3 on secretion of matrix metalloproteinases in human normal and carcinoma trophoblasts

The invasion of cytotrophoblast cells into the maternal endometrium during embryonic implantation is very similar to the metastasis of carcinoma cells. However, the significant difference is that the former is a highly controlled process. In this report, the effects of integrin αvβ3 on the secretion of matrix metalloproteinase (MMP) were compared between normal cytotrophoblast cells and choriocarcinoma cells by RT-PCR, gelatin zymography and immunocytochemistry. The results reveal that both normal cytotrophoblast cells and choriocarcinoma cells can express integrin αvβ3. The secretion of MMPs in normal cytotrophoblast ceils is up-regulated by anti-αvβ3 antibody, whereas, decreased in choriocarcinoma cells. It was suggested that αvβ3 can modulate the expression of MMPs in trophoblasts, and this action is carried out through distinct mechanisms in normal and carcinoma cytotrophoblast cells.     

Comparative study on the effect of integrin αvβ3 on secretion of matrix metalloproteinases in human normal and carcinoma trophoblasts

The invasion of cytotrophoblast cells into the maternal endometrium during embryonic implantation is very similar to the metastasis of carcinoma cells. However, the significant difference is that the former is a highly controlled process. In this report, the effects of integrin αvβ3 on the secretion of matrix metalloproteinase (MMP) were compared between normal cytotrophoblast cells and choriocarcinoma cells by RT-PCR, gelatin zymography and immunocytochemistry. The results reveal that both normal cytotrophoblast cells and choriocarcinoma cells can express integrin αvβ3. The secretion of MMPs in normal cytotrophoblast cells is up-regulated by anti-αvβ3 antibody, whereas, decreased in choriocarcinoma cells. It was suggested that αvβ3 can modulate the expression of MMPs in trophoblasts, and this action is carried out through distinct mechanisms in normal and carcinoma cytotrophoblast cells.     

A sperm-mediated GFP gene transfer in amphioxus（Branchiostoma belcheri tsingtaoense）

The cephalochordate amphioxus is the closest living invertebrate relative of the vertebrate and has emerged as an important animal model for evolutionary developmental biology research. Many insights into the evolution of the vertebrate body plan can be gained by comparing vertebrate embryogenesis with that of the amphioxus. Investigation of the gene regulation during amphioxus embryogenesis will provide us clues to clarify how changes of gene regulation affect the body plan evolution at this turning point and thus will throw light on the origin and evolution of the vertebrate.