Standardized ileal digestibility of amino acids in extruded full-fat soybean fed to non-gestating, gestating, and lactating sows.

This study was conducted to determine and compare the apparent ileal digestibility and standardized ileal digestibility (SID) of crude protein (CP) and amino acids (AA) in extruded full-fat soybean (EFSB) fed to nongestating, midgestating, late-gestating, and lactating sows. Six EFSB samples were collected from different sources. Fourteen nongestating sows (Landrace × Yorkshire; parity 3 to 5) were fitted with a T-cannula at the distal ileum. After recovery, sows were assigned to a replicated 7 × 3 incomplete Latin square design. The diets included a nitrogen-free (NF) diet and six experimental diets (EFSB 1 to 6). Eight midgestating sows (Landrace × Yorkshire; parity 3; day 48 of gestation), eight late-gestating sows (Landrace × Yorkshire; parity 3; day 90 of gestation), and eight lactating sows (Landrace × Yorkshire; parity 3; day 6 of lactation) were all assigned to four dietary treatments in a repeated 4 × 3 incomplete Latin square design. The diets included a NF diet and three experimental diets (EFSB 4 to 6). Results showed that there were significant differences in the AID and SID of CP and other AA in nongestating sows (P < 0.05), the AID and SID values of EFSB 1 to 3 were higher than those of EFSB 4 to 6, and the value of EFSB 5 was the lowest. For midgestating sows, there were differences in the AID of methionine (EFSB 5 had a lower value than EFSB 4 and 6) (P < 0.01). For late-gestating sows, only the AID of methionine (EFSB 5 had a lower value than EFSB 4 and 6), tryptophan (EFSB 5 had a higher value than EFSB 4 and 6), and proline (EFSB 5 had a higher value than EFSB 4) was different (P < 0.05), and the SID of methionine (EFSB 4 had a higher value than EFSB 5) and tryptophan (EFSB 5 had a higher value than EFSB 4 and 6) was different (P < 0.05). The SID of histidine and valine was greater in lactation than in nongestation (P = 0.045 and P = 0.02, respectively). The SID of isoleucine was greater in lactation than in nongestation and gestation (P < 0.01). The SID of methionine in nongestation was lower than in gestation and lactation (P < 0.01). The SID of cysteine was the lowest in midgestation (P = 0.045), and the SID of proline was greater in midgestation than in lactation and nongestation (P < 0.01). In conclusion, the AA ileal digestibility of six EFSB samples from different sources was different, and the ileal digestibility of CP and most AA was not affected by the physiological stage of sows.

Extruded full-fat soybean (EFSB) is produced by treating soybean with high temperature and pressure. It is rich in oil, protein, and energy. The standardized ileal digestibility (SID) of amino acids in EFSB has been studied in growing or finishing pigs, but not in sows. The ileal digestibility of amino acids in EFSB may be different between sows and growing pigs and even between sows at different physiological stages. Therefore, the SID of amino acids of six EFSB samples from different sources was evaluated in nongestating, midgestating, late-gestating, and lactating sows. Results indicate that the amino acid ileal digestibility of six EFSB samples from different sources was different, and the ileal digestibility of crude protein and most amino acids was not affected by the physiological stage of sows.

Standardized ileal digestibility of amino acids in cottonseed meal fed to pregnant and non-pregnant sows.

This study determined the apparent ileal digestibility (AID) and standard ileal digestibility (SID) of crude protein (CP) and amino acids (AA) of six cottonseed meal (CSM) samples in pregnant and non-pregnant sows. Two CSM samples were processed by expelling with a CP level of 40.67% (ECSM41) and 44.64% (ECSM45), and four samples were processed by solvent-extracted which contained graded CP levels of 45% (SECSM45), 51.16% (SECSM51), 56.44% (SECSM56), and 59.63% (SECSM60). Landrace ×Yorkshire third parity sows, 7 at gestation and 14 non-pregnant, were fitted with T-cannula in the distal ileum. Pregnant sows were allotted to a 7 × 6 Latin square design with a cornstarch-based nitrogen-free (NF) diet and the six CSM diets, and non-pregnant sows were allotted to a replicated 7 × 3 Latin square design with seven diets and three periods, respectively, resulting in a total of six replicates per treatment. All experimental sows were fed 3.0 kg/d of the experimental diets. The AID of CP in ECSM41 (75.58%) was lower than in SECSM51 (80.42%), SECSM56 (80.50%), and SECSM60 (82.44%) diets for pregnant sows (P < 0.05). The AID of CP in ECSM41 (77.88%) was significantly lower than in SECSM60 (81.87%) diets for non-pregnant sows (P < 0.05). The physiological phase did not affect the AID of CP (P > 0.05). The SID of CP was affected by diets for both pregnant (P < 0.01) and non-pregnant sows (P = 0.06). The physiological phase also affected the SID of CP (P < 0.01). The AID of histidine, leucine, methionine, threonine, and tryptophan significantly differed between different CSM samples in both pregnant (P < 0.05) and non-pregnant sows (P < 0.05). The AID of dispensable AA aspartic acid, cysteine, glutamic acid, serine, and tyrosine differed between different CSM samples of both pregnant (P < 0.05) and non-pregnant sows (P < 0.05). For pregnant sows, the indispensable AA cysteine, glycine, proline, and tyrosine had significantly different SID between different groups (P < 0.05). For non-pregnant sows, the SID of arginine, lysine, methionine, threonine, aspartic acid, cysteine, and serine had different values among different diets (P < 0.05). In conclusion, the current study presented that the ileal AA digestibility of CSM fed to pregnant and non-pregnant sows increased with the decreased of fiber content, and the current findings can contribute to a precise formulation of diets for sows using CSM.

As a protein-rich cottonseed byproduct, cottonseed meal (CSM) is considered a vegetable protein source that can substitute soybean meal in the feed of livestock animals. However, the presence of free gossypol and high fiber levels in CSM have been limiting factors for its use in growing and finishing pigs, yet its nutritive value is still uncertain for sows. There is a lack of standard ileal digestibility (SID) of amino acids (AA) for plant proteins because fitting a T-cannula in the distal ileum is difficult. Therefore, this study evaluated the apparent ileal digestibility and SID of 18 AA of CSM in sows at two physiological stages (gestation and non-pregnancy). We found that CSM with different chemical compositions impacted the SID of AA when fed to pregnant and non-pregnant sows. Additionally, the physiological stage of the sow has a substantial impact on the SID of some AA. The current findings of this study provided a basis for the precise formulation of sow diets with CSM.

Nutritional values of cottonseed meal from different sources fed to gestating and non-pregnant sows.

This study set out to determine the apparent total tract digestibility (ATTD) of the nutrients and energy in six cottonseed meal (CSM) feedstuffs fed to pregnant and non-pregnant sows. The six types of CSM were: two expelled CSMs with crude protein (CP) levels of 40.67% and 44.64%, and four solvent-extracted CSMs with CP levels of 45.18%, 51.16%, 56.44%, and 59.63%. Fourteen gestating sows (at the fourth parity with body weights of 220.6 ± 18.4 kg at days 30 of gestation) and 14 non-pregnant sows (after the third parity with body weights of 219 ± 14.6 kg) were assigned to a replicated 7 × 3 Youden square design with seven diets and three periods. The seven diets included an entirely corn-based diet and six diets each containing 20.0% of the six CSMs tested. Each period included a 5-d acclimation to the experimental diets, followed by a 5-d period during which urine and feces were collected. Significant differences were found among the six CSM diets, regardless of reproductive stage, regarding 1) the ATTD of neutral detergent fiber (NDF) (P < 0.05) and 2) the ATTD of dry matter (DM), organic matter (OM), and CP and the gross energy (GE) (P < 0.01). Non-pregnant sows had a greater ATTD of OM and CP (P < 0.01) compared with gestating sows. The digestible energy (DE) and metabolizable energy (ME) of the six CSM samples ranged from 12.48 to 17.15 MJ/kg and 11.35 to 15.88 MJ/kg, respectively, for non-pregnant sows, and from 12.86 to 16.41 MJ/kg and 12.43 to 14.72 MJ/kg, respectively, for gestating sows. However, the DE, ME, and ME:DE ratios of each CSM were similar between gestating and non-pregnant sows. DE and ME were negatively correlated with NDF and ADF, respectively, but were positively corrected with CP level (P < 0.01). Collectively, the DE, ME, and nutrient digestibility of CSM varied greatly according to the chemical compositions, and CSMs with higher protein and lower fiber levels had greater DE and ME levels.

Cottonseed meal (CSM) is a high-protein feedstuff produced as a by-product of cottonseed processing. Concerns regarding the negative effects of antinutritional factors such as gossypol toxicity have limited the use of CSM as sow feed. As genetic breeding and feed processing technology improve, the potential for CSM as a supplementary protein in sow diets has increased. However, precise evaluation of the nutritive value of CSM for sows is key to accurate diet formulation. We evaluated the nutritional values of six CSMs prepared using different processing methods with different chemical compositions, fed to pregnant and non-pregnant sows, and investigated the relationships between their chemical composition and digestible (DE) and metabolizable energy (ME). We found no differences in DE and ME between gestating and non-pregnant sows for the six CSMs tested. However, there were large variations in DE and ME among the six CSMs tested, and most DEs and MEs were higher than the values published by the NRC (2012), which were measured from pigs in the growth and finishing stages. These findings demonstrate the different DE and ME values of CSM with different chemical compositions and provide a basis for precise diet formulation for sows.

The nutritive value of soybean meal from different sources for sows during mid- and late gestation.

A precise understanding of the nutritive value of soybean meal (SBM) for pregnant sow is required for accurate feeding. Hence, we evaluated the nutritive value of 11 SBM samples from different sources for sows during mid and late gestation. In total, 24 mid-gestating sows (parity three; 230.3 ± 12.0 kg on day 37 of gestation) and 24 late-gestating sows (parity three; 238.8 ± 20.9 kg on day 72 of gestation) were assigned to a replicated 12 × 3 Youden square design with 12 diets and 3 periods. The 12 diets included a corn-based diet and 11 diets containing 25.50% SBMs from different sources. After 5-d adaptation, urine and feces were collected for 5 d. Although the chemical characteristics of SBM varied between samples, no differences were observed in digestible energy (DE), metabolizable energy (ME), apparent total tract digestibility (ATTD) of dry matter, gross energy, crude fiber, and neutral detergent fiber values in SBMs fed to both animal groups. However, de-hulled SBM 4 from Brazil displayed greater ATTD for nitrogen (N) in late-gestating sows (P < 0.05); animals displayed significantly (P < 0.01) greater ME, ME:DE ratio, and N net utilization values when compared with mid-gestating sows. The chemical composition of SBMs can be used to predict DE and ME values. In conclusion, ME, ME:DE ratio, and N net utilization SBM values for late-gestating sows were greater than in mid-gestating sows. Therefore, we should consider differences in ME values for SBMs when formulating diets for sows in mid and late gestation periods.

Soybean meal (SBM) is the most commonly used protein source in swine diets, with high available energy. Sows have crucial roles in the pig industry, therefore precise knowledge of actual SBM nutritive values at different gestation stages is vital for efficient livestock production and management. In our study, we evaluated the nutritive value of SBMs from different sources in mid- and late-gestating sows, and generated prediction equations for digestible energy (DE) and metabolizable energy (ME) values. We identified no differences in DE and ME values in SBMs from different sources when fed to sows during mid- and late gestation. However, regardless of the pregnancy stage, DE and ME SBM values for sows, identified in this study, were greater than values published by NRC (2012). Also, ME, ME:DE ratio, and nitrogen (N) net utilization SBM values for late-gestating sows were greater than in mid-gestating sows. The chemical composition of SBMs can be used to predict DE and ME values. Our study data can be used to accurately formulate diets for pregnant sows.

The potential role of Annexin 3 in diapause embryo restart of Artemia sinica and in response to stress of low temperature.

Annexins are highly conserved and ubiquitous in various somatic cell types. They are involved in membrane transport and a range of calcium-regulated activities on the cell membrane surface, including vesicular transport, membrane fusion in exocytosis, signal transduction, and formation of calcium channels. They also regulate inflammatory response, cell differentiation, and interaction between cytoskeletal proteins. In this study, for the first time, an ANX3 gene from Artemia sinica ( As-anx3) was cloned. The As-anx3 full-length complementary DNA comprises 1,024 bp and has a 948 bp open reading frame encoding a 315-amino-acid polypeptide with four ANX domains. The profiles of both As-ANX3 mRNA and protein expression exhibited peaks at the 0 hr stage and had the same significant downregulation trend throughout the post-diapause embryo development stage. The ERK1/2, the phosphorylation levels of ERK1/2, and cell cycle-related protein (CDK4) expressions were analyzed by western blot analysis. The results showed that CDK4 presented a significantly ascending trend from 0 and 40 hr, although the phosphorylation levels of ERK1/2 did not increase significantly. The transcriptional and protein expressions of As-ANX3 were highly upregulated when the temperature was lowered from 25 to 15°C, but the expressions showed a gradual downward trend when the temperature was further lowered to 5°C. These results indicated that As-ANX3 plays a crucial role in restarting diapause and low-temperature stress in A. sinica.

Fucoxanthin inhibits tumour-related lymphangiogenesis and growth of breast cancer.

Tumour lymphangiogenesis plays an important role in promoting the growth and lymphatic metastasis of tumours. The process is associated with cell proliferation, migration and tube-like structure formation in lymphatic endothelial cells (LEC), but no antilymphangiogenic agent is currently used in clinical practice. Fucoxanthin is a material found in brown algae that holds promise in the context of drug development. Fucoxanthin is a carotenoid with variety of pharmacological functions, including antitumour and anti-inflammatory effects. The ability of fucoxanthin to inhibit lymphangiogenesis remains unclear. The results of experiments performed as part of this study show that fucoxanthin, extracted from Undaria pinnatifida (Wakame), inhibits proliferation, migration and formation of tube-like structures in human LEC (HLEC). In this study, fucoxanthin also suppressed the malignant phenotype in human breast cancer MDA-MB-231 cells and decreased tumour-induced lymphangiogenesis when used in combination with a conditional medium culture system. Fucoxanthin significantly decreased levels of vascular endothelial growth factor (VEGF)-C, VEGF receptor-3, nuclear factor kappa B, phospho-Akt and phospho-PI3K in HLEC. Fucoxanthin also decreased micro-lymphatic vascular density (micro-LVD) in a MDA-MB-231 nude mouse model of breast cancer. These findings suggest that fucoxanthin inhibits tumour-induced lymphangiogenesis in vitro and in vivo, highlighting its potential use as an antilymphangiogenic agent for antitumour metastatic comprehensive therapy in patients with breast cancer.

The Potential Roles of the Apoptosis-Related Protein PDRG1 in Diapause Embryo Restarting of Artemia sinica.

High salinity and low temperatures can induce Artemia sinica to enter the diapause stage during embryonic development. Diapause embryos stop at the gastrula stage, allowing them to resist apoptosis and regulate cell cycle activity to guarantee normal development after diapause termination. P53 and DNA damage-regulated gene 1 (pdrg1) is involved in cellular physiological activities, such as apoptosis, DNA damage repair, cell cycle regulation, and promotion of programmed cell death. However, the role of pdrg1 in diapause and diapause termination in A. sinica remains unknown. Here, the full-length A. sinica pdrg1 cDNA (As-pdrg1) was obtained and found to contain 1119 nucleotides, including a 228 bp open reading frame (ORF), a 233 bp 5'-untranslated region (UTR), and a 658-bp 3'-UTR, which encodes a 75 amino acid protein. In situ hybridization showed no tissue specific expression of As-pdrg1. Quantitative real-time PCR and western blotting analyses of As-pdrg1 gene and protein expression showed high levels at 15-20 h of embryo development and a subsequent downward trend. Low temperatures upregulated As-pdrg1 expression. RNA interference for the pdrg1 gene in Artemia embryos caused significant developmental hysteresis. Thus, PDRG1 plays an important role in diapause termination and cell cycle regulation in early embryonic development of A. sinica.

Cloning, expression pattern, and potential role of apoptosis inhibitor 5 in the termination of embryonic diapause and early embryo development of Artemia sinica.

During the embryonic development of Artemia sinica, the diapause phenomenon can be induced by high salinity or low temperature conditions. The diapause embryo at the gastrula stage is maintained under the threat of apoptosis to guarantee the embryo's normal development. In this process, apoptosis inhibitor proteins play vital roles in protecting embryos against apoptosis. Apoptosis inhibitor5 (API5) plays a pivotal role in regulating the cell cycle and preventing programmed cell death after growth factor starvation. In the present study, we cloned the full-length cDNA representing the api5 gene from A. sinica (As-api5), which encodes a 372-amino acid protein. In situ hybridization experiments revealed that As-api5 expression is not tissue or organ specific. Quantitative real-time PCR analyses of the developmental expression of As-api5 showed that it reached its highest level at 10h, after which its expression decreased. High salinity and low temperature treatments increased the expression of As-api5. Western blotting was used to assess the abundance of As-API5 and related proteins (As-CyclinA, As-CyclinE, As-E2F1, As-CDK2, As-APAF1, and As-Caspase9). Downregulation of As-api5 expression using a short interfering RNA resulted in increased mortality and embryo malformation of A. sinica. Taken together, the results indicated that API5 plays a crucial role in embryonic diapause termination and early embryo development of A. sinica.

Protective Effects of Fucoidan on Aß25-35 and d-Gal-Induced Neurotoxicity in PC12 Cells and d-Gal-Induced Cognitive Dysfunction in Mice.

Alzheimer's disease (AD) is a chronic neurodegenerative disease which contributes to memory loss and cognitive decline in the elderly. Fucoidan, extracted from brown algae, is a complex sulfated polysaccharide and potential bioactive compound. In this study, we investigated whether fucoidan protects PC12 cells from apoptosis induced by a combination of beta-amyloid 25-35 (Aß25-35) and d-galactose (d-Gal), and improves learning and memory impairment in AD model mice. The results indicated that fucoidan could inhibit the release of cytochrome c from the mitochondria to cytosol and activation of caspases, and increase the expression of apoptosis inhibitor proteins (IAPs), including livin and X-linked IAP (XIAP) in PC12 cells damaged by Aß25-35 and d-Gal-induction. Fucoidan reversed the decreased activity of acetylcholine (ACh) and choline acetyl transferase (ChAT), as well as the increased activity of acetylcholine esterase (AChE), in AD model mice induced by infusion of d-Gal. Furthermore, fucoidan improved antioxidant activity in vitro and in vivo by activation of superoxide dismutase (SOD) and glutathione (GSH). These results suggested that fucoidan could protect PC12 cells from apoptosis and ameliorate the learning and memory impairment in AD model mice, which appeared to be due to regulating the cholinergic system, reducing oxidative stress, and inhibiting the caspase-dependent apoptosis pathway.

Identification, expression pattern and functional characterization of As-kip2 in diapause embryo restarting process of Artemia sinica.

Proper control of the cellular processes requires a variety of regulatory proteins that are involved in the cell cycle, proliferation and apoptosis. Cyclin-dependent kinase inhibitor (CKI) negatively regulates transcription and arrests the cell cycle in G1 phase. KIP2 is a member of CKI family, which could inhibit proliferation by tight-binding with several cyclin-CDK complexes. During the embryonic development of the brine shrimp, Artemia sinica, KIP2 plays a key role in the cell cycle regulation, but the specific mechanisms remain unknown. Herein, the 1023bp full-length cDNA of kip2 from A. sinica was cloned. The mRNA expression patterns of As-kip2, As-carp-1 in different development stages and pattern of As-kip2 under environmental stresses were investigated. In situ hybridization of As-kip2 mRNA and immunofluorescence of As-CARP-1 protein showed no tissue or organ specificity. Furthermore, western blotting showed the expressions levels of As-KIP2, As-E2F1, As-p53, As-cyclin E, As-SODD protein, and pattern of As-KIP2 under environmental stresses. Our research revealed that As-KIP2 plays crucial role in the restarting process of diapause embryo in Artemia sinica.

Involvement of PGRP-SC2 from Artemia sinica in the innate immune response against bacteria and expression pattern at different developmental stages.

Peptidoglycan-recognition protein-SC2 precursor-like protein (PGRP-SC2) is a vital protein in innate immunity with a vita role in response to bacteria challenge in invertebrates. Here, a 678-bp full-length cDNA of pgrp-sc2 from A. sinica was obtained containing a 558-bp open reading frame encoding 185 amino acids with a calculated molecular mass of 19.6 kDa. The predicted protein contains a PGRP and an Amidase2 domain, indicating that PGRP-SC2 is a PGRP family member and has N-acetylmuramoyl-l-alanine amidase activity. The expression and localization of pgrp-sc2/PGRP-SC2 in A.sinica during embryonic development and bacterial challenge were determined by qPCR, WB and ISH. During different A. sinica embryonic development stages, the expression level of pgrp-sc2/PGRP-SC2 was most highly expressed at 0 and 5 h and after challenge by Gram-positive bacteria, it increased with increasing bacterial concentrations, indicating that it plays a vital role in A. sinica early embryonic development and innate immunity.

APC/CCDC20 and APC/C play pivotal roles in the process of embryonic development in Artemia sinica.

Anaphase Promoting Complex or Cyclosome (APC/C) is a representative E3 ubiquitin ligase, triggering the transition of metaphase to anaphase by regulating degradation and ensures the exit from mitosis. Cell division cycle 20 (CDC20) and Cell division cycle 20 related protein 1 (CDH1), as co-activators of APC/C, play significant roles in the spindle assembly checkpoint, guiding ubiquitin-mediated degradation, together with CDC23. During the embryonic development of the brine shrimp, Artemia sinica, CDC20, CDH1 and CDC23 participate in cell cycle regulation, but the specific mechanisms of their activities remain unknown. Herein, the full-length cDNAs of cdc20 and cdc23 from A. sinica were cloned. Real-time PCR analyzed the expression levels of As-cdc20 and As-cdc23. The locations of CDH1, CDC20 and CDC23 showed no tissue or organ specificity. Furthermore, western blotting showed that the levels of As-CDC20, securin, cyclin B, CDK1, CDH1, CDC14B, CDC23 and geminin proteins conformed to their complicated degradation relationships during different embryo stages. Our research revealed that As-CDC20, As-CDH1 and APC mediate the mitotic progression, downstream proteins degradation and cellular differentiation in the process of embryonic development in A. sinica.

The Potential Roles of the G1LEA and G3LEA Proteins in Early Embryo Development and in Response to Low Temperature and High Salinity in Artemia sinica.

Late embryogenesis abundant proteins (LEA) are stress resistance-related proteins that play crucial roles in protecting against desiccation, cold and high salinity in a variety of animals and plants. However, the expression pattern, distribution and functions of LEA proteins in the post-diapause period of Artemia sinica, and under high salinity and low temperature stresses, remain unknown. In this study, the complete cDNA sequences of the group 1 LEA (As-g1lea) and group 3 LEA (As-g3lea) genes from A. sinica were cloned. The expression patterns and location of As-G1LEA and As-G1LEA were investigated. The protein abundances of As-G1LEA, As-G3LEA and Trehalase were analyzed during different developmental stages of the embryo and under low temperature and high salinity stresses in A. sinica. The full-length cDNA of As-g1lea was 960 bp, encoding a 182 amino acid protein, and As-g3lea was 2089 bp, encoding a 364 amino acid protein. As-g1lea and As-g3lea showed their highest expressions at 0 h of embryonic development and both showed higher relative expression in embryonic, rather than adult, development stages. The abundances of As-G1LEA, As-G3LEA and trehalose were upregulated under low temperature and downregulated under high salinity stress. These two genes did not show any tissue or organ specific expression. Our results suggested that these LEA proteins might play a pivotal role in stress tolerance in A. sinica.

Cloning and expression of retinoblastoma-binding protein 4 gene in embryo diapause termination and in response to salinity stress from brine shrimp Artemia sinica.

Retinoblastoma binding protein 4 (RBBP4) is a nuclear protein with four WD-repeat sequences and thus belongs to a highly conserved subfamily of proteins with such domains. This retinoblastoma-binding protein plays an important role in nucleosome assembly and histone modification, which influences gene transcription and regulates cell cycle and proliferation. Artemia sinica (brine shrimp) undergoes an unusual diapause process under stress conditions of high salinity and low temperature. However, the role of RBBP4 in diapause termination of embryo development in A. sinica remains unknown. Here, the full-length cDNA of the As-rbbp4 gene was obtained from A. sinica and found to contain 1411 nucleotides, including a 1281 bp open reading frame (ORF), 63 bp 5'-untranslated region (UTR) and a 67-bp 3'-UTR, which encodes a 427 amino acid (48 kDa) protein. Bioinformatic analysis indicated As-RBBP4 to be mainly located in the nucleus, with a theoretical isoelectric point of 4.79. Protein sequence domain analysis showed that As-RBBP4 is a conserved protein, especially in the WD40 domain. No specificity in expression of this gene was observed in tissues or organs by in situ hybridization. Real-time quantitative PCR and Western blot analyses of As-RBBP4 gene and protein expression, respectively, showed notably high levels at 10 h and a subsequent downward trend. Obvious trends in upregulation of As-RBBP4 were observed under conditions of low temperature and high salinity stress. As-E2F1 and As-CyclinE also presented similar trends as that of As-RBBP4 in Western blots. Analysis of the RBBP4 expression in early embryonic development of A. sinica indicated that this protein plays an important role in diapause termination and cell cycle regulation.

Fucoidan inhibits lymphangiogenesis by downregulating the expression of VEGFR3 and PROX1 in human lymphatic endothelial cells.

Lymphangiogenesis is one of the promoters of tumor lymphatic metastasis. Fucoidan which is a fucose-enriched sulfated polysaccharide has effect on various pharmacological activities including anti-metastasis activity. However, the inhibitory effect of fucoidan on lymphangiogenesis remains unclear. Here, fucoidan extracted from U. pinnatifida sporophylls suppressed HLECs proliferation, migration and tube-like structure formation, and had inhibitory effect of tumor-induced lymphangiogenesis in vitro. Additionally, we found that fucoidan had a dose-dependent depressive effect on the expressions of PROX1, vascular endothelial growth factor receptor 3 (VEGFR3), NF-κB, phospho-PI3K and phospho-Akt in HLECs. Moreover, anti-lymphangiogenesis effect of fucoidan was assessed by using mouse tumor model. In summary, fucoidan inhibit tumor lymphangiogenesis and lymphatic metastasis by suppressing the NF-κB/PI3K/Akt signaling pathway through reduced levels of PROX1 and VEGFR3.

Fucoidan Suppresses Hypoxia-Induced Lymphangiogenesis and Lymphatic Metastasis in Mouse Hepatocarcinoma.

Metastasis, the greatest clinical challenge associated with cancer, is closely connected to multiple biological processes, including invasion and adhesion. The hypoxic environment in tumors is an important factor that causes tumor metastasis by activating HIF-1α. Fucoidan, extracted from brown algae, is a sulfated polysaccharide and, as a novel marine biological material, has been used to treat various disorders in China, Korea, Japan and other countries. In the present study, we demonstrated that fucoidan derived from Undaria pinnatifida sporophylls significantly inhibits the hypoxia-induced expression, nuclear translocation and activity of HIF-1α, the synthesis and secretion of VEGF-C and HGF, cell invasion and lymphatic metastasis in a mouse hepatocarcinoma Hca-F cell line. Fucoidan also suppressed lymphangiogenesis in vitro and in vivo. In addition, accompanied by a reduction in the HIF-1α nuclear translocation and activity, fucoidan significantly reduced the levels of p-PI3K, p-Akt, p-mTOR, p-ERK, NF-κB, MMP-2 and MMP-9, but increased TIMP-1 levels. These results indicate strongly that the anti-metastasis and anti-lymphangiogenesis activities of fucoidan are mediated by suppressing HIF-1α/VEGF-C, which attenuates the PI3K/Akt/mTOR signaling pathways.

Molecular cloning, characterization and expression analysis of the protein arginine N-methyltransferase 1 gene (As-PRMT1) from Artemia sinica.

Protein arginine N-methyltransferase 1 (PRMT1) is an important epigenetic regulation factor in eukaryotic genomes. PRMT1 is involved in histone arginine loci methylation modification, changes in eukaryotic genomes' chromatin structure, and gene expression regulation. In the present paper, the full-length 1201-bp cDNA sequence of the PRMT1 homolog of Artemia sinica (As-PRMT1) was cloned for the first time. The putative As-PRMT1 protein comprises 346 amino acids with a SAM domain and a PRMT5 domain. Multiple sequence alignments revealed that the putative sequence of As-PRMT1 protein was relatively conserved across species, especially in the SAM domain. As-PRMT1 is widely expressed during embryo development of A. sinica. This is followed by a dramatic upregulation after diapause termination and then downregulation from the nauplius stage. Furthermore, As-PRMT1 transcripts are highly upregulated under conditions of high salinity and low temperature stress. These findings suggested that As-PRMT1 is a stress-related factor that might promote or inhibit the expression of certain genes, play a critical role in embryonic development and in resistance to low temperature and high salinity stress.

Identification, expression pattern and potential role of variable lymphocyte receptor Aj-VLRA from Apostichopus japonicus in response to bacterial challenge.

The variable lymphocyte receptors (VLRs) are found in jawless vertebrates (agnathans), and specifically recognize bacteria and viruses via their leucine-rich repeats (LRRs). VLRs are believed to be adaptive immune response molecules. Echinoderms do not have adaptive immune systems; however, in the present study, a VLR cDNA named Aj-VLRA was cloned and characterized from sea cucumber, Apostichopus japonicus. The complete cDNA of Aj-VLRA was 3072 bp, including a 1995 bp open reading frame encoding 664 amino acids comprising LRR domains, a predicted transmembrane helix and an N-terminal signal peptide. As determined by quantitative real-time reverse transcription polymerase chain reaction, Aj-VLRA transcripts are ubiquitously expressed in the body wall, longitudinal muscles, intestine and respiratory tree of A. japonicus. The expression level of Aj-VLRA was upregulated after challenge with four common marine bacteria. In situ hybridization showed that the expression of Aj-VLRA was widely distributed in the four tissues, particularly in the cytoplasm of epidermal cells. Recombinantly expressed Aj-VLRA (including the LRR domains) could bind to bacteria including Micrococcus lysodeikticus (Gram+) and Vibrio anguillarum (Gram-). Collectively, the results suggested that Aj-VLRA is related to an innate immune response of A. japonicus.

Identification and expression patterns of extracellular matrix-associated genes fibropellin-ia and tenascin involved in regeneration of sea cucumber Apostichopus japonicus.

Sea cucumbers have a strong regenerative capacity. Many important genes involved in the molecular mechanism of regeneration and associated with intercellular signaling pathways of regeneration have been identified. The product of the fibropellin-ia gene forms a layer known as the apical lamina that surrounds the sea cucumber embryo throughout development. Meanwhile, the tenascin gene displays highly restricted and dynamic patterns of expression in the embryo and is expressed in the adult during normal processes such as wound healing, nerve regeneration and tissue involution. In this study, we cloned for the first time full-length cDNAs of fibropellin-ia (1390 bp, encoding a 199 amino acid protein) and tenascin (1366 bp, encoding a 179 amino acid protein) from Apostichopus japonicus (designated Aj-fnia and Aj-tenascin, respectively) using rapid amplification of cDNA ends. The structures and characteristics of these two genes were analyzed bioinformatically, and their expression patterns associated with extracellular matrix remodeling in regeneration of A. japonicus were investigated by real-time PCR and in situ hybridization (ISH). Expression levels of Aj-fnia and Aj-tenascin in the regeneration tissues were higher than those in normal tissues. The highest expression levels of Aj-fnia and Aj-tenascin were shown in the intestine and respiratory tree on the 15th and 20th days after sea cucumbers were eviscerated. In the body wall, the highest expression levels of Aj-fnia and Aj-tenascin occurred at 35 and 45 min during early regeneration and then emerged between 5 and 7 days again during late regeneration after the body wall was injured. ISH analysis revealed expression of these genes in the body wall, longitudinal muscle, intestine and respiratory tree. These findings suggest that Aj-fnia and Aj-tenascin are crucial genes that play important roles in the regeneration of the sea cucumber.

Identification, expression pattern and functional characterization of As-MyD88 in bacteria challenge and during different developmental stages of Artemia sinica.

Myeloid differentiation factor 88 (MYD88), a key adapter protein in Toll-like receptor signaling, affects the immune response and the formation of the dorsal-ventral axis. Here, the 1555bp full-length cDNA of MyD88 from Artemia sinica (As-MyD88) was obtained. Molecular characterization revealed that the sequence includes an 1182bp open reading frame encoding a predicted protein of 393 amino acids. The predicted protein contains a death domain in the N-terminus, and box1 and 2 motifs of the TIR domain in the C-terminus. Real-time quantitative PCR, Western blotting and immunohistochemistry were used to determine the expression level, protein production and location of As-MYD88 during embryonic development and bacterial challenge. The highest expression level during embryonic development was at the 0h and 5h stages of A. sinica. As-MYD88 was remarkably upregulated after bacterial challenge. Our results suggested that As-MYD88 plays a vital role in response to bacterial challenge, and during post-diapause embryonic development of A. sinica.