Proteomic analysis of rice (Oryza sativa) seeds during germination

Although seed germination is a major subject in plant physiological research, there is still a long way to go to elucidate the mechanism of seed germination. Recently, functional genomic strategies have been applied to study the germination of plant seeds. Here, we conducted a proteomic analysis of seed germination in rice (Oryza sativa indica cv. 9311) - a model monocot. Comparison of 2-DE maps showed that there were 148 proteins displayed differently in the germination process of rice seeds. Among the changed proteins, 63 were down-regulated, 69 were up-regulated (including 20 induced proteins). The down-regulated proteins were mainly storage proteins, such as globulin and glutelin, and proteins associated with seed maturation, such as "early embryogenesis protein" and "late embryogenesis abundant protein", and proteins related to desiccation, such as "abscisic acid-induced protein" and "cold-regulated protein". The degradation of storage proteins mainly happened at the late stage of germination phase II (48 h imbibition), while that of seed maturation and desiccation associated proteins occurred at the early stage of phase II (24 h imbibition). In addition to alpha-amylase, the up-regulated proteins were mainly those involved in glycolysis such as UDP-glucose dehydrogenase, fructokinase, phosphoglucomutase, and pyruvate decarboxylase. The results reflected the possible biochemical and physiological processes of germination of rice seeds.     

Adiponectin decreases plasma glucose and improves insulin sensitivity in diabetic Swine

To investigate the effects of recombinant human adiponectin on the metabolism of diabeticswine induced by feeding a high-fat/high-sucrose diet (HFSD),diabetic animal models were constructedby feeding swine with HFSD for 6 months.The effects of recombinant adiponectin were assessed bydetecting the change of plasma glucose levels by commercially available enzymatic method test kits andevaluating the insulin sensitivity by oral glucose tolerance test (OGTT). About 1.5 g purified recombinantadiponectin was produced using a 15-liter fermenter.A single injection of purified recombinant humanadiponectin to diabetic swine led to a 2- to 3-fold elevation in circulating adiponectin,which triggered atransient decrease in basal glucose level (P<0.05).This effect on glucose was not associated with anincrease in insulin level.Moreover,after adiponectin injection,swine also showed improved insulin sensitivitycompared with the control (P<0.05).Adiponectin might have the potential to be a glucose-lowering agentfor metabolic disease.Adiponectin as a potent insulin enhancer linking adipose tissue and glucose metabolismcould be useful to treat insulin resistance.     

A Lectin from Chinese Mistletoe Increases γδ T Cell-mediated Cytotoxicity through Induction of Caspase-dependent Apoptosis

In this study, a mistletoe lectin (ML) was purified from Chinese mistletoe and the effect of this 60 kDa Chinese ML on human γδ T cell cytotoxicity, apoptosis and modulation of the cytokine network was studied. The cytotoxic properties of δ T cells was evaluated by using a ~(51)Cr release test and employed fluorescence-activated cell sorting and enzyme-linked immunosorbent assay analysis to quantify translocation of the cell membrane phospholipid, phosphatidylserine and nuclear DNA fragmentation during apoptosis. It was found that: (ⅰ) ML effectively stimulated γδ T cell proliferation in a dose- and time-dependent manner; (ⅱ) ML increased γδ T cell cytotoxicity; (ⅲ) ML could modulate lipopolysaccharide-induced cytokine release in a pro-inflammatory manner by increasing tumor necrosis factor (TNF)-α release and inhibiting the release of anti-inflammatory interleukin (IL)-10; (ⅳ) ML induced apoptosis in caspase-dependent and CD95-independent manner. The results indicated that ML is a potent immunomodulator to human γδ T cell cytotoxicity, apoptosis and cytokine production.     

Two New Alkamides from Spilanthes callimorpha

Two new alkamides, 8,11-dihydroxy-dodeca-2E,4E,9E-triensaureisobutylamid （Compound 1） and 7-hydroxy-trideca-2E,8E- dien-10,12-diynoic acid isobutylamide （Compound 2）, as well as eight known compounds were isolated from the whole plants of Spilanthes callimorpha. The structures of the alkamides were characterized on the basis of their spectral data.     

Defining the optimal parameters for hairpin-based knockdown constructs

Induction of gene silencing using intracellularly expressed silencing triggers has been explored for large-scale loss-of-function screening, creation of knockdown cell lines or knockdown animals, and disease intervention. In all of these applications, the use of highly potent silencing constructs can maximize the possibility of obtaining target knockdown and thereby is intrinsically important for the chance of success. Several attempts have been made to improve the potency of a silencing construct. Results published in high profile journals such as Nature Biotechnology and Nature Genetics suggest that shRNAs with a 29-nucleotide (nt) stem is much more potent than shRNAs with a 19-nt stem, and miR30-based silencing constructs are much more potent than shRNA-based constructs. In this study, we systematically investigated several parameters, including the use of shRNA- or miR30-based scaffolds, the length of shRNA, and the selection of shRNA sequences for their impact on the knockdown efficiency of a silencing construct. Our studies revealed that the optimal configurations for a potent silencing trigger could be an shRNA with a 19-nt stem and a 9-nt loop. By comparing properties that favor the functional shRNAs and siRNAs using a set of 190 shRNAs against 19 targets and 360 siRNAs against four targets, we found that the functional shRNAs and siRNAs displayed similar but not identical nucleotide preferences. Based on the characteristic nucleotide preferences in the functional versus the nonfunctional shRNAs, we developed a computer program that outperforms an advanced siRNA selection algorithm for the enrichment of highly functional shRNAs.     

Plant uptake and the leaching of metals during the hot EDDS-enhanced phytoextraction process

Using pot experiments, the effect of the application of the biodegradable chelating agent S,S-ethylenediaminedisuccinic acid (EDDS) in hot solutions at 90 degrees C on the uptake of Cu, Pb, Zn, and Cd by corn (Zea mays L. cv. Nongda No. 108) and beans (P vulgaris L. white bean), and the potential leaching of metals from soil, were studied. When EDDS was applied as a hot solution at the rate of 1 mmol kg(-1), the concentrations and total phytoextraction of metals in plant shoots exceeded or approximated those in the shoots of plants treated with normal EDDS at the rate of 5 mmol kg(-1). On the other hand, the leaching of Cu, Pb, Zn, and Cd after the application of the hot EDDS solution at the rate of 1 mmol kg(-1) was reduced by 46%, 21%, 57%, and 35% in comparison with that from the application of normal EDDS at 5 mmol kg(-1), respectively. For treatment with 1 mmol kg(-1) of EDDS, the leached metals decreased to the levels of the control group (that without EDDS amendment) 14 d after the application of EDDS. The soil amendment with biodegradable EDDS in hot solutions may provide a good alternative to chelate-enhanced phytoextraction in enhancing metal uptake by plants and limiting metals from leaching out of the soil.     

Genetic analysis of attractin homologs

Attractin (ATRN) and Attractin-like 1 (ATRNL1) are highly similar type I transmembrane proteins. Atrn null mutant mice have a pleiotropic phenotype including dark fur, juvenile-onset spongiform neurodegeneration, hypomyelination, tremor, and reduced body weight and adiposity, implicating ATRN in numerous biological processes. Bioinformatic analysis indicated that Atrn and Atrnl1 arose from a common ancestral gene early in vertebrate evolution. To investigate the genetics of the ATRN system and explore potential redundancy between Atrn and Atrnl1, we generated and characterized Atrnl1 loss- and gain-of-function mutations in mice. Atrnl1 mutant mice were grossly normal with no alterations of pigmentation, central nervous system pathology or body weight. Atrn null mutant mice carrying a beta-actin promoter-driven Atrnl1 transgene had normal, agouti-banded hairs and significantly delayed onset of spongiform neurodegeneration, indicating that over-expression of ATRNL1 compensates for loss of ATRN. Thus, the two genes are redundant from the perspective of gain-of-function but not loss-of-function mutations.     

Taxol induced apoptosis regulates amino acid transport in breast cancer cells

A major outcome from Taxol treatment is induction of tumor cell apoptosis. However, metabolic responses to Taxol-induced apoptosis are poorly understood. In this study, we hypothesize that alterations in specific amino acid transporters may affect the Taxol-induced apoptosis in breast cancer cells. In this case, the activity of the given transporter may serve as a biomarker that could provide a biological assessment of response to drug treatment. We have examined the mechanisms responsible for Taxol-induced neutral amino acid uptake by breast cancer cells, such as MCF-7, BT474, MDAMB231 and T47D. The biochemical and molecular studies include: (1) growth-inhibition (MTT); (2) transport kinetics: (3) substrate-specific inhibition; (4) effect of thiol-modifying agents NEM and NPM; (5) gene expression of amino acid transporters; and (6) apoptotic assays. Our data show that Taxol treatment of MCF-7 cells induced a transient increase in Na⁺-dependent transport of the neutral amino acid transporter B0 at both gene and protein level. This increase was attenuated by blocking the transporter in the presence of high concentrations of the substrate amino acid. Other neutral amino acid transporters such as ATA2 (System A) and ASC were not altered. Amino acid starvation resulted in the expected up-regulation of System A (ATA2) gene, but not for B0 and ASC. B0 was significantly down regulated. Taxol treatment had no significant effect on the uptake of arginine and glutamate as measured by System y⁺ and X⁻ _GC respectively. Tunel assays and FACS cell cycle analysis demonstrated that both Taxol- and doxorubicin-induced upregulation of B0 transporter gene with accompanying increase in cell apoptosis, could be reversed partially by blocking the B0 transporter with high concentration of alanine, and/or by inhibiting the caspase pathway. Both Taxol and doxorubicin treatment caused a significant decrease in S-phase of the cell cycle. However, Taxol-induced an increase primarily in the G2 fraction while doxorubicin caused increase in G1/G0 together with a small increase in G2. In summary, our study showed that Taxol induced apoptosis in several breast cancer cells results in activation of amino acid transporter System B0 at both gene and protein level. Similar response was observed with another chemotherapeutic agent Doxorubicin, suggesting that this increase is in response to apoptosis, and not only due to changes in cell cycle related events. Drs. Wu and Shen contributed equally to this study.     

The carboxyl-terminal SH3 domain of the mammalian adaptor CrkII promotes internalization of Listeria monocytogenes through activation of host phosphoinositide 3-kinase

The intracellular bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to gastroenteritis, meningitis or abortion. Listeria induces its internalization into some mammalian cells through binding of the bacterial surface protein InlB to its host receptor, the Met Receptor Tyrosine Kinase. InlB-induced activation of Met stimulates host signal transduction pathways that culminate in cell surface changes driving pathogen engulfment. One mammalian protein with the potential to couple Met to downstream signalling is the adaptor CrkII. CrkII contains an unusual carboxyl-terminal SH3 domain (SH3C) that promotes entry of Listeria. However, binding partners or downstream effectors of SH3C remain unknown. Here, we use RNA interference and overexpression studies to demonstrate that SH3C affects bacterial uptake, at least in part, through stimulation of host phosphatidylinositide (PI) 3-kinase. Experiments with latex beads coated with InlB protein indicated that one potential role of SH3C and PI 3 kinase is to promote changes in the F-actin cytoskeleton necessary for particle engulfment. Taken together, our results indicate that the CrkII SH3C domain engages a cellular ligand that regulates PI 3 kinase activity and host cell surface rearrangements.