Summary1. The Janzen-Connell hypothesis provides explanations for species coexistence and predicts that recruitment of tree juveniles is reduced by host-specific enemies, particularly soil biota. Previous studies, however, have not fully addressed the aspect of host specificity. Using a legume tree (Ormosia glaberrima) in a subtropical monsoon forest as a model, we experimentally investigated the mechanisms underlying a Janzen-Connell effect. 2. A negative plant-soil feedback was identified by a field census and confirmed by planting surface-sterilized seeds at different densities around focal trees. Fungicides were applied to test whether the effects were caused by fungi. In a growth-room inoculation experiment, effects of collected soil samples on seedling survival were examined and compared to soil samples treated with fungicides. Sequencing of the internal transcribed spacer region (ITS) sequence of the 28S ribosomal RNA gene was used for pathogen identification. The fungus causing seedling mortality was isolated and characterized by ITS rDNA sequencing and inoculation experiments. 3. A Janzen-Connell effect was identified at a field site, in which O. glaberrima was a locally common species. In situ treatments with fungicides and a corresponding growth-room simulation experiment showed that seedling survival depended on the distance to focal adult trees and that a fungal pathogen attacked seeds and seedlings. No negative plant-soil feedback was observed at another field site with a single O. glaberrima tree, indicating a locally rare species advantage. 4. The disease-inducing fungus was identified as Fusarium oxysporum. Inoculation experiments showed that the isolated fungus was pathogenic on O. glaberrima seedlings, but non-pathogenic on seedlings from other tree species co-occurring with O. glaberrima. Moreover, susceptibility of O. glaberrima depended on seed provenance (likely genotype). 5. Synthesis. We demonstrate that an observed negative plant-soil feedback on a locally common legume tree is caused by a host-specific pathogen. Our data fully support the criteria of spatially unequal pathogen distribution and host specificity proposed in the Janzen-Connell model. Taken the interaction between O. glaberrima and F. oxysporum as a paradigm, we suggest that host-specific pathogens, locally accumulated around parent trees, are important determinants of tree community structure.
Glycoside hydrolases are often members of a multigene family, suggesting individual roles for each isoenzyme. Various extracellular glycoside hydrolases have an important but poorly understood function in remodelling the cell wall during plant growth. Here, MsXyl1, a concanavalin A-binding protein from alfalfa (Medicago sativa L.) belonging to the glycoside hydrolase family 3 (beta-D-xylosidase branch) is characterized. Transcripts of MsXyl1 were detected in roots (particularly root tips), root nodules, and flowers. MsXyl1 under the control of the CaMV 35S promoter was expressed in the model legume Medicago truncatula (Gaertner). Concanavalin A-binding proteins from the transgenic plants exhibited 5-8-fold increased activities towards three p-nitrophenyl (PNP) glycosides, namely PNP-beta-D-xyloside, PNP-alpha-L-arabinofuranoside, and PNP-alpha-L-arabinopyranoside. An antiserum raised against a synthetic peptide recognized MsXyl1, which was processed to a 65 kDa form. To characterize the substrate specificity of MsXyl1, the recombinant protein was purified from transgenic M. truncatula leaves by concanavalin A and anion chromatography. MsXyl1cleaved beta-1,4-linked D-xylo-oligosaccharides and alpha-1,5-linked L-arabino-oligosaccharides. Arabinoxylan (from wheat) and arabinan (from sugar beet) were substrates for MsXyl1, whereas xylan (from oat spelts) was resistant to degradation. Furthermore, MsXyl1 released xylose and arabinose from cell wall polysaccharides isolated from alfalfa roots. These data suggest that MsXyl1 is a multifunctional beta-xylosidase/alpha-L-arabinofuranosidase/alpha-L-arabinopyranosidase implicated in cell wall turnover of arabinose and xylose, particularly in rapidly growing root tips. Moreover, the findings of this study demonstrate that stable transgenic M. truncatula plants serve as an excellent expression system for purification and characterization of proteins.
While regulatory policy is well defined for orphan drug development in the United States and Europe, rare disease policy in China is still evolving. Many Chinese patients currently pay out of pocket for international treatments that are not yet approved in China. The lack of a clear definition and therefore regulatory approval process for rare diseases has, until now, de-incentivized pharmaceutical companies to pursue rare disease drug development in China. In turn, many grassroots movements have begun to support rare disease patients and facilitate drug discovery through research. Recently, the Chinese FDA set new regulatory guidelines for drugs being developed in China, including an expedited review process for life-saving treatments. In this review, we discuss the effects of these new policy changes on and suggest potential solutions to innovate orphan drug development in China.
A gas chromatography mass spectrometry (GC–MS) method has been developed and fully validated for the simultaneous determination of natural borneol (NB) and its metabolite, camphor, in rat plasma. Following a single liquid–liquid extraction, the analytes were separated using an HP-5MS capillary column (0.25 mm×30 m×0.25 μm) and analyzed by MS in the selected ion monitoring mode. Selected ion monitor (m/z) of borneol, camphor and internal standard was 95, 95 and 128, respectively. Linearity, accuracy, precision and extraction recovery of the analytes were all satisfactory. The method was successfully applied to pharmacokinetic studies of NB after oral administration to Wistar rats.
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