Two-dimensional gel electrophoresis-based analysis provides global insights into the cotton ovule and fiber proteomes

Proteomic analysis of upland cotton was performed to profile the global detectable proteomes of ovules and fibers using two-dimensional electrophoresis (2DE). A total of 1,203 independent protein spots were collected from representative 2DE gels, which were digested with trypsin and identified by matrix-assisted laser desorption and ionization-time-offlight/ time-of-flight (MALDI-TOF/TOF) mass spectrometry. The mass spectrometry or tandem mass spectrometry (MS or MS/MS) data were then searched against a local database constructed from Gossypium hirsutum genome sequences, resulting in successful identification of 975 protein spots (411 for ovules and 564 for fibers). Functional annotation analysis of the 975 identified proteins revealed that ovule-specific proteins were mainly enriched in functions related to fatty acid elongation, sulfur amino acid metabolism and post-replication repair, while fiber-specific proteins were enriched in functions related to root hair elongation, galactose metabolism and D-xylose metabolic processes. Further annotation analysis of the most abundant protein spots showed that 28.96% of the total proteins in the ovule were mainly located in the Golgi apparatus, endoplasmic reticulum, mitochondrion and ribosome, whereas in fibers, 27.02% of the total proteins were located in the cytoskeleton, nuclear envelope and cell wall. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses of the ovule-specific protein spots P61, P93 and P198 and fiber-specific protein spots 230, 477 and 511 were performed to validate the proteomics data. Protein-protein interaction network analyses revealed very different network cluster patterns between ovules and fibers. This work provides the largest protein identification dataset of 2DE-detectable proteins in cotton ovules and fibers and indicates potentially important roles of tissue-specific proteins, thus providing insights into the cotton ovule and fiber proteomes on a global scale.     

Paeoniflorin Inhibits Migration and Invasion of Human Glioblastoma Cells via Suppression Transforming Growth Factor β-Induced Epithelial–Mesenchymal Transition

Paeoniflorin (PF) is a polyphenolic compound derived from Radix Paeoniae Alba thathas anti-cancer activities in a variety of human malignancies including glioblastoma. However, the underlying mechanisms have not been fully elucidated. Epithelial to mesenchymal transition (EMT), characterized as losing cell polarity, plays an essential role in tumor invasion and metastasis. TGFβ, a key member of transforming growth factors, has been demonstrated to contribute to glioblastoma aggressiveness through inducing EMT. Therefore, the present studies aim to investigate whether PF suppresses the expression of TGFβ and inhibits EMT that plays an important role in anti-glioblastoma. We found that PF dose-dependently downregulates the expression of TGFβ, enhances apoptosis, reduces cell proliferation, migration and invasion in three human glioblastoma cell lines (U87, U251, T98G). These effects are enhanced in TGFβ siRNA treated cells and abolished in cells transfected with TGFβ lentiviruses. In addition, other EMT markers such as snail, vimentin and N-cadherin were suppressed by PF in these cell lines and in BALB/c nude mice injected with U87 cells. The expression of MMP2/9, EMT markers, are also dose-dependently reduced in PF treated cells and in U87 xenograft mouse model. Moreover, the tumor sizes are reduced by PF treatment while there is no change in body weight. These results indicate that PF is a potential novel drug target for the treatment of glioblastoma by suppression of TGFβ signaling pathway and inhibition of EMT.

     

Pharmacokinetics and liver uptake of three Schisandra lignans in rats after oral administration of liposome encapsulating β-cyclodextrin inclusion compound of Schisandra extract

Schisandra chinensis fructus (SCF) is widely used traditional Chinese medicine, which possesses hepato-protective potential. Schisandrin (SD), schisantherin (ST), and γ-schizandrin (SZ) are the major bioactive lignans. The main problem associated with the major bioactive lignans oral administration is low oral bioavailability due to the lignans’ poor aqueous solubility and taste. The aim of the present research work was to develop liposome (SCL) encapsulated β-cyclodextrin (β-CD) inclusion complex loaded with SCF extract (SCF-E). The SD, ST, and SZ were selected as effective candidates to perform comparisons of liver targeting among the solution (SES), β-cyclodextrin inclusion compound (SCF-E-β-CD), liposome (SEL), and SCL of SCF-E to characterize the pharmacokinetic behaviors and liver targeting in rats. The β-CD inclusion complex (SCF-E-β-CD) was used to improve the solubility. The concentrations were determined using high-performance liquid chromatography (HPLC) and analyzed by DAS3.0. The pharmacokinetic results indicate that the plasma concentration-time courses were fitted well to the one-compartment model with the first weighing factor. The half-life period (t_1/2) and area under the concentration-time curve (AUC) of the three components in SCL were the largest. The SCL exhibit a relatively high liver targeting effect. The results would be helpful for guiding the clinical application of this herbal medicine.     

Cell cycle-dependent subcellular distribution of ClC-3 in HeLa cells

Chloride channel-3 (ClC-3) is suggested to be a component and/or a regulator of the volume-activated Cl(-) channel in the plasma membrane. However, ClC-3 is predominantly located inside cells and the role of intracellular ClC-3 in tumor growth is unknown. In this study, we found that the subcellular distribution of endogenous ClC-3 varied in a cell cycle-dependent manner in HeLa cells. During interphase, ClC-3 was distributed throughout the cell and it accumulated at various positions in different stages. In early G1, ClC-3 was mainly located in the nucleus. In middle G1, ClC-3 gathered around the nuclear periphery as a ring. In late G1, ClC-3 moved back into the nucleus, where it remained throughout S phase. In G2, ClC-3 was concentrated in the cytoplasm. When cells progressed from G2 to the prophase of mitosis, ClC-3 from the cytoplasm translocated into the nucleus. During metaphase and anaphase, ClC-3 was distributed throughout the cell except for around the chromosomes and was aggregated at the spindle poles and in between two chromosomes, respectively. ClC-3 was then again concentrated in the nucleus upon the progression from telophase to cytokinesis. These results reveal a cell cycle-dependent change of the subcellular distribution of ClC-3 and strongly suggest that ClC-3 has nucleocytoplasmic shuttling dynamics that may play key regulatory roles during different stages of the cell cycle in tumor cells.     

Natural products as a gold mine for selective matrix metalloproteinases inhibitors

Nineteen natural compounds with diverse structures are identified as potential MMPIs using structure-based virtual screening from 4000 natural products. Hydroxycinnamic acid or analogs of natural products are important for potent inhibitory and selectivity against MMPs, and the solvent effect in the S1' pocket can affect the hydrophobic interactions and hydrogen bonds between MMPIs and MMPS, making MMPIs exhibit certain selectivity for a specific MMP isoenzyme. Furthermore, compound 5 can reduce the expression of both MMP-2 and active-MMP-9, and suppress the migration of MDA-MB-231 tumor cell in a wound healing assay, which may be further developed as an anticancer agent.     

A chloroplast envelope membrane protein containing a putative LrgB domain related to the control of bacterial death and lysis is required for chloroplast development in Arabidopsis thaliana

? A protein encoded by At1g32080 was consistently identified in proteomic studies of Arabidopsis chloroplast envelope membranes, but its function remained unclear. The protein, designated AtLrgB, may have evolved from a gene fusion of lrgA and lrgB. In bacteria, two homologous operons, lrgAB and cidAB, participate in an emerging mechanism to control cell death and lysis. ? We aim to characterize AtLrgB using reverse genetics and cell biological and biochemical analysis. ? AtLrgB is expressed in leaves, but not in roots. T-DNA insertion mutation of AtLrgB produced plants with interveinal chlorotic and premature necrotic leaves. Overexpression of full-length AtLrgB (or its LrgA and LrgB domains, separately), under the control of CaMV 35S promoter, produced plants exhibiting veinal chlorosis and delayed greening. At the end of light period, the T-DNA mutant had high starch and low sucrose contents in leaves, while the 35S:AtLrgB plants had low starch and high sucrose contents. Metabolite profiling revealed that AtLrgB appeared not to directly transport triose phosphate or hexose phosphates. In yeast cells, AtLrgB could augment nystatin-induced membrane permeability. ? Our work indicates that AtLrgB is a new player in chloroplast development, carbon partitioning and leaf senescence, although its molecular mechanism remains to be established.     

Hypertrophic response to angiotensin II is mediated by protein kinase D-extracellular signal-regulated kinase 5 pathway in human aortic smooth muscle cells

Angiotensin II plays a critical role in hypertrophy of vascular smooth muscle cells, however, the molecular underpinnings remain unclear. The present study indicated that AT₁/PKC/PKD pathway was able to regulate downstream ERK5, affecting pro-hypertrophic responses to Ang II. Ang II-stimulated phosphorylation of ERK5 in a time- and dose-dependent manner in human aortic smooth muscle cells (HASMCs). The pharmacological inhibitors for AT₁ and PKCs significantly inhibited Ang II-induced ERK5 activation, suggesting the involvement of the AT₁/PKC pathway. In particular, PKD was critical for Ang II-induced ERK5 activation since silencing PKD by siRNA markedly inhibited Ang II-induced ERK5 activation. Consequently, we found that Losartan, Gö 6983 and PKD siRNA significantly attenuated ERK5 activated translocation and hypertrophy of HASMCs by Ang II. Taken together, we demonstrated for the first time that Ang II activates ERK5 via the AT₁/PKC/PKD pathway and revealed a critical role of ERK5 in Ang II-induced HASMCs hypertrophy.     

A glycine site‐specific NMDA receptor antagonist protects retina ganglion cells from ischemic injury by modulating apoptotic cascades

Glutamate neurotoxicity is one of the causative factors leading to neural degeneration including retina. Inhibition of NMDA receptors has been shown neuroprotective effects. However, specifically inhibition of glycine subunit in NMDA receptors and its effects on retina neural protection has not been tested. In this study, using a glycine site‐specific NMDA receptor antagonist, we investigated its neuroprotective effects on rat retinal ganglion cells (RGCs) from a transient ischemic injury and its possible underlying mechanisms. Following an ischemia/reperfusion injury the structural damages of rat retinas were assessed by an immunofluorescence method and the apoptosis of retinal neural cells was evaluated by using a terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) method. The survived RGCs were labeled by retrograde manner and counted on whole‐mounted retinas. In the presence of glycine site‐specific NMDA receptor antagonist, the thickness of retina was sustained, especially in the inner nuclear layers compared with mock controls. While a significantly higher numbers of TUNEL‐positive apoptotic cells and fewer of RGCs were observed in the retina without the glycine antagonist, indicating its strong protective roles. Some apoptotic factors such as Bax, Bcl‐2, CAMK II, COX1, COX4, Caspase‐3, and GRIN1 gene have been tested from retinal samples with or without the glycine antagonist. A significantly lower of expressions of Bax, CAMK II, COX1, COX4, Caspase‐3, and GRIN1 have been shown in the retinas with the antagonist. Bcl‐2/Bax ratio was significantly higher with the antagonist, suggested that the glycine site‐specific NMDA receptor antagonist protecting RGC death might through inhibition of apoptotic signaling. J. Cell. Physiol. 223:819–826, 2010. © 2010 Wiley‐Liss, Inc.     

A versatile tool for tracking the differentiation of human embryonic stem cells

The ability of human embryonic stem cells (hESCs)to undergo indefinite self-renewal in vitro and to produce lineages derived from all three embryonic germ layers both in vitro and in vivo makes such cells extremely valuable in both clinical and research settings.However,the generation of specialized cell lineages from a mixture of differentiated hESCs remains technically difficult.Tissue specific promoter-driven reporter genes are powerful tools for tracking cell types of interest in differentiated cell populations.Here,we describc the construction of modular lentivectors containing different tissue-specific promoters(Tαl of α-tubulin:αP2 of adipocyte Protein 2;and AFP of alpha fetoprotein)driving expression of humanized Renilla green fluorescent protein(hrGFP).To this end,we used MultiSite gateway technology and employed the novel vectors to successfully monitor hESC differentiation.We present a versatile method permitting target cells to bc traced.Our system will facilitate research in developmental biology,transplantation,and in vivo stem cell tracking.