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Purification and characterization of a novel anticoagulant peptide from marine echiuroid worm, Urechis unicinctus
Abstracts / Journal of Biotechnology 136S (2008) S527–S540
VI1-P-002
S529
Sequences analysis of mitochondrial tRNA-thr, tRNA-pro and control region from spotted seals (Phoca Largha) in liaodong gulf
results suggest that UAP bound to FIXa prolongs blood-clotting time by inhibiting the conversion of FX to FXa in the intrinsic tenase complex. It is possible to provide biochemical properties and health benefits of a novel nutraceutical or pharmaceutical material with an anticoagulant activity.
Xianggang Gao 1,∗ , Chongbo He 1 , Jiabo Han 1 , Xing Liu
References
1
Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China E-mail address: [email protected] (C. He). Liaodong Gulf of Bohai Sea is one of the eight breeding concentrations of spotted seals (Phoca Largha) around the world (Han et al., 2005; Rugh et al., 1997). The sequences of mtDNA control region, adjacent proline and a portion of the threonine tRNA genes of 46 individual spotted seals was sequenced using PCR-based sequencing method and 17 haplotypes with 717 bp were obtained from the 46 sequences. Compared with its closely related harbor seal (Phoca vitulina), all the 17 haplotype sequences from spotted seals have two deletions in the mtDNA control region, which could be use as a species-specific marker between spotted seals and harbor seals (Stanley et al., 1996; Mizuno et al., 2003). Compared with spotted seals along Sea of Japan and Sea of Okhotsk, spotted seals of Liaodong Gulf have relative less haplotypes and low genetic diversity, and have one insertion at site 16,296 of mtDNA, which could be a marker between these populations (Mizuno et al., 2003). The result from these sequences analysis show that the spotted seals of Liaodong Gulf maybe belong to an independent population and no gene flow with those along Sea of Japan and Sea of Okhotsk. References Han, J.B., Wang, W., Ma, Z.Q., 2005. Spotted seals in the Estuary of Shuangtaizi River of Liaodong Bay. Mar. Environ. Sci. 24 (1), 51–53 (in Chinese, with English abstract). Mizuno, A.W., Onuma, M., Takahashi, M., et al., 2003. Population genetic structure of the spotted seals Phoca largha along the coast of Hokkaido, based on mitochondrial DNA sequences. Zool. Sci. 20, 783–788. Rugh, D.J., Shelden, K.E., et al., 1997. Soptted seals, Phoca largha, in Alaska. Mar. Fish. 59 (1), 1–17. Stanley, H.F., Casey, S., Cornahan, J.M., et al., 1996. Worldwide patterns of mitochondrial DNA differentiation in the Harbor Seal (Phoca vitulina). Mol. Biol. Evol. 13 (2), 368–382.
doi:10.1016/j.jbiotec.2008.07.1242 VI1-P-005 Purification and characterization of a novel anticoagulant peptide from marine echiuroid worm, Urechis unicinctus Won-Kyo Jung 1,∗ , Se-Kwon Kim 1 1
Department of Marine Life Science, and Marine Bio Research Center, Chosun University, Gwangju 501-759, Republic of Korea E-mail address: [email protected] (W.-K. Jung).
A novel inhibitory peptide against blood coagulation factor IXa (FIXa) was isolated from the marine echiuroid worm (Urechis unicinctus, order Urechidae). U. unicinctus anticoagulant peptide (UAP), GELTPESGPDLFVHFLDGNPSYSLYADAVPR (Mw : 3343 kDa) potently prolonged the activated partial thromboplastin time (APTT), corresponding to inhibition of an endogenous blood coagulation factor in the intrinsic pathway. In the specific factor inhibitory assay, FIXa activity in normal plasma was significantly (P < 0.05) decreased by addition of UAP in dose-dependant manner (IC50 = 42.6 g ml−1 ). Binding affinity assay using a surface plasmon resonance (SPR) spectrometer showed that UAP binding to FIXa could inhibit the interaction between FIXa and FX. The present
Athukorala, Y., Jung, W.K., Vasanthan, T., Jeon, Y.J., 2006. An anticoagulative polysaccharide from an enzymatic hydrolysate of Ecklonia cava. Carbohydr. Polym. 66, 184–191. Davie, E.W., Fujikawa, K., Kisel, W., 1991. The coagulation cascade: initiation maintenance and regulation. Biochemistry 30, 10363–10370. Jung, W.K., Je, J.Y., Kim, H.J., Kim, S.K., 2001. A novel anticoagulant protein from Scapharca broughtonii. J. Biochem. Mol. Biol. 35, 199–205.
doi:10.1016/j.jbiotec.2008.07.1243 VI1-P-006 Genetic variations analysis for the wild and culturede populations of Paralichthys olivaceus in Dalian sea area by microsatellite markers Xuemei Qiu 1,2,∗ , Shuang Du 1 , Xiuli Wang 1 , Zhiqiang Jiang 1,2 1
College of Life Sciences and Biotechnology, Dalian Fisheries University, Dalian 116023, China 2 Key Laboratory of Mariculture and Biotechnology of Agriculture Ministry, Dalian Fisheries University, Dalian 116023, China E-mail address: [email protected] (X. Qiu).
Paralichthys olivaceus belongs to Pleuronectiformes, Bothidae, Paralichthys. The resource of P. olivaceus population has decreased due to over fishing and degrated environment, the artificial breeding has been adopted for 10 years. So polymorphism analysis and quality evaluation of the wild and cultured populations of P. olivaceus in china is necessary. For these reasons microsatellite DNA markers method (Sekino and Hrara, 2000) were used to detect the genetic diversity the wild and cultured populations of P. olivaceus, it is important to provide more information and data for the protection and sustainable utilization of the P. olivaceus genome. Thirty primer pairs were used to estimate genetic diversity and genetic differentiation in two populations of the wild (n = 32) and cultured (n = 32) of P. olivaceus. The PCR products were isolated by electrophoresis in polyacrylamide gel. The data of 15 loci were analyzed by PopGene software. The results showed that the average numbers of the allele in the wild and cultured populations is 5.400 and 4.6667, the average population heterozygosity in the wild and cultured populations is 0.7396 and 0.6746, the polymorphism information content value in the wild and cultured populations is 0.6582 and 0.6331. The results are consistent with previous reports (Wei et al., 2004). It is show that the wild population has much genetic diversity and acclimation. These indicated that the wild and cultured populations of P. olivaceus in Dalian have relatively medium discrimination power or better genetic diversity. Acknowledgement This study was supported by The Liao-ning Province Natural Science Funds (20062128). References Sekino, M., Hrara, M., 2000. Isolation and characterization microsatellite DNA loci in Japan flounder Paralichthys olivaceus. Mol. Ecol. 9, 2201–2203. Wei, W., Feng, Y., Tianxiang, G., et al., 2004. Genetic variations at ten microsatellite loci in natural and cultured stocks of left-eyed flounder Paralichthys olivaceus in Shandong Coastal Waters. Oceanol. Limnol. Sin. 6, 531–537.
doi:10.1016/j.jbiotec.2008.07.1244
VI1-P-002
S529
Sequences analysis of mitochondrial tRNA-thr, tRNA-pro and control region from spotted seals (Phoca Largha) in liaodong gulf
results suggest that UAP bound to FIXa prolongs blood-clotting time by inhibiting the conversion of FX to FXa in the intrinsic tenase complex. It is possible to provide biochemical properties and health benefits of a novel nutraceutical or pharmaceutical material with an anticoagulant activity.
Xianggang Gao 1,∗ , Chongbo He 1 , Jiabo Han 1 , Xing Liu
References
1
Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China E-mail address: [email protected] (C. He). Liaodong Gulf of Bohai Sea is one of the eight breeding concentrations of spotted seals (Phoca Largha) around the world (Han et al., 2005; Rugh et al., 1997). The sequences of mtDNA control region, adjacent proline and a portion of the threonine tRNA genes of 46 individual spotted seals was sequenced using PCR-based sequencing method and 17 haplotypes with 717 bp were obtained from the 46 sequences. Compared with its closely related harbor seal (Phoca vitulina), all the 17 haplotype sequences from spotted seals have two deletions in the mtDNA control region, which could be use as a species-specific marker between spotted seals and harbor seals (Stanley et al., 1996; Mizuno et al., 2003). Compared with spotted seals along Sea of Japan and Sea of Okhotsk, spotted seals of Liaodong Gulf have relative less haplotypes and low genetic diversity, and have one insertion at site 16,296 of mtDNA, which could be a marker between these populations (Mizuno et al., 2003). The result from these sequences analysis show that the spotted seals of Liaodong Gulf maybe belong to an independent population and no gene flow with those along Sea of Japan and Sea of Okhotsk. References Han, J.B., Wang, W., Ma, Z.Q., 2005. Spotted seals in the Estuary of Shuangtaizi River of Liaodong Bay. Mar. Environ. Sci. 24 (1), 51–53 (in Chinese, with English abstract). Mizuno, A.W., Onuma, M., Takahashi, M., et al., 2003. Population genetic structure of the spotted seals Phoca largha along the coast of Hokkaido, based on mitochondrial DNA sequences. Zool. Sci. 20, 783–788. Rugh, D.J., Shelden, K.E., et al., 1997. Soptted seals, Phoca largha, in Alaska. Mar. Fish. 59 (1), 1–17. Stanley, H.F., Casey, S., Cornahan, J.M., et al., 1996. Worldwide patterns of mitochondrial DNA differentiation in the Harbor Seal (Phoca vitulina). Mol. Biol. Evol. 13 (2), 368–382.
doi:10.1016/j.jbiotec.2008.07.1242 VI1-P-005 Purification and characterization of a novel anticoagulant peptide from marine echiuroid worm, Urechis unicinctus Won-Kyo Jung 1,∗ , Se-Kwon Kim 1 1
Department of Marine Life Science, and Marine Bio Research Center, Chosun University, Gwangju 501-759, Republic of Korea E-mail address: [email protected] (W.-K. Jung).
A novel inhibitory peptide against blood coagulation factor IXa (FIXa) was isolated from the marine echiuroid worm (Urechis unicinctus, order Urechidae). U. unicinctus anticoagulant peptide (UAP), GELTPESGPDLFVHFLDGNPSYSLYADAVPR (Mw : 3343 kDa) potently prolonged the activated partial thromboplastin time (APTT), corresponding to inhibition of an endogenous blood coagulation factor in the intrinsic pathway. In the specific factor inhibitory assay, FIXa activity in normal plasma was significantly (P < 0.05) decreased by addition of UAP in dose-dependant manner (IC50 = 42.6 g ml−1 ). Binding affinity assay using a surface plasmon resonance (SPR) spectrometer showed that UAP binding to FIXa could inhibit the interaction between FIXa and FX. The present
Athukorala, Y., Jung, W.K., Vasanthan, T., Jeon, Y.J., 2006. An anticoagulative polysaccharide from an enzymatic hydrolysate of Ecklonia cava. Carbohydr. Polym. 66, 184–191. Davie, E.W., Fujikawa, K., Kisel, W., 1991. The coagulation cascade: initiation maintenance and regulation. Biochemistry 30, 10363–10370. Jung, W.K., Je, J.Y., Kim, H.J., Kim, S.K., 2001. A novel anticoagulant protein from Scapharca broughtonii. J. Biochem. Mol. Biol. 35, 199–205.
doi:10.1016/j.jbiotec.2008.07.1243 VI1-P-006 Genetic variations analysis for the wild and culturede populations of Paralichthys olivaceus in Dalian sea area by microsatellite markers Xuemei Qiu 1,2,∗ , Shuang Du 1 , Xiuli Wang 1 , Zhiqiang Jiang 1,2 1
College of Life Sciences and Biotechnology, Dalian Fisheries University, Dalian 116023, China 2 Key Laboratory of Mariculture and Biotechnology of Agriculture Ministry, Dalian Fisheries University, Dalian 116023, China E-mail address: [email protected] (X. Qiu).
Paralichthys olivaceus belongs to Pleuronectiformes, Bothidae, Paralichthys. The resource of P. olivaceus population has decreased due to over fishing and degrated environment, the artificial breeding has been adopted for 10 years. So polymorphism analysis and quality evaluation of the wild and cultured populations of P. olivaceus in china is necessary. For these reasons microsatellite DNA markers method (Sekino and Hrara, 2000) were used to detect the genetic diversity the wild and cultured populations of P. olivaceus, it is important to provide more information and data for the protection and sustainable utilization of the P. olivaceus genome. Thirty primer pairs were used to estimate genetic diversity and genetic differentiation in two populations of the wild (n = 32) and cultured (n = 32) of P. olivaceus. The PCR products were isolated by electrophoresis in polyacrylamide gel. The data of 15 loci were analyzed by PopGene software. The results showed that the average numbers of the allele in the wild and cultured populations is 5.400 and 4.6667, the average population heterozygosity in the wild and cultured populations is 0.7396 and 0.6746, the polymorphism information content value in the wild and cultured populations is 0.6582 and 0.6331. The results are consistent with previous reports (Wei et al., 2004). It is show that the wild population has much genetic diversity and acclimation. These indicated that the wild and cultured populations of P. olivaceus in Dalian have relatively medium discrimination power or better genetic diversity. Acknowledgement This study was supported by The Liao-ning Province Natural Science Funds (20062128). References Sekino, M., Hrara, M., 2000. Isolation and characterization microsatellite DNA loci in Japan flounder Paralichthys olivaceus. Mol. Ecol. 9, 2201–2203. Wei, W., Feng, Y., Tianxiang, G., et al., 2004. Genetic variations at ten microsatellite loci in natural and cultured stocks of left-eyed flounder Paralichthys olivaceus in Shandong Coastal Waters. Oceanol. Limnol. Sin. 6, 531–537.
doi:10.1016/j.jbiotec.2008.07.1244