Differential proteomic analysis of platelets suggested possible signal cascades network in platelets treated with salvianolic acid B

Background

Salvianolic acid B (SB) is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2β1. But, the signal cascades in platelets after SB binding are still not clear.

Methodology/Principal Findings

In the present study, a differential proteomic analysis (two-dimensional electrophoresis) was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2β1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2β1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca(2+) and reactive oxygen species (ROS) were checked with or without pre-treatment of platelets using antibody against integrin α2β1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets.

Conclusions/Significance

Integrin α2β1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2β1 might include regulation of intracellular Ca(2+) level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets.     

Mth10b, a unique member of the Sac10b family, does not bind nucleic acid

The Sac10b protein family is regarded as a group of nucleic acid-binding proteins that are highly conserved and widely distributed within archaea. All reported members of this family are basic proteins that exist as homodimers in solution and bind to DNA and/or RNA without apparent sequence specificity in vitro. Here, we reported a unique member of the family, Mth10b from Methanobacterium thermoautotrophicum ΔH, whose amino acid sequence shares high homology with other Sac10b family proteins. However, unlike those proteins, Mth10b is an acidic protein; its potential isoelectric point is only 4.56, which is inconsistent with the characteristics of a nucleic acid-binding protein. In this study, Mth10b was expressed in Escherichia coli and purified using a three-column chromatography purification procedure. Biochemical characterization indicated that Mth10b should be similar to typical Sac10b family proteins with respect to its secondary and tertiary structure and in its preferred oligomeric forms. However, an electrophoretic mobility shift analysis (EMSA) showed that neither DNA nor RNA bound to Mth10b in vitro, indicating that either Mth10b likely has a physiological function that is distinct from those of other Sac10b family members or nucleic acid-binding ability may not be a fundamental factor to the actual function of the Sac10b family.     

Caveolin-1 plays a crucial role in inhibiting neuronal differentiation of neural stem/progenitor cells via VEGF signaling-dependent pathway

In the present study, we aim to elucidate the roles of caveolin-1(Cav-1), a 22 kDa protein in plasma membrane invaginations, in modulating neuronal differentiation of neural progenitor cells (NPCs). In the hippocampal dentate gyrus, we found that Cav-1 knockout mice revealed remarkably higher levels of vascular endothelial growth factor (VEGF) and the more abundant formation of newborn neurons than wild type mice. We then studied the potential mechanisms of Cav-1 in modulating VEGF signaling and neuronal differentiation in isolated cultured NPCs under normoxic and hypoxic conditions. Hypoxic embryonic rat NPCs were exposed to 1% O₂ for 24 h and then switched to 21% O₂ for 1, 3, 7 and 14 days whereas normoxic NPCs were continuously cultured with 21% O₂. Compared with normoxic NPCs, hypoxic NPCs had down-regulated expression of Cav-1 and up-regulated VEGF expression and p44/42MAPK phosphorylation, and enhanced neuronal differentiation. We further studied the roles of Cav-1 in inhibiting neuronal differentiation by using Cav-1 scaffolding domain peptide and Cav-1-specific small interfering RNA. In both normoxic and hypoxic NPCs, Cav-1 peptide markedly down-regulated the expressions of VEGF and flk1, decreased the phosphorylations of p44/42MAPK, Akt and Stat3, and inhibited neuronal differentiation, whereas the knockdown of Cav-1 promoted the expression of VEGF, phosphorylations of p44/42MAPK, Akt and Stat3, and stimulated neuronal differentiation. Moreover, the enhanced phosphorylations of p44/42MAPK, Akt and Stat3, and neuronal differentiation were abolished by co-treatment of VEGF inhibitor V1. These results provide strong evidence to prove that Cav-1 can inhibit neuronal differentiation via down-regulations of VEGF, p44/42MAPK, Akt and Stat3 signaling pathways, and that VEGF signaling is a crucial target of Cav-1. The hypoxia-induced down-regulation of Cav-1 contributes to enhanced neuronal differentiation in NPCs.     

Stability analysis of liver cancer-related microRNAs

MicroRNAs (miRNAs) are non-coding, single-stranded RNAs of approximately 22 nt and constitute a novel class of gene regulators that are found in both plants and animals. Several studies have demonstrated that serum miRNAs could serve as potential biomarkers for the detection of various cancers and other diseases. A few documents regarding the stability of liver cancer-related miRNAs in serum are available. A systemic analysis of the stability of miRNA in serum is quite necessary. The purpose of this study was to evaluate the stability of miRNAs from three different sources, cultured liver cancer Huh-7 cell line, clinical liver cancer, and serum under different experimental conditions, including different temperature, time duration, pH values, RNase A digestion, DNase I digestion, and various freeze-thaw cycles. The qRT-PCR analysis demonstrated that liver cancer-related miRNAs were detectable under each of test conditions, indicating that miRNAs were extremely stable and resistant to destruction and degradation under harsh environmental conditions. However, ribosomal RNA was fragile and easily degraded by demonstrating sharp decrease of relative expression under the non-physiological test conditions. We also established a robust procedure for serum RNA extraction, which is greatly important not only for the miRNA profiling studies but also for the disease prognosis based on abnormal miRNA expression.     

Evaluation of Beauveria bassiana (Hyphomycetes) isolates as potential agents for control of Dendroctonus valens

Abstract The red turpentine beetle (RTB), Dendroctonus valens LeConte, as a destructive invasive pest, has become one of the most economically important forest pest in China. Effective control measures are desperately needed. Entomopathogenic fungi, such as Beauveria bassiana, have shown great potential for the management of some bark beetle species. In this study, 12 isolates of B. bassiana from bark beetle were examined for biological characteristics and virulence, to assess their potential as biocontrol agents for RTB. There were significant differences (at P= 0.05) in colony growth rate, conidial yield, conidial germination, tolerance to UV light and extracellular proteases activity among the tested B. bassiana isolates. Isolates, including Bb1801, Bb1906, Bb789 and Bb773, exhibited the best characteristics, because they have faster hyphal growth rate, higher spore production and faster spore germination, higher UV tolerance and protease (Pr1) production. The results of a pathogenicity test of B. bassiana on RTB larvae showed that most isolates of B. bassiana have demonstrated high efficacy and the highest virulent isolate was Bb1801, which killed 100% of the treated insects and had a median lethal time (LT₅₀) of 4.60 days at a concentration of 1×10⁷ conidia/mL. Therefore, isolate Bb1801 has a great potential for sustainable control of RTB in the forest. The correlation between biological characteristics and virulence of the fungal isolates is discussed and the possibility of combination of entomopathogenic fungi with semiochemicals, as one of the promising strategy for RTB control, is considered.     

Isolation and characterization of the anthocyanidin genes PAL, F3H and DFR of Scutellaria viscidula (Lamiaceae)

Anthocyanidin is a group of flavonoid compounds used as a vegetable pigment and plays an important role in flower coloration and environmental adaptations of the Chinese ornamental plant Scutellaria viscidula. We determined the cDNA sequences of phenylalanine ammonia-lyase (SvPAL), flavanone 3-hydroxylase (SvF3H) and dihydroflavonol 4-reductase (SvDFR) genes in S. viscidula. Comparative analysis showed that the protein products of these three genes did not have a transit peptide at their N-terminal portion, which indicated that these enzymes were directly involved in the substrate conversion in the cytoplasmic matrix. Bioinformatic analysis further revealed that Svpal, Svf3h and Svdfr were the members of flavonoid biosynthetic genes with highly conserved motifs. Based on phylogenetic tree analysis, it appears that PAL, F3H or DFR from different plants might have originated from the same ancestor. This study can help to map and regulate the important stages involved in anthocyanidin biosynthesis by genetic engineering to diversify flower color and improve the ornamental value of S. viscidula.     

Comparison of alternative extraction methods for secretome profiling in human hepatocellular carcinoma cells

Secreted proteins are important sources for early detection and diagnosis of disease, and as such have received considerable attention. The extraction of low concentration proteins from large volumes of culture media, which are rich in salts and other compounds that interfere with most proteomics techniques, presents a problem for secretome studies. Ultrafiltration, precipitation, and dialysis are three major extraction methods that can be used to overcome this problem. The present study for the first time, compared the merits and shortcomings of these three methods, without bias. Centrifugal ultrafiltration provided the best extraction efficiency, and precipitation provided the highest number of identifiable proteins. The three methods yielded closely related, but different, information on the secretome; thus, they should be considered complementary or, at least, supplementary methods. Three hundred and sixty unique proteins were identified, including 211 potential secreted proteins. Compared with previous studies, this study also identified 42 new secreted proteins. The present study not only offers a reference for the selection of secretome extraction methods, but also expands the secretome database for the investigation of hepatocellular carcinoma.     

Effects of osteoprotegerin from transfection of pcDNA3.1(+)/chOPG on bioactivity of chicken osteoclasts

Background  

Osteoprotegerin (OPG) has been reported to prevent bone resorption by inhibiting the formation, function, and survival of osteoclasts in a variety of animal models. However, the effects of OPG on bone metabolism in avian species have not been described. The objective of this study was to investigate the effects of chicken OPG (chOPG) expressed in chicken embryo fibroblasts (CEFs) on chicken osteoclast function in vitro.