Expression and bioactivity of recombinant segments of human perforin.

The aim of the study was to prepare an active recombinant human perforin by comparing 5 candidate segments of human perforin. Full-length perforin, MAC1 (28-349 aa), MAC2 (166-369 aa), C-100, and N-60 of human perforin were selected as candidate active segments and designated, respectively, HP1, HP2, HP3, HP4, and HP5. The target genes were amplified by PCR and the products were individually subcloned into pGEM-T. The genes for HP1, HP2, HP3, and HP5 were subcloned into pET-DsbA, whereas pET-41a (+) was used as the expression vector of HP4. The fusion proteins were expressed in Escherichia coli BL21pLysS(DE3) and purified using nickel nitrilotriacetic acid (NTA) agarose affinity chromatography. The hemolysis microassay was used as an activity assay of fusion protein. From this study, we obtained the recombinant plasmids pGEM-T-HP1, -HP2, -HP3, -HP4 and -HP5, consisting of 1600, 960, 600, 300bp, and 180, respectively. From these recombinant plasmids, expression plasmids were successfully constructed and expressed in E. coli BL21pLysS(DE3). The resultant fusion proteins, affinity purified using Ni-NTA, were approximately 80, 58, 45, 44, and 30 kDa, respectively. The recombinant proteins were assayed for activity on hemolysis. HP2 and HP5 were the only recombinant proteins that were active in hemolysis, and the hemolytic function was concentration dependent. These results demonstrate that active recombinant forms of perforin can be synthesized in a prokaryote model. The recombinant N-60 and MAC1 (28-349 aa) of human perforin have the function of forming pores. Our study provides the experimental basis for further investigation on the application of perforin.     

Crystal structure of the Leishmania major phosphodiesterase LmjPDEB1 and insight into the design of the parasite-selective inhibitors

Human leishmaniasis is a major public health problem in many countries, but chemotherapy is in an unsatisfactory state. Leishmania major phosphodiesterases (LmjPDEs) have been shown to play important roles in cell proliferation and apoptosis of the parasite. Thus LmjPDE inhibitors may potentially represent a novel class of drugs for the treatment of leishmaniasis. Reported here are the kinetic characterization of the LmjPDEB1 catalytic domain and its crystal structure as a complex with 3-isobutyl-1-methylxanthine (IBMX) at 1.55 A resolution. The structure of LmjPDEB1 is similar to that of human PDEs. IBMX stacks against the conserved phenylalanine and forms a hydrogen bond with the invariant glutamine, in a pattern common to most inhibitors bound to human PDEs. However, an extensive structural comparison reveals subtle, but significant differences between the active sites of LmjPDEB1 and human PDEs. In addition, a pocket next to the inhibitor binding site is found to be unique to LmjPDEB1. This pocket is isolated by two gating residues in human PDE families, but constitutes a natural expansion of the inhibitor binding pocket in LmjPDEB1. The structure particularity might be useful for the development of parasite-selective inhibitors for the treatment of leishmaniasis.     

Triterpenoids from <i>Uncaria rhynchophylla</i> and Their PTP1B Inhibitory Activity

Uncaria rhynchophylla (Gouteng) is a famous traditional Chinese medicine used for psychiatric and hypotensive purposes in China. In this study, the ethyl acetate (EtOAc) part of U. rhynchophylla was revealed with protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. Subsequent investigation on the EtOAc part yielded one new triterpenoid, 3β-formyloxy-6β,19α-dihydroxyurs-12-en-28-oic acid (1) and four known ones, 3β,6β,19α-trihydroxyurs-12-en-28-oic acid (2), 2-oxopomolic acid (3), 3β,19α-dihydroxy-6-oxo-olean-12-en-28-oic acid (4) and sumaresinolic acid (5). The structure of compound 1 was determined by extensive HRESIMS, IR, 1D and 2D NMR spectroscopic analyses. Two ursane-type triterpenoids (2 and 3) showed selective inhibition on PTP1B with IC₅₀ values of 48.2 and 178.7 μM. The enzyme kinetic study suggested that compounds 2 and 3 were mix-type inhibitors on PTP1B with K_i values of 15.6 and 132.5 μM. This investigation manifests the antidiabetic potency of U. rhynchophylla with triterpenoids as the active constituents.     

Assembly of rhizosphere microbial communities in Artemisia annua: recruitment of plant growth‐promoting microorganisms and inter‐kingdom interactions between bacteria and fungi

Plant and Soil - Plant roots assemble unique microbial communities in rhizosphere, which are critical for plant adapting to natural environment. Given the pivotal importance of plant-microbe...     

Oriented cell division: new roles in guiding skin wound repair and regeneration

Tissue morphogenesis depends on precise regulation and timely co-ordination of cell division and also on the control of the direction of cell division. Establishment of polarity division axis, correct alignment of the mitotic spindle, segregation of fate determinants equally or unequally between daughter cells, are essential for the realization of oriented cell division. Furthermore, oriented cell division is regulated by intrinsic cues, extrinsic cues and other cues, such as cell geometry and polarity. However, dysregulation of cell division orientation could lead to abnormal tissue development and function. In the present study, we review recent studies on the molecular mechanism of cell division orientation and explain their new roles in skin repair and regeneration.     

The structure and component characteristics of partial nitrification biofilms under autotrophic and heterotrophic conditions

The differences in the structure and component characteristics of partial nitrification biofilms between autotrophic and heterotrophic conditions were investigated in this work. Three-dimensional excitation–emission matrix fluorescence spectroscopy (EEM), fluorescence staining, and confocal laser scanning microscopy (CLSM) were used to determine differences in the architecture and extracellular polymeric substance (EPS) distribution of the autotrophic and heterotrophic biofilms. Partial nitrification was successfully achieved, and the results demonstrated that an appropriate amount of organic carbon (chemical oxygen demand (COD)/N?=?2.6) is advantageous for obtaining better partial nitrification. The final ammoniation and nitrosation rates achieved were 97 and 99 %, respectively. Proteins (PN) and polysaccharides (PS) were dominant in the tightly bound EPS (TB-EPS) of autotrophic and heterotrophic biofilms, with PN/PS ratios of 0.96 and 0.69, respectively. Proteins, lipids, α-d-glucopyranose polysaccharides, and nucleic acids were mostly present within the layers of biofilms, but they were distributed in the upper-middle portion of the autotrophic biofilm and increased with depth from the upper layer in the heterotrophic biofilms.     

<Emphasis Type="Italic">Paradevosia shaoguanensis</Emphasis> gen. nov., sp. nov., Isolated from a Coking Wastewater

A Gram staining negative, rod-shaped, aerobic bacterial strain J5-3^T with a single polar flagellum was isolated from coking wastewater collected from Shaoguan, Guangdong, China. It was motile and capable of optimal growth at pH 6–8, 30 °C, and 0–2 % (w/v) NaCl. Its predominant fatty acids were 11-methyl C_18:1 ω7c (29.2 %), C_16:0 (20.6 %), C_19:0 cyclo ω8c (18.2 %), C_18:0 (11.0 %), and C_18:1 ω7c/C_18:1 ω6c (10.9 %) when grown on trypticase soy agar. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two unknown glycolipids (GL1, GL2), and two unknown phospholipid (PL1, PL2). The predominant ubiquinone was Q-10, and the genome DNA G+C content was 61.7 mol %. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain J5-3^T belonged to the family Hyphomicrobiaceae in Alphaproteobacteria. It shared the 16S rRNA gene sequence similarities of 93.8–96.1 % with the genus Devosia, 94.5–94.8 % with the genus Pelagibacterium, and <92.0 % with all the other type strains in family Hyphomicrobiaceae. It can be distinguished from the closest phylogenetic neighbors based on several phenotypic and genotypic features, including α-galactosidase activity, tetracycline susceptibility, major fatty acid composition, polar lipid profile, DNA gyrase B subunit (gyrB) gene sequence, and random-amplified polymorphic DNA profile. Therefore, we consider strain J5-3^T to represent a novel species of a novel genus within the family Hyphomicrobiaceae, for which the name Paradevosia shaoguanensis gen. nov., sp. nov. is proposed. The type strain of Paradevosia shaoguanensis is J5-3^T (=CGMCC 1.12430^T =LMG 27409^T).