Study of the Structure and Bioactivity of Polysaccharides from Different Parts of Stemona tuberosa Lour.

The polysaccharides from Stemona tuberosa Lour, a kind of plant used in Chinese herbal medicine, have various pharmacological activities, such as anti-inflammatory and antioxidant properties. However, the effects of the extraction methods and the activity of polysaccharides from different parts are still unknown. Therefore, this study aimed to evaluate the effects of different extraction methods on the yields, chemical compositions, and bioactivity of polysaccharides extracted from different parts of Stemona tuberosa Lour. Six polysaccharides were extracted from the leaves, roots, and stems of Stemona tuberosa Lour through the use of hot water (i.e., SPS-L1, SPS-R1, and SPS-S1) and an ultrasound-assisted method (i.e., SPS-L2, SPS-R2, and SPS-S2). The results showed that the physicochemical properties, structural properties, and biological activity of the polysaccharides varied with the extraction methods and parts. SPS-R1 and SPS-R2 had higher extraction yields and total sugar contents than those of the other SPSs (SPS-L1, SPS-L2, SPS-S1, and SPS-S2). SPS-L1 had favorable antioxidant activity and the ability to downregulate MUC5AC expression. An investigation of the anti-inflammatory properties showed that SPS-R1 and SPS-R2 had greater anti-inflammatory activities, while SPS-R2 demonstrated the strongest anti-inflammatory potential. The results of this study indicated that SPS-L1 and SPS-L2, which were extracted from non-medicinal parts, may serve as potent natural antioxidants, but further study is necessary to explore their potential applications in the treatment of diseases. The positive anti-inflammatory effects of SPS-R1 and SPS-R2 in the roots may be further exploited in drugs for the treatment of inflammation.

The involvement of AMP-activated protein kinase α in regulating glycolysis in Yesso scallop Patinopecten yessoensis under high temperature stress.

AMP-activated protein kinase α subunit (AMPKα), the central regulatory molecule of energy metabolism, plays an important role in maintaining energy homeostasis and helping cells to resist the influence of various adverse factors. In the present study, an AMPKα was identified from Yesso scallop Patinopecten yessoensis (PyAMPKα). The open reading frame (ORF) of PyAMPKα was of 1599 bp encoding a putative polypeptide of 533 amino acid residues with a typical KD domain, a α-AID domain and a α-CTD domain. The deduced amino acid sequence of PyAMPKα shared 59.89-74.78% identities with AMPKαs from other species. The mRNA transcripts of PyAMPKα were found to be expressed in haemocytes and all the examined tissues, including gill, mantle, gonad, adductor muscle and hepatopancreas, with the highest expression level in adductor muscle. PyAMPKα was mainly located in cytoplasm of scallop haemocytes. At 3 h after high temperature stress treatment (25 °C), the mRNA transcripts of PyAMPKα, the phosphorylation level of PyAMPKα at Thr170 and the lactic acid (LD) content in adductor muscle all increased significantly, while the glycogen content decreased significantly. The activity of pyruvate kinase (PyPK) and the relative mRNA expression level of phosphofructokinase (PyPFK) were significantly up-regulated at 3 h after high temperature stress treatment (25 °C). Furthermore, the PyAMPKα activator AICAR could effectively upregulate the phosphorylation level of PyAMPKα, and increase activities of PyPFK and pyruvate kinase (PyPK). Meanwhile the glycogen content also declined under AICAR treatment. These results collectively suggested that PyAMPKα was involved in the high temperature stress response of scallops by enhancing glycolysis pathway of glycogen. These results would be helpful for understanding the functions of PyAMPKα in maintaining energy homeostasis under high temperature stress in scallops.

The expression profile of a multi-stress inducible transient receptor potential vanilloid 4 (TRPV4) in Pacific oyster Crassostrea gigas.

Transient receptor potential vanilloid 4 (TRPV4) is one of the major non-selective cation channel proteins, which plays a crucial role in sensing biotic and abiotic stresses, such as pathogen infection, temperature, mechanical pressure and osmotic pressure changes by regulating Ca2+ homeostasis. In the present study, a TRPV4 homologue was identified in Pacific oyster Crassostrea gigas, designated as CgTRPV4. The open reading frame (ORF) of CgTRPV4 was of 2298 bp encoding a putative polypeptide of 765 amino acid residues with three typical ankyrin domains and six conserved transmembrane domains of TRPV4 subfamily proteins, as well as multiple N-glycosylation sites, cAMP- and cGMP-dependent protein kinase phosphorylation sites, protein kinase C phosphorylation sites, casein kinase II phosphorylation sites, and prokaryotic membrane lipoprotein lipid attachment site. The deduced amino acid sequence of CgTRPV4 shared 20.5%-26.2% similarity with TRPV4s from other species. During the early ontogenesis stages of oyster, the mRNA transcripts of CgTRPV4 were detectable in all the stages with the highest expression level in fertilized eggs and the lowest in D-hinged larvae. In adult oyster, the CgTRPV4 mRNA could be detected in all the examined tissues, including gill, hepatopancreas, adductor muscle, labial palp, mantle and haemocyte, with the highest expression level in gill (45.08-fold of that in hepatopancreas, p < 0.05). In immunocytochemical assay, the CgTRPV4 positive signals were distributed in both endoplasmic reticulum and cytoplasmic membrane of oyster haemocytes. The mRNA expression of CgTRPV4 in gill was significantly up-regulated after high temperature stress at 30°C (p < 0.05) and after Vibrio splendidus stimulation (p < 0.05). These results indicated that CgTRPV4 was a classical member of TRPV4 family in oyster, which was induced by either biotic or abiotic stimulations and involved in mediating the stress response of oysters.

Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device.

Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect posttranslational modifications with single-molecule sensitivity. Here, we demonstrate single-molecule protein sequencing using a dynamic approach in which single peptides are probed in real time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. We annotate amino acids and identify the peptide sequence by measuring fluorescence intensity, lifetime, and binding kinetics on an integrated semiconductor chip. Our results demonstrate the kinetic principles that allow recognizers to identify multiple amino acids in an information-rich manner that enables discrimination of single amino acid substitutions and posttranslational modifications. With further development, we anticipate that this approach will offer a sensitive, scalable, and accessible platform for single-molecule proteomic studies and applications.

The involvement of PyBeclin 1 and PyLC3 in regulating the activation of autophagy in scallop Patinopecten yessoensis after acute high temperature stress.

Beclin 1 and LC3 are important autophagy regulation proteins involved in vesicle nucleation and extension stage, respectively. In the present study, a Beclin 1 and a LC3 were identified from Yesso scallop Patinopecten yessoensis (PyBeclin 1 and PyLC3). The open reading frame (ORF) of PyBeclin 1 was of 1335 bp encoding a putative polypeptide of 444 amino acid residues with an N-terminal BCL-2 homology 3 (BH3) domain, a central coiled-coil domain (CCD), and a C-terminal evolutionarily conserved domain (ECD). The ORF of PyLC3 was of 369 bp encoding a putative polypeptide of 122 amino acid residues with an APG12 domain. The deduced amino acid sequences of PyBeclin 1 and PyLC3 shared 31.92-74.09% and 68.38-79.50% identities with Beclin 1s and LC3s from other species, respectively. The mRNA transcripts of PyBeclin 1 and PyLC3 were found to be expressed in all the examined tissues, including adductor muscle, gonad, gill, haemocytes and mantle, with the highest expression level in gill and haemocytes. The mRNA expression level of PyBeclin 1 in haemocytes increased significantly at 1, 3, 6, 12 and 24 h (2.98-4.07 fold of that in the Blank group, p < 0.05), and returned to normal level at 48 h after acute high temperature stress at 25 °C. Unlike PyBeclin 1, the mRNA transcripts of PyLC3 in haemocytes were significantly up-regulated at1, 3, 6 and 12 h (1.80-2.54 fold of that in the Blank group, p < 0.05), then decreased to blank level at 24 h (p > 0.05), and increased significantly again at 48 h (3.70 fold of that in the Blank group, p < 0.05) after high temperature. PyBeclin 1 and PyLC3 were mainly located in the cytoplasm and a small amount in the nucleus with few puncta, and the numbers of PyBeclin 1 and PyLC3 puncta increased at 3 h after acute high temperature stress. The LC3-II levels in gill and haemocytes were significantly up-regulated at 1 h and 3 h after acute high temperature stress. These results collectively suggested that PyBeclin 1 and PyLC3 were conserved members of Beclin 1 and LC3 family in scallops, and involved in regulating the activation of autophagy in scallops after acute high temperature stress.

The expression profile of calnexin in Patinopecten yessoensis after acute high temperature stress.

Calnexin (CNX) is one of the major calcium-binding proteins in endoplasmic reticulum (ER) and plays a crucial role in regulating Ca2+ homeostasis and unfolded protein response (UPR). In the present study, PyCNX was identified in Yesso Scallop Patinopecten yessoensis. The open reading frame (ORF) of PyCNX was of 1794 bp encoding a putative polypeptide of 598 amino acid residues with an N-terminal signal peptide, a typical calreticulin (CRT) domain and a C-terminal transmembrane domain. The deduced amino acid sequence of PyCNX shared 47.91%-70.16% identities with CNXs from other species. The mRNA transcripts of PyCNX were constitutively expressed in all the examined tissues, including gills, gonad, hepatopancreas, mantle and haemocytes, with the highest expression level in gills. The mRNA transcripts of PyCNX in gills were significantly up-regulated at 1 h (p < 0.01), down-regulated to similar level of Blank group at 3 h (p > 0.05) and 6 h (p > 0.05), and decreased significantly from 12 to 48 h (p < 0.05) after acute high temperature stress (25 °C). PyCNX was mainly located in the ER of haemocytes. The expression profiles of PyATF6, PyIRE1 and PyGRP78 in the gills of scallops after acute high temperature stress were investigated using quantitative real-time PCR. The mRNA transcripts of PyATF6 increased significantly at 1 h, 3 h and 6 h after acute high temperature stress (p < 0.01), then down-regulated to similar level of Blank group at 12 h (p > 0.05) and 24 h (p > 0.05), and finally significantly up-regulated again at 48 h (p < 0.05). Similar to PyATF6, the mRNA transcripts of PyIRE1 were significantly up-regulated at 1 h (p < 0.05), 3 h (p < 0.01), 6 h (p < 0.05) and 48 h (p < 0.05) after acute high temperature stress. The mRNA transcripts of PyGRP78 were significantly up-regulated at 3 h (p < 0.05), reached the highest level at 6 h (p < 0.01) after acute high temperature stress, and kept significant higher level at 12-48 h (p < 0.05). These results indicated that PyCNX was involved in the response upon the acute high temperature stress of scallops probably by regulating UPR.