Structure of human cytosolic X-prolyl aminopeptidase: a double Mn(II)-dependent dimeric enzyme with a novel three-domain subunit

X-prolyl aminopeptidases catalyze the removal of a penultimate prolyl residue from the N termini of peptides. Mammalian X-prolyl aminopeptidases are shown to be responsible for the degradation of bradykinin, a blood pressure regulator peptide, and have been linked to myocardial infarction. The x-ray crystal structure of human cytosolic X-prolyl aminopeptidase (XPN-PEP1) was solved at a resolution of 1.6 angstroms. The structure reveals a dimer with a unique three-domain organization in each subunit, rather than the two domains common to all other known structures of X-prolyl aminopeptidase and prolidases. The C-terminal catalytic domain of XPNPEP1 coordinates two metal ions and shares a similar fold with other prolyl aminopeptidases. Metal content analysis and activity assays confirm that the enzyme is double Mn(II) dependent for its activity, which contrasts with the previous notion that each XPNPEP1 subunit contains only one Mn(II) ion. Activity assays on an E41A mutant demonstrate that the acidic residue, which was considered as a stabilizing factor in the protonation of catalytic residue His498, plays only a marginal role in catalysis. Further mutagenesis reveals the significance of the N-terminal domain and dimerization for the activity of XPNPEP1, and we provide putative structural explanations for their functional roles. Structural comparisons further suggest mechanisms for substrate selectivity in different X-prolyl peptidases.     

<Emphasis Type="Italic">TfPLC1</Emphasis>, a gene encoding phosphoinositide-specific phospholipase C,is predominantly expressed in reproductive organs in <Emphasis Type="Italic">Torenia fournieri</Emphasis>

Phosphoinositide-specific phospholipase C (PI-PLC) is an important enzyme, which is a key player involved in eukaryotic signal transduction pathways. In plants, it plays a key role in growth and development as well as environmental stress. However, little is known about its roles in signal transduction during sexual reproduction process. In this study, we cloned and characterized a gene of full-length PI-PLC from ovules of Torenia fournieri, designated as TfPLC1. It was 2,171 bp in length, including an open reading frame encoding a polypeptide of 583 amino acids with molecular mass of 66.02 kDa. The amino acid sequence deduced from the cDNA sequence shows 40–76% similarity to other plant PI-PLCs and contains the characteristic X, Y and C2 domains. Northern blot analysis demonstrated it was predominantly expressed in ovules and flowers. Furthermore, TfPLC1 promoter::GUS transgenic analysis indicated it specifically expressed in ovule, stigma and mature pollen grain. Immunohistochemical staining showed that, in mature stigma, TfPLC1 protein was principally localized in the cells of stigmatic receptive surface. Together, our data suggest that TfPLC1 may play an important role in plant sexual reproduction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the fungus Penicillium marneffei

Penicillium marneffei is an opportunistic fungal pathogen of humans, causing respiratory, skin, and systemic mycosis in south-east Asia. Here we describe the transformation of P. marneffei with Agrobacterium tumefaciens, and the optimization of the transformation procedure. Transformations in different combinations between A. tumefaciens stains (LBA4404 and EHA105) and binary vectors (pCB309A, pBI129A, and pCaMBIA1312A) showed that EHA105/pBI129A were the most efficient partners. Southern blot analysis suggested that 87.5 % of transformants obtained with this protocol displayed single hybridization bands, indicating a single insert of T-DNA in each of the transformants. Unique hybridization patterns, along with thermal asymmetric interlaced PCR (TAIL-PCR) analysis of T-DNA insertion sites, suggested that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in P. marneffei. Several mutants with altered phenotypes were obtained during the construction of the mutant library, indicating the usefulness of the approach for functional genetic analysis in this important fungal pathogen.     

Flow-induced structural transition in the beta-switch region of glycoprotein Ib

The impact of fluid flow on structure and dynamics of biomolecules has recently gained much attention. In this article, we present a molecular-dynamics algorithm that serves to generate stable water flow under constant temperature, for the study of flow-induced protein behavior. Flow simulations were performed on the 16-residue β-switch region of platelet glycoprotein Ibα, for which crystal structures of its N-terminal domain alone and in complex with the A1 domain of von Willebrand factor have been solved. Comparison of the two structures reveals a conformational change in this region, which, upon complex formation, switches from an unstructured loop to a β-hairpin. Interaction between glycoprotein Ibα and von Willebrand factor initiates platelet adhesion to injured vessel walls, and the adhesion is enhanced by blood flow. It has been hypothesized that the loop to β-hairpin transition in glycoprotein Ibα is induced by flow before binding to von Willebrand factor. The simulations revealed clearly a flow-induced loop→β-hairpin transition. The transition is dominated by the entropy of the protein, and is seen to occur in two steps, namely a dihedral rotation step followed by a side-group packing step.     

Evolution of conditional dispersal: a reaction–diffusion–advection model

To study evolution of conditional dispersal, a Lotka-Volterra reaction-diffusion-advection model for two competing species in a heterogeneous environment is proposed and investigated. The two species are assumed to be identical except their dispersal strategies: both species disperse by random diffusion and advection along environmental gradients, but one species has stronger biased movement (i.e., advection along the environmental gradients) than the other one. It is shown that at least two scenarios can occur: if only one species has a strong tendency to move upward the environmental gradients, the two species can coexist since one species mainly pursues resources at places of locally most favorable environments while the other relies on resources from other parts of the habitat; if both species have such strong biased movements, it can lead to overcrowding of the whole population at places of locally most favorable environments, which causes the extinction of the species with stronger biased movement. These results provide a new mechanism for the coexistence of competing species, and they also imply that selection is against excessive advection along environmental gradients, and an intermediate biased movement rate may evolve.     

Reaction chemistry and phase behavior of lignin in high-temperature and supercritical water

Decomposition of organosolve lignin in water/phenol solutions was studied in a 50 nL micro-reactor coupled with optical, Raman and infrared microscopies at temperatures up to 600 degrees C and water densities up to 1165 kg/m3. It was found that when phenol was used with {lignin+water} mixtures that a homogenous phase was formed that seemed to promote the decomposition of lignin into phenolic fragments by hydrolysis and pyrolysis. Phenol, along with the homogenous reaction conditions also inhibited re-polymerization of the phenolics and promoted oil formation. On the other hand, in the absence of phenol, lignin remained as a heterogeneous phase with water over the range of conditions studied. The homogeneous conditions and conditions for inhibiting char formation by phenol were elucidated and it was found that mixtures of phenol and lignin become homogeneous at 400-600 degrees C and high water densities of 428-683 kg/m3, corresponding to maximum pressures of 93 MPa. These results were further used to propose reaction paths.     

Efficient production of biodiesel from high free fatty acid-containing waste oils using various carbohydrate-derived solid acid catalysts

In the present study, such carbohydrate-derived catalysts have been prepared from various carbohydrates such as d-glucose, sucrose, cellulose and starch. The catalytic and textural properties of the prepared catalysts have been investigated in detail and it was found that the starch-derived catalyst had the best catalytic performance. The carbohydrate-derived catalysts exhibited substantially higher catalytic activities for both esterification and transesterification compared to the two typical solid acid catalysts (sulphated zirconia and Niobic acid), and gave markedly enhanced yield of methyl esters in converting waste cooking oils containing 27.8wt% high free fatty acids (FFAs) to biodiesel. In addition, under the optimized reaction conditions, the starch-derived catalyst retained a remarkably high proportion (about 93%) of its original catalytic activity even after 50 cycles of successive re-use and thus displayed very excellent operational stability. Our results clearly indicate that the carbohydrate-derived catalysts, especially the starch-derived catalyst, are highly effective, recyclable, eco-friendly and promising solid acid catalysts that are highly suited to the production of biodiesel from waste oils containing high FFAs.     

Care of HIV-infected patients in China

Compared with high infection areas of the world, the total HIV infection rate in China is relatively low. Nonetheless,because of China‘s vast territory and large population, the potential infection risk must be taken seriously. In the next few years, needle sharing among injection drug users will remain the most common route of transmission for the HIV/AIDS epidemic in China. Unprotected sex is gradually becoming a major route of transmission. China began to implement HAART in 1999 according to international standards. Prior to 2003, there were only about 150 HIV/AIDS patients were treated with HAART in some clinical trials and about 100 HIV/AIDS patients were treated by private sources.Results of those treatments are the scientific basis for development of the therapeutic strategies in China. In March of 2003, the Chinese government initiated China CARES program. In November of 2003, the Chinese Ministry of Health announced a national policy of free ARV treatment to all HIV Chinese citizens who were in poverty and required ARV therapy. There are total of 19,456 HIV/AIDS patients received free ARV drugs to date in 159 regions and 441 towns.Current challenges are how to follow-up and evaluate those patients in the clinical settings. The longer the therapy is postponed, the more side effects and the higher probability of drug resistance are going to occur. It remains unclear,therefore, when HAART regimen should be started in the HIV/AIDS population in China.