A novel tumor necrosis factor ligand superfamily member (CsTL) from Ciona savignyi: Molecular identification and expression analysis

A novel invertebrate TNF ligand was identified and characterized in Ciona savignyi. The CsTL cDNA consisted of 995 nucleotides and encoded 281 amino acids. A conserved TNF family signature and several motifs of TNF ligand superfamily were identified in deduced amino acid sequence of CsTL. Phylogenetic analysis grouped CsTL, CiTNF (predicted TNF ligand superfamily homolog in Ciona intestinalis) and urchin TL1A with their own cluster apart from mammalian TNF alpha, LTA, TNFSF15 and fish TNFa proteins. Expression studies demonstrated that CsTL mRNA is present in all tested tissues from unchallenged ascidians and its expression was significantly upregulated in hemocytes following LIPS injection. The recombinant CsTL protein expressed using a baculovirus expression system showed potential cytotoxic activity in L929 cells. Present results indicated that TNF ligand superfamity molecules are present in marine invertebrates. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus

Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular structure and expression patterns of flounder (Paralichthys olivaceus) Myf-5, a myogenic regulatory factor

Myf-5, a member of the myogenic regulatory factors (MRF), has been shown to be expressed in muscle precursors in early stage zebrafish embryos. The MRFs, including MyoD, Myf-5, Myogenin and MR-F4, belong to the basic Helix-Loop-Helix transcription factors that contain a conserved basic Helix-Loop-Helix (bHLH) domain. To better understand the role of Myf-5 in the development of fish muscles, we have isolated the Myf-5 genomic sequence and cDNA from Flounder (Paralichthys olivaceus), and analyzed its structures and patterns of expression. Promoter analysis identified several putative transcription factor binding sites such as an E-box, NF-Y sites that might confer muscle-specific expression. Myf-5 transcripts were first detected in the paraxial mesoderm that gives rise to slow muscles. During somitogenesis, Myf-5 expression was found in developing somites. Myf-5 expression decreased gradually in somites in the anterior region, but remained strong in the newly formed somites. In the hatching stage, the expression was also detected in other muscle cells such as head muscle and fin muscle. In the growing fish, RT-PCR results showed that Myf-5 was expressed in the skeletal muscle and intestine. (c) 2006 Elsevier Inc. All rights reserved.

Clay surface modification and its coagulation of red tide organisms

Surface modification of montmorillonite by means of Mg2+ insertion reaction has been studied and a positively charged montmorillonite has been prepared. The effects of preparation temperature and Mg2+ concentration on the positive charge property of the clay and on the clay coagulating Heterosigma akashiwo have been studied. The results showed that the modified clay enhanced the coagulation and the used amount decreased to 1/5-1/10 of the original. The removal rates of Heterosigma akashiwo were correlated positively with positive charge on the clay in accordance with theoretical model.

Hypoxia-inducible factor 1 alpha cDNA cloning and its mRNA and protein tissue specific expression in domestic yak (Bos grunniens) from Qinghai-Tibetan plateau

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated a-subunit and a constitutively expressed beta-subunit. How animals living on Qinghai-Tibetan plateau adapt to the extreme hypoxia environment is known indistinctly. In this study, the Qinghai yak which has been living at 3000-5000 m attitude for at least two millions of years was selected as the model of high hypoxia-tolerant adaptation species. The HIF-1 alpha ORFs (open reading frames) encoding for two isoforms of HIF-1 alpha have been cloned from the brain of the domestic yak. Its expression of HIF-1 alpha was analyzed at both mRNA and protein levels in various tissues. Both its HIF-1 alpha mRNA and protein are tissue specific expression. Its HIF-1 alpha protein's high expression in the brain, lung, and kidney showed us that HIF-1 alpha protein may play an important role in the adaptation to hypoxia environment. (c) 2006 Elsevier Inc. All rights reserved.

Cloning of hypoxia-inducible factor 1 alpha cDNA from a high hypoxia tolerant mammal - plateau pika (Ochotona curzoniae)

Hypoxia-inducible factor I is a transcription factor composed of HIF-1alpha and HIF-1beta. It plays an important role in the signal transduction of cell response to hypoxia. Plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant and cold adaptation species livin only at 3000-5000m above sea level on the Qinghai-Tibet Plateau. In this study, HIF-1alpha cDNA of plateau pika was cloned and its expression in various tissues was studied. The results indicated that plateau pika HIF-1alpha cDNA was highly identical to those of the human (82%), bovine (89%), mouse (82%), and Norway rat (77%). The deduced amino acid sequence (822 bp) showed 90%, 92%, 86%, and 86% identities with those of the human, bovine, house mouse, and Norway rat, respectively. Northern blot analyses detected two isoforms named pLHIF-1alpha and pSHIF-1alpha. The HIF-1alpha mRNA was highly expressed in the brain and kidney, and much less in the heart, lung, liver, muscle, and spleen, which was quite different from the expression pattern of mouse mRNA. Meanwhile, a new variant of plateau pika HIF-1alpha mRNA was identified by RT-PCR and characterized. The deduced protein, composed of 536 amino acids, lacks a part of the oxygen-dependent degradation domain (ODD), both transactivation domains (TADs), and the nuclear localization signal motif (NLS). Our results suggest that HIF-1alpha may play an important role in the pika's adaptation to hypoxia, especially in brain and kidney, and pika HIF-1alpha function pattern may be different from that of mouse HIF-1alpha. Further-more, for the high ratio of HIF-1alpha homology among the animals, the HIF-1alpha gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa. (C) 2004 Elsevier Inc. All rights reserved.

Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation

It has been suggested that endothelial apoptosis is a primary lesion in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). We tested this hypothesis by examining the phenotypic signatures of endothelial microparticles (EMP) in TTP patients. In addition, the effect of TTP plasma on microvascular endothelial cells (MVEC) in culture was further delineated. EMP released by endothelial cells (EC) express markers of the parent EC; EMP released in activation carry predominantly CD54 and CD62E, while those in apoptosis CD31 and CD105. We investigated EMP release in vitro and in TTP patients. Following incubation of MVEC with TTP plasma, EMP and EC were analysed by flow cytometry for the expression of CD31, CD51, CD54, CD62E, CD105, CD106 and von Willebrand factor (VWF) antigen. EMP were also analysed in 12 TTP patients. In both EC and EMP, CD62E and CD54 expression were increased 3- to 10-fold and 8- to 10-fold respectively. However, CD31 and CD105 were reduced 40-60% in EC but increased twofold in EMP. VWF expression was found in 55 +/- 15% of CD62E(+) EMP. Markers of apoptosis were negative. In TTP patients, CD62E(+) and CD31(+)/CD42b(-) EMP were markedly elevated, and preceded and correlated well with a rise in platelet counts and a fall in lactate dehydrogenase. CD62E(+) EMP (60 +/- 20%) co-expressed VWF and CD62E. The ratio of CD31(+)/42b(-) to CD62E(+) EMP exhibited a pattern consistent with activation. In conclusion, our studies indicate endothelial activation in TTP. EMP that co-express VWF and CD62E could play a role in the pathogenesis of TTP.

Capillary zone electrophoresis investigation of interactions between granulocyte-colony stimulating factor and dextran sulfate/carrageenan oligosaccharide

The interactions between granulocyte-colony stimulating factor (G-CSF) and dextran sulfate/kappa-carrageenan oligosaccharide were studied by capillary zone electrophoresis. Dextran sulfate could strongly interact with G-CSF and the complex was detected. The binding constant and stoichiometry were determined to be 1.2x10(6) (mol/L)(-1) and 3:1, respectively. However, the interaction between K-carrageenan oligosaccharide and G-CSF was not found.