Identification and characterization of a MBP isoform specific to hypothalamus in orange-spotted grouper (Epinephelus coioides)

Myelin basic protein (MBP), as a major component of the myelin sheath, has been revealed to play an important role informing and maintaining myelin structure in vertebrate nervous system. In teleost, hypothalamus is an instinctive brain center and plays significant roles in many physiological functions, such as energy metabolism, growth, reproduction, and stress response. In comparison with other MBP identified in vertebrates, a smallest MBP is cloned and identified from the orange-spotted grouper hypothalamic cDNA plasmid library in this study. RT-PCR analysis and Western blot detection indicate that the EcMBP is specific to hypothalamus, and expresses mainly in the tuberal hypothalamus in adult grouper. Immunofluorescence localization suggests that EcMBP should be expressed by oligodendrocytes, and the expressing cells should be concentrated in hypothalamus and the area surrounding hypothalamus, such as NPOpc, VC, DP, NLTm, and NDLI The studies on EcMBP expression pattern and developmental behaviour in the brains of grouper embryos and larvae reveal that the EcMBP-expressing cells are only limited in a defined set of cells on the border of hypothalamus, and suggest that the EcMBP-expressing cells might be a subpopulation of oliaodendrocyte progenitor cells. This study not only identifies a smallest MBP isoform specific to hypothalamus that can be used as a molecular marker of oligodendrocytes in fish, but also provides new insights for MBP evolution and cellular distribution. (C) 2007 Elsevier B.V.. All rights reserved.

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. (c) 2006 Published by Elsevier B.V.

Inductive expression and characterization analysis of Paralichthys olivaceus pigment epithelium-derived factor in a virally infected cell line

Pigment epithelium-derived factor (PEDF) is acknowledged to be a non-inhibitory member of the serine protease inhibitor (serpin) superfamily, with antiangiogenesis, and neuroprotective and immumoregulatory function, mainly in the tissues of nervous system. Here, A PEDF gene homolog, Paralichthys olivaceus PEDF (PoPEDF), was isolated from flounder embryonic cells (FEC) treated with UV-inactivated Grass carp hemorrhage virus (GCHV) and subsequently identified as a differentially expressed gene. The full length of PoPEDF cDNA is 1803 bp with an open reading frame of 1212 bp encoding a 403-amino-acid protein. This deduced protein contains an N-terminal signal peptide, a glycosylation site, a consensus serpin motif, and a 34-mer and a 44-mer fragment, all of which are very conserved in the PEDF family. PoPEDF gene exhibits a conserved exon-intron arrangement with 8 exons and 7 introns. This conserved evolutionary relationship was further confirmed by a phylogenetic analysis, where fish PEDFs and mammalian members formed a well-supported clade. Constitutive expression of PoPEDF was widely detected in many tissues. In response to UV-inactivated GCHV or poly(I:C), PEDF mRNA was upregulated in FEC cells with time. This is the first report on the transcriptional induction of PEDF in virally infected cells. (C) 2005 Elsevier Inc. All rights reserved.

Early Embryonic Exposure to Polychlorinated Biphenyls Disrupts Heat-Shock Protein 70 Cognate Expression in Zebrafish

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that have documented neurological effects in children exposed in utero. To better define neuronally linked molecular targets during early development, zebrafish embryos were exposed to Aroclor 1254, a mixture of PCB congeners that are common environmental contaminants. Microarray analysis of the zebrafish genome revealed consistent significant changes in 38 genes. Of these genes, 55% (21) are neuronally related. One gene that showed a consistent 50% reduction in expression in PCB-treated embryos was heat-shock protein 70 cognate (Hsc70). The reduction in Hsc70 expression was confirmed by real-time polymerase chain reaction (PCR), revealing a consistent 30% reduction in expression in PCB-treated embryos. Early embryonic exposure to PCBs also induced structural changes in the ventro-rostral cluster as detected by immunocytochemistry. In addition, there was a significant reduction in dorso-rostral neurite outgrowth emanating from the RoL1 cell cluster following PCB exposure. The serotonergic neurons in the developing diencephalon showed a 34% reduction in fluorescence when labeled with a serotonin antibody following PCB exposure, corresponding to a reduction in serotonin concentration in the neurons. The total size of the labeled neurons was not significantly different between treated and control embryos, indicating that the development of the neurons was not affected, only the production of serotonin within the neurons. The structural and biochemical changes in the developing central nervous system following early embryonic exposure to Aroclor 1254 may lead to alterations in the function of the affected regions.

Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

Amphi-Eomes/Tbr1: An amphioxus cognate of vertebrate Eomesodermin and T-Brain1 genes whose expression reveals evolutionarily distinct domain in amphioxus development

A cDNA for a novel T-box containing gene was isolated from the amphioxus Branchiostoma belcheri. A molecular phylogenetic tree constructed from the deduced amino acid sequence of the isolated cDNA indicates that this gene belongs to the T-Brain subfamily. In situ hybridization reveals that the expression is first detected in the invaginating archenteron at the early gastrula stage and this expression is down-regulated at the neurula stage. In early larvae, the expression appears again and transcripts are detected exclusively in the pre-oral pit (wheel organ-Hatschek's pit of the adult). In contrast to the vertebrate counterparts, no transcripts are detected in the brain vesicle or nerve cord throughout the development. These results are interpreted to mean that a role of T-Brain products in vertebrate forebrain development was acquired after the amphioxus was split from the lineage leading to the vertebrates. On the other hand, comparison of the tissue-specific expression domain of T-Brain genes and other genes between amphioxus and vertebrates revealed that the pre-oral pit of amphioxus has several molecular features which are comparable to those of the vertebrate olfactory and hypophyseal placode. (C) 2002 Wiley-Liss, Inc.

Phenoloxidase, a marker enzyme for differentiation of the neural ectoderm and the epidermal ectoderm during embryonic development of amphioxus Branchiostoma belcheri tsingtaunese

The development of phenoloxidase during amphioxus embryogenesis was spectrophotometrically and histochemically studied for the first time in the present study. It was found that (1) PO activity initially appeared in the general ectoderm including the neural ectoderm and the epidermal ectoderm at the early neurala stage but not in the mesoderm or the endoderm, and (2) PO activity disappeared in the neural plate cells but remained unchanged in the epidermal cells when the neural plate was morphologically quite distinct from the rest of the ectoderm. It is apparent that PO could serve as a marker enzyme for differentiation of the neural ectoderm from the epidermal ectoderm during embryonic development of amphioxus. (C) 2000 Elsevier Science ireland Ltd. All rights reserved.

Expression of a twist-related gene, Bbtwist, during the development of a lancelet species and its relation to cephalochordate anterior structures

Mesoderm formation plays a crucial role in the establishment of the chordate body plan. In this regard, lancelet embryos develop structures such as the anteriorly extended notochord and the lateral divertecula in their anterior body. To elucidate the developmental basis of these structures, we examined the expression pattern of a lancelet twist-related gene, Bbtwist, from the late gastrula to larval stages. In late-gastrula embryos, the transcripts of Bbtwist were detected in the presumptive first pair of somites and the middorsal wall of the primitive gut. The expression of Bbtwist was then upregulated in the lateral wall of somites and the notochord. At the late-neurula stage, it was also expressed in the anterior wall of the primitive gut, as well as in the evaginating lateral diverticula. No signal was detected in the left lateral diverticulum when it was separated from the gut, while in the right one, the gene was expressed later during the formation of the head coelom in knife-shaped larvae, and in the anterior part of the notochord in the same larvae. In 36-h larvae, only faint expression was detected in the differentiating notochordal and paraxial mesoderm in the caudal region. These expression patterns suggest that Bbtwist is involved in early differentiation of mesodermal subsets as seen in Drosophila and vertebrates. The expression in the anterior notochord may be related to its anterior expansion. The expression in the anterior wall of the primitive gut and its derivative, the lateral diverticula, suggests that lancelets share the capability to produce a mesodermal population from the tip of the primitive gut with nonchordate deuterostome embryos. (C) 1998 Academic Press.

A close phylogenetic relationship between Sipuncula and Annelida evidenced from the complete mitochondrial genome sequence of Phascolosoma esculenta

Background: There are many advantages to the application of complete mitochondrial (mt) genomes in the accurate reconstruction of phylogenetic relationships in Metazoa. Although over one thousand metazoan genomes have been sequenced, the taxonomic sampling is highly biased, left with many phyla without a single representative of complete mitochondrial genome. Sipuncula (peanut worms or star worms) is a small taxon of worm-like marine organisms with an uncertain phylogenetic position. In this report, we present the mitochondrial genome sequence of Phascolosoma esculenta, the first complete mitochondrial genome of the phylum. Results: The mitochondrial genome of P. esculenta is 15,494 bp in length. The coding strand consists of 32.1% A, 21.5% C, 13.0% G, and 33.4% T bases (AT = 65.5%; AT skew = -0.019; GC skew = -0.248). It contains thirteen protein-coding genes (PCGs) with 3,709 codons in total, twenty-two transfer RNA genes, two ribosomal RNA genes and a non-coding AT-rich region (AT = 74.2%). All of the 37 identified genes are transcribed from the same DNA strand. Compared with the typical set of metazoan mt genomes, sipunculid lacks trnR but has an additional trnM. Maximum Likelihood and Bayesian analyses of the protein sequences show that Myzostomida, Sipuncula and Annelida (including echiurans and pogonophorans) form a monophyletic group, which supports a closer relationship between Sipuncula and Annelida than with Mollusca, Brachiopoda, and some other lophotrochozoan groups. Conclusion: This is the first report of a complete mitochondrial genome as a representative within the phylum Sipuncula. It shares many more similar features with the four known annelid and one echiuran mtDNAs. Firstly, sipunculans and annelids share quite similar gene order in the mitochondrial genome, with all 37 genes located on the same strand; secondly, phylogenetic analyses based on the concatenated protein sequences also strongly support the sipunculan + annelid clade (including echiurans and pogonophorans). Hence annelid "key-characters" including segmentation may be more labile than previously assumed.

VTA-NAc内orexin A在吗啡和性奖赏相关行为中作用的研究

There are a lot of differences in the neural mechanisms underlying between drug reward and natural reward despite the common neual basis. Undoubtedly, revealing the common and the different mechanisms underlying drug reward and natural reward will promote the development of research on drug addiction. Among diversified natural rewards, sex is often compared to drug because sexual reward has more similarities to drug. The mesolimbic dopamine system (VTA-NAc pathway) is a common pathway activated by natural reinforcers and addictive drugs, mediating reward, emotion and motivation under physiological conditions. The neuroadaptations taking place in the central nervous system including the mesolimbic dopamine system after repeatedly drug taking leads to persistent drug craving, Orexin, a neuropeptide produced in the lateral hypothalamus, plays an important role in reward-associated, motivated behaviors. Orexin neurons have extensive projections to the mesolimbic dopamine system. In order to further investigate the roles of orexin A in drug reward, this study examined the regulatory roles of orexin A in the VTA and NAcSh on drug reinforcement (acqusition of morphine CPP) and drug-seeking behavior (expression of morphine CPP). Moreover, the roles of orexin A on drug reward were compared with sexual reward. The main results are as follows: 1. The expression of morphine CPP was inhibited by intracerebroventricularly (i.c.v.) administered OX1R antagonist SB334867; 2. The male unconditioned sexual motivation was not affected by i.c.v. administered SB334867. However, i.c.v. given orexin A inhibited unconditioned sexual motivation in sexually high-motivated rats but did not affect sexual motivation in low-motivated rats; 3. The acquisition and expression of morphine CPP was inhibited by SB334867 microinjected into the VTA. SB334867 or orexin A injected into the NAcSh did not influence the acquisition of morphine CPP, but orexin A increased the locomotor activity in rats treated with morphine (3mg/kg); 4. SB334867 microinjected into the VTA did not affect male copulatory behavior, neither affect the acqusition of copulatory CPP; 5. The expression of copulatory CPP was associated with increased Fos protein expression in hypothalamic orexin A neurons, and SB334867 microinjected into the VTA inhibited expression of copulatory CPP. These results suggest that, (1) endogenous orexin A is not involved in male unconditioned sexual motivation, but involved in drug craving; (2) orexin A in the VTA instead of in the NAc is involved in drug reinforcement; (3) orexin A in the VTA is critical for drug-seeking behavior, but it is still unclear for the role of orexin A in the NAcSh; (4) in contrast to drug reinforcement, orexin A in the VTA is not involved in reinforcing effect of sexual reward. Orexin A plays a role both in drug-seeking behavior and in sexual reward-seeking behavior, but the different orexin A neuron populations may be responsible for the roles of orexin A in two types of reward. In a word, the differential roles of orexin A in drug and sexual reward are found in the present study, which provides some evidence for further research on the mechanisms of drug addiction.