Structural Properties of the Human Protease-Activated Receptor 1 Changing by a Strong Antagonist

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Truncated tyrosine kinase B brain-derived neurotrophic factor receptor directs cortical neural stem cells to a glial cell fate by a novel signaling mechanism

During development of the mammalian cerebral cortex neural stem cells (NSC) first generate neurons and subsequently produce glial cells. The mechanism(s) responsible for this developmental shift from neurogenesis to gliogenesis is unknown. Brain-derived neurotrophic factor (BDNF) is believed to play important roles in the development of the mammalian cerebral cortex; it enhances neurogenesis and promotes the differentiation and survival of newly generated neurons. Here, we provide evidence that a truncated form of the BDNF receptor tyrosine kinase B (trkB-t) plays a pivotal role in directing embryonic mouse cortical NSC to a glial cell fate. Expression of trkB-t promotes differentiation of NSC toward astrocytes while inhibiting neurogenesis both in cell culture and in vivo. The mechanism by which trkB-t induces astrocyte genesis is not simply the result of inhibition of full-length receptor with intrinsic tyrosine kinase activity signaling. Instead, binding of BDNF to trkB-t activates a signaling pathway (involving a G-protein and protein kinase C) that induced NSC to become glial progenitors and astrocytes. Thus, the increased expression of trkB-t in the embryonic cerebral cortex that occurs coincident with astrocyte production plays a pivotal role in the developmental transition from neurogenesis to gliogenesis. Our findings suggest a mechanism by which a single factor (BDNF) regulates the production of the two major cell types in the mammalian cerebral cortex.     

Enantioselective hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide by immobilized cells of an R-amidase-producing bacterium, Delftia tsuruhatensis CCTCC M 205114, on an alginate capsule carrier

Immobilized cells of Delftia tsuruhatensis CCTCC M 205114 harboring R-amidase were applied in asymmetric hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide (R − 1) from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate (S)-2, 2-dimethylcyclopropane carboxamide (S − 1). Maximum R-amidase activity of 13.1 U/g wet cells (0.982 U/g beads) was obtained under conditions of 3% sodium alginate, 2.5% CaCl₂, 15 h crosslinking and 2 mm bead size, which was 53.9% of that of free cells (24.3 U/g wet cells). In addition, characterization of the immobilized cells was examined. The optimum R − 1 hydrolysis conditions were identified as follows: substrate concentration 10 mM, pH 8.5, temperature 35°C and time course 40 min. Under optimum conditions, the maximum yield and enantiomeric excess of (R)-2, 2-dimethylcyclopropanecarboxylic acid were 49.5% and >99%, respectively. This afforded S − 1 with a yield >49% and an e.e. of 97.7%. With good operational stability and excellent enanotioselectivity, the immobilized cells could be potentially utilized in industrial production of S − 1.     

Genomic structure,gene expression,and promoter analysis of human multidrug resistance-associated protein 7

The multidrug resistance-associated protein (MRP) subfamily transporters associated with anticancer drug efflux are attributed to the multidrug-resistance of cancer cells. The genomic organization of human multidrug resistance-associated protein 7 (MRP7) was identified. The human MRP7 gene, consisting of 22 exons and 21 introns, greatly differs from other members of the human MRP subfamily. A splicing variant of human MRP7, MRP7A, expressed in most human tissues, was also characterized. The 1.93-kb promoter region of MRP7 was isolated and shown to support luciferase activity at a level 4- to 5-fold greater than that of the SV40 promoter. Basal MRP7 gene expression was regulated by 2 regions in the 5'-flanking region at -1,780-1,287 bp, and at -611 to -208 bp. In Madin-Darby canine kidney (MDCK) cells, MRP7 promoter activity was increased by 226% by genotoxic 2-acetylaminofluorene and 347% by the histone deacetylase inhibitor, trichostatin A. The protein was expressed in the membrane fraction of transfected MDCK cells.     

Duvelisib attenuates bleomycin-induced pulmonary fibrosis via inhibiting the PI3K/Akt/mTOR signalling pathway

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that seriously threatens the health of patients. The pathogenesis of IPF is still unclear, and there is a lack of effective therapeutic drugs. Myofibroblasts are the main effector cells of IPF, leading to excessive deposition of extracellular matrix (ECM) and promoting the progression of fibrosis. Inhibiting the excessive activation and relieving autophagy blockage of myofibroblasts is the key to treat IPF. PI3K/Akt/mTOR pathway plays a key regulatory role in promoting fibroblast activation and autophagy inhibition in lung fibrosis. Duvelisib is a PI3K inhibitor that can simultaneously inhibit the activities of PI3K-δ and PI3K-γ, and is mainly used for the treatment of relapsed/refractory chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma tumour (SLL). In this study, we aimed to examine the effects of Duvelisib on pulmonary fibrosis. We used a mouse model of bleomycin-induced pulmonary fibrosis to evaluate the effects of Duvelisib on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of Duvelisib in lung fibroblasts in vitro. The in vivo experiments showed that Duvelisib significantly alleviated bleomycin-induced collagen deposition and improved pulmonary function. In vitro and in vivo pharmacological experiments showed that Duvelisib dose-dependently suppressed lung fibroblast activation and improved autophagy inhibition by inhibiting the phosphorylation of PI3K, Akt and mTOR. Our results indicate that Duvelisib can alleviate the severity of pulmonary fibrosis and provide potential drugs for the treatment of pulmonary fibrosis.     

FGF21 impedes peripheral myelin development by stimulating p38 MAPK/c‐Jun axis

Fibroblast growth factor 21 (FGF21) as a metabolic stress hormone, is mainly secreted by the liver. In addition to its well‐defined roles in energy homeostasis, FGF21 has been shown to promote remyelination after injury in the central nervous system. In the current study, we sought to examine the potential roles of FGF21 in the peripheral nervous system (PNS) myelination. In the PNS myelin development, Fgf21 expression was reversely correlated with myelin gene expression. In cultured primary Schwann cells (SCs), the application of recombinant FGF21 greatly attenuates myelination‐associated gene expression, including Oct6, Krox20, Mbp, Mpz, and Pmp22. Accordingly, the injection of FGF21 into neonatal rats markedly mitigates the myelination in sciatic nerves. On the contrary, the infusion of the anti‐FGF21 antibody accelerates the myelination. Mechanistically, both extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK) were stimulated by FGF21 in SCs and sciatic nerves. Following experiments including pharmaceutical intervention and gene manipulation revealed that the p38 MAPK/c‐Jun axis, rather than ERK, is targeted by FGF21 for mediating its repression on myelination in SCs. Taken together, our data provide a new aspect of FGF21 by acting as a negative regulator for the myelin development process in the PNS via activation of p38 MAPK/c‐Jun.     

Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions

We have determined the structure of a mutant (Q237W) of HhaI DNA methyltransferase, complexed with the methyl-donor product AdoHcy. The Q237W mutant proteins were crystallized in the monoclinic space group C2 with two molecules in the crystallographic asymmetric unit. Protein-protein interface calculations in the crystal lattices suggest that the dimer interface has the specific characteristics for homodimer protein-protein interactions, while the two active sites are spatially independent on the outer surface of the dimer. The solution behavior suggests the formation of HhaI dimers as well. The same HhaI dimer interface is also observed in the previously characterized binary (M.HhaI-AdoMet) and ternary (M.HhaI-DNA-AdoHcy) complex structures, crystallized in different space groups. The dimer is characterized either by a non-crystallographic two-fold symmetry or a crystallographic symmetry. The dimer interface involves three segments: the amino-terminal residues 2-8, the carboxy-terminal residues 313-327, and the linker (amino acids 179-184) between the two functional domains--the catalytic methylation domain and the DNA target recognition domain. Both the amino- and carboxy-terminal segments are part of the methylation domain. We also examined protein-protein interactions of other structurally characterized DNA MTases, which are often found as a 2-fold related 'dimer' with the largest dimer interface area for the group-beta MTases. A possible evolutionary link between the Type I and Type II restriction-modification systems is discussed.     

恶性疟多抗原表位融合基因在不同载体中的克隆与表达

通过PCR方法扩增并分析了恶性疟原虫多抗原表位融合基因awte序列,再分别克隆于温度诱导型融合表达载体pEX31b和IPTG诱导型融合型表达载体pGEMEX1中,并进行了诱导表达,并对表达产物MS2－AWTE和T7φ10－AWTE融合蛋白进行间接ELISA检测,证实MS2－AWTE和T7φ10－AWTE都具有免疫学活性。另外,同样将awte基因分别克隆于温度诱导型和IPTG诱导型非融合表达载体pBV200、pKEN中并进行诱导表达,但在SDS－PAGE中并未检测到可见的AWTE表达带。