Identification of CD4 and transferrin receptor antibodies by CXCR4 antibody-guided Pathfinder selection.

To generate human antibodies against CXCR4, a seven-transmembrane chemokine receptor and a principal coreceptor for HIV-1, several rounds of Pathfinder and Step-back selection from a large phage display antibody library were performed on Jurkat cells. A mAb against CXCR4 or biotinyated phage antibodies were used as guide molecules. Over 100 pan-Jurkat-cell-positive antibodies were characterized, but none were CXCR4 specific. However, several antibodies against CD4 and the transferrin receptor were identified. Our results indicate that, although Pathfinder and Step-back selection can be used to select phage antibodies on whole cells, the successful selection of certain targets is still complex and limited. The reason is probably, in part, due to the inaccessibility of the targeted extracellular structures and the range of the horseradish peroxidase-labeled guide molecule. Refinements of these techniques are required to improve target specificity and selectivity.     

Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato over-expressing glycerol-3-phosphate acyltransferase gene

Over-expression of chloroplastic glycerol-3-phosphate acyltransferase gene (LeGPAT) increased unsaturated fatty acid contents in phosphatidylglycerol (PG) of thylakoid membrane in tomato. The effect of this increase on the xanthophyll cycle and chloroplast antioxidant enzymes was examined by comparing wild type (WT) tomato with the transgenic (TG) lines at chilling temperature (4 °C) under low irradiance (100 μmol m⁻² s⁻¹). Net photosynthetic rate and the maximal photochemical efficiency of photosystem (PS) 2 (F_v/F_m) in TG plants decreased more slowly during chilling stress and F_v/F_m recovered faster than that in WT plants under optimal conditions. The oxidizable P700 in both WT and TG plants decreased during chilling stress under low irradiance, but recovered faster in TG plants than in the WT ones. During chilling stress, non-photochemical quenching (NPQ) and the de-epoxidized ratio of xanthophyll cycle in WT plants were lower than those of TG tomatoes. The higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in TG plants resulted in the reduction of O₂ ^−· and H₂O₂ contents during chilling stress. Hence the increase in content of unsaturated fatty acids in PG by the over-expression of LeGPAT could alleviate photoinhibition of PS2 and PS1 by improving the de-epoxidized ratio of xanthophyll cycle and activities of SOD and APX in chloroplast.     

Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai–Tibet plateau and in other zones of China

Qinghai-Tibet plateau is the highest place in the world and the environment in that plateau is hard for animals and plants, with low temperature, low concentration of oxygen and high solar radiation. In this study, 61 root nodule isolates from Vicia, Oxytropis, Medicago, Melilotus and Onobrychis species grown in Qinghai-Tibet plateau and in loess plateau were comparatively characterized. Based upon the results of numerical taxonomy, ARDRA, AFLP, DNA-DNA hybridization and 16S rDNA sequencing, the isolates were classified as Rhizobium leguminosarum, Sinorhizobium meliloti, Sinorhizobium fredii, Mesorhizobium sp., Phyllobacterium sp., Stenotrophomonas sp. and two non-symbiotic groups related to Agrobacterium and Enterobacteriaceae. The strains isolated from Qinghai-Tibet plateau and from the loess plateau were mixed in these species or groups. Oxytropis spp. and Medicago archiducis-nicolai grown in Qinghai-Tibet plateau were recorded as new hosts for R. leguminosarum, as well as Oxytropis glabra and Medicago lupulina for S. fredii. In addition, strains resistant to high alkaline (pH 11) and high concentration of NaCl (3-5%, w/v) were found in each of the rhizobial species. This was the first systematic study of rhizobia isolated from Qinghai-Tibet plateau.     

Overexpression of complexin in PC12 cells inhibits exocytosis by preventing SNARE complex recycling

Complexin is an important protein that functions during Ca2+-dependent neurotransmitter release. Substantial evidence supports that complexin performs its role through rapid interaction with SNARE complex with high affinity. However, alpha-SNAP/NSF, which can disassemble the cis-SNARE complex in the presence of MgATP, competes with complexin to bind to SNARE complex. In addition, injection of alpha-SNAP into chromaffin cells enhances the size of the readily releasable pool, and mutation disrupting the ATPase activity of NSF results in the accumulation of SNARE complex. Thus, whether high concentrations of complexin could result in a reverse result is unclear. In this paper, we demonstrate that when stably overexpressed in PC12 cells, high levels of complexin result in the accumulation of SNARE complex. This in turn leads to a reduction in the size of the readily releasable pool of large dense core vesicles. These results suggest that high levels of complexin seem to prevent SNARE complex recycling, presumably by displacing NSF and alpha-SNAP from SNARE complex.     

Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro-1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay

We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.     

PPARbeta/delta activation inhibits angiotensin II-induced collagen type I expression in rat cardiac fibroblasts

Peroxisome proliferator-activated receptors (PPARalpha, beta/delta and gamma) are nuclear receptors and PPARgamma activation was previously reported to inhibit collagen expression in the heart, but whether PPARbeta/delta also regulates collagen expression in the heart remains unclear. In this study, we investigated the effect of PPARbeta/delta activation on angiotensin II (Ang II)-induced collagen type I expression in adult rat cardiac fibroblasts. The results showed that PPARbeta/delta was expressed at the moderate level in cardiac fibroblasts. GW501516, a selective PPARbeta/delta agonist, depressed Ang II-stimulated collagen type I expression and collagen synthesis in cardiac fibroblasts in a concentration-dependent manner. Furthermore, these inhibitory effects of GW501516 were completely reversed by the knockdown of PPARbeta/delta via RNA interference. In summary, we find that PPARbeta/delta is present in cardiac fibroblasts and PPARbeta/delta activation inhibits Ang II-induced collagen type I expression at least in part via decreasing collagen synthesis. PPARbeta/delta may be a promising therapeutic target for myocardial fibrosis.     

Small logging roads do not restrict movements of forest rats in Bornean logged forests

Selective logging is driving the proliferation of roads throughout tropical rain forests, particularly narrow, unpaved logging roads. However, little is known about the extent of road edge effects or their influence on the movements of tropical understory animal species. Here, we used forest rats to address the following questions: (a) Does the occupancy of rats differ from road edges to forest interior within logged forests? (b) Do roads inhibit the movements of rats within these forests? We established trapping grids along a road edge‐to‐forest interior gradient at four roads and in three control sites within a logged forest in Sabah, Malaysia. To quantify the probability of road crossing, rats were captured, translocated across a road, and then recaptured on subsequent nights. We caught 216 individuals of eight species on 3,024 trap nights. Rat occupancy did not differ across the gradient from road edge to interior, and 48 percent of the 105 translocated individuals crossed the roads and were recaptured. This proportion was not significantly different from that of rats returning in control sites (38% of 60 individuals), suggesting that small roads were not barriers to rat movements within logged forests. Subadults were significantly more likely to return from translocation than adults in both road and control sites. Our results are encouraging for the ecology of small mammal communities in heavily logged forests, because small logging roads do not restrict the movements of rats and therefore are unlikely to create an edge effect or influence habitat selection.