Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny

The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.     

A novel pathway of rapid TLR-triggered activation of integrin-dependent leukocyte adhesion that requires Rap1 GTPase

Rapid β2-integrin activation is indispensable for leukocyte adhesion and recruitment to sites of infection and is mediated by chemokine- or P-selectin glycoprotein ligand-1–induced inside-out signaling. Here we uncovered a novel pathway for rapid activation of integrin-dependent leukocyte adhesion, triggered by toll-like receptor (TLR)–mediated signaling. TLR2 or TLR5 ligation rapidly activated integrin-dependent leukocyte adhesion to immobilized ICAM-1 and fibronectin. Consistently, in vivo administration of the TLR2-ligand Pam3CSK4 increased integrin-dependent slow rolling and adhesion to endothelium within minutes, as identified by intravital microscopy in the cremaster model. TLR2 and TLR5 ligation increased β2-integrin affinity, as assessed by the detection of activation-dependent neoepitopes. TLR2- and TLR5-triggered integrin activation in leukocytes required enhanced Rap1 GTPase activity, which was mediated by Rac1 activation and NADPH oxidase-2–dependent reactive oxygen species production. This novel direct pathway linking initial pathogen recognition by TLRs to rapid β2-integrin activation may critically regulate acute leukocyte infiltration to sites of pathogen invasion.     

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.     

Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival

The protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4- to 7.5-fold the size of its rpoS⁺ wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH₂O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log₁₀ in sterile ddH₂O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log₁₀ in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derived rpoS⁺ and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.     

Immunosensor with a controlled orientation of antibodies by using NeutrAvidin-protein A complex at immunoaffinity layer

The orientation of antibody was controlled by using NeutrAvidin-protein A complex on the gold surface of SPR biosensor. The surface density of receptor antibody (anti-hIgG) was compared by treatment of receptor antibody to the layer of avidin, NeutrAvidin, protein A, NeutrAvidin-protein A complex and bare gold surface of SPR biosensor. The ligand antibody (hIgG) was injected to each IA layer and the binding ratio of ligand antibody per unit receptor was estimated as a parameter of orientation control. The NeutrAvidin-protein A complex on gold surface of SPR biosensor showed the highest surface density of receptor antibody as well as the binding ratio of ligand antibody per receptor antibody. The NeutrAvidin-protein A complex was also prepared on biotin-labelled SAM, and the binding ratio of ligand per receptor was found to be significantly improved in comparison to the IA layer prepared by chemical coupling of receptor antibody to the SAM layer. The NeutrAvidin-protein A complex which showed the highest efficiency for the binding of ligand antibodies, was applied for the detection of a cancer marker called CEA. By using NeutrAvidin-protein A complex and sandwich assay for signal amplification, sensitivity was improved to be 1.5-fold higher than bare gold surface and the detection of CEA with the detection limit of 30 ng/ml was achieved.     

The synthesis of an analogue of the locust CRF-like diuretic peptide, and the biological activities of this and some C-terminal fragments

The synthesis is described of an analogue of the locust CRF-like diuretic peptide in which methionine in positions 1,3, and 13 is replaced by isosteric methyl-homoserine residues. This analogue has been tested for biological activity on Malpighian tubules in vitro, and feeding behavior in vivo. It is highly active in stimulating fluid secretion and accumulation of cAMP in tubules, and on increasing the latency to feed and reducing meal duration. A 15 residue fragment from the C-terminus of the CRF-like peptide, Locmi-DP(32-46), is fully active in the feeding assay, but has only weak ability to stimulate the accumulation of cAMP in tubules. Two smaller fragments, Locmi-DP(32-37) and Locmi-DP(41-46), were tested but neither had consistent biological activity in any of the assays used here. None of the peptides tested have any substantive activity in increasing cGMP in tubules.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Regulation of gene expression in rat prostate by androgen and beta-adrenergic receptor pathways

Denervation of rat ventral prostate has been accomplished by excising prostatic tissue fragments and implanting them under the renal capsules of intact syngeneic rats. This resulted in a substantial reduction of expression of a major organ-specific secretory protein, prostatic binding protein (PBP). The depressed level of PBP and its subunits and mRNAs could be restored, however, to as much as 80% of control levels by the administration of a pharmacological dose of exogenous androgen, testosterone propionate (TP), and/or a beta-adrenergic agonist, isoproterenol (ISO). Furthermore, compared to ascorbate-treated controls, TP and ISO increased the synthesis of total cellular protein and PBP by the prostatic renal implants. TP and/or ISO also remodelled the luminal epithelial structure and elevated secretory functions. ISO alone had no effect, however, in castrated animals, indicating that androgen plays a dominant role in the restoration of tissue PBP content. Concomitant to increased PBP content and remodelling of prostatic histomorphology, androgen was also found to raise the depressed levels of beta 2-adrenergic and androgen receptors in the prostatic isografts maintained in intact hosts. In contrast, although an established rat prostatic epithelial cell line (NbE-1) contains high affinity androgen receptor, androgen failed to restore beta-adrenergic receptor as well as PBP content in this cultured cell line. These results, taken together, suggest that a tight coupling between androgen receptor and beta 2-adrenergic receptor pathways may be a prerequisite for PBP expression and functional differentiation in the rat ventral prostate gland.     

Involvement of Matrix Metalloproteinases on the Inhibition of Cells Invasion and Migration by Emodin in Human Neuroblastoma SH-SY5Y Cells

Emodin (1,3,8-trihydroxy-6-methylanthaquinone), an active component present in the root and rhizome of Rheum palmatum L. (Polygonaceae) has anti-bacterial, anti-tumor, diuretic and vasorelaxant effects. However, its mechanism of action on the cell migration and invasion of human neuroblastoma cancer SH-SY5Y cells is not fully understood. In this study, firstly, the effects of emodin on the percentage of viable cells were examined by using MTT assay and it was found that emodin induced dose-and time-dependent inhibition in human neuroblastoma SH-SY5Y cells. Second, the effects of emodin on the migration and invasion of SH-SY5Y cells were examined by using wound assay and matrigel counting and the results showed that emodin suppressed the migration and invasion of SH-SY5Y cells. Third, we examined the effect of emodin on the levels of associated proteins by using Western blotting and the results indicated that emodin inhibited the levels of GRB2, RhoA, HIF-1α, VEGF, FAK, iNOS, COX2, p-p38, p-c-jun, MMP2, MMP9 and MMP7 but promoted the levels of PKC, PI3K, MEKK3 and NF-κB p65 that led to the inhibition of migration and invasion of SH-SY5Y cells in vitro.