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Objective To investigate the effect of rhoptry protein 38 (ROP38) from Toxoplasma gondii (T. gondii) on the maturation of dendritic cells (DCs) by Toll-like receptor 4 (TLR4) induction in vitro. Methods The total RNA from T. gondii RH strain was extracted by guanidine thiocyanate method, and then cDNA was synthesized with reverse transcription reaction. After ROP38 gene was amplified by PCR, the recombinant pGEX-4T-ROP38 was constructed and expressed under IPTG induction. The recombinant ROP38 protein was detected by SDS-PAGE and Western blot analysis. The secondary structure and antigenicity of the ROP38 were predicted through DNAStar8.0 and ProtScale. In vitro, DCs were isolated and cultured for 6 days, then reacted with ROP38 antigen for 2 hours. The CD11c was detected by flow cytometry, and the data were analyzed with ANOVA by SPSS 23.0 software. Results The amplified gene was about 516 bp as expected. The sequence analysis showed that its homology was 99% compared with the reported sequence (XM_002366710.2) from GenBank. It was found that the relative molecular mass (M r ) of recombinant ROP38 was 45 kD. The prediction of structure indicated that there were 5 α-helices, 5 β-sheets, 5 hydrophilic regions and 8 potential epitopes in ROP38 protein. In vitro, the expression of CD11c on DCs was significantly up-regulated after stimulated with ROP38, and the expression level of CD11c in ROP38 infection group was significantly higher than that of the other groups. Conclusion ROP38 promotes the maturation of DCs mediated by TLR4.

As a kind of mitochondrial membrane protein with protein kinase activity, phosphatase and tensin homolog deleted on chromosome ten induced kinase 1 (PINK1) is involved in many biological metabolic processes. Since PINK1 had been found to be associated with Parkinson's disease, researchers have been exploring its biological function. PINK1 localizes in the outer mitochondrial membrane and regulates cell function through phosphorylating proteins. PINK1 is involved in mitochondrial function, mitochondrial morphology and mitochondrial autophagy, but the regulatory pathway is not yet clear. PINK1 is expressed widely in many tissues with a variety of biological activity, especially in tissues with high energy consumption. It may therefore be involved in the development and regulation of many diseases. Mutations in PINK1 were originally discovered to cause autosomal recessive Parkinson's disease. Recently some research has revealed that PINK1 is related to the development of neonatal hypoxic-ischemic encephalopathy, cancer, diabetes and other diseases. Studying and exploring the biological functions of PINK1 will facilitate the identification of the targets for therapeutic intervention for its related diseases. This review article mainly focuses on recent studies about the biological function and related diseases of PINK1.

Objective: To clone calcium-dependent protein kinase gene (camk) from Puccinia striiformis f. sp. tritici (Pst) and analyze its function., Methods: The cDNA full-length of Pscamk was isolated by using reverse transcriptional-PCR (RT-PCR), and gene expression profile at different morphological stages was analyzed via quantitative real-time--PCR (qRT-PCR). Pst urediospores were treated with CaMK suppressor KN-93 and germination rate was investigated., Results: A gene cDNA full-length with 1 620 bp was obtained and designated as Pscamk. qRT-PCR analysis showed Pscamk expression was highly induced in the early stages of Pst infection and reached the maximum at 6 h post inoculation (hpi) as 20.74-fold as that in the control (0 hpi). With increasing of the concentration of CaMK suppressor KN-93, germination rate of Pst urediospores was gradually decreased. The germination rate was reduced to 8.02%, only 12% of the control, under 1.4 μmol/L KN-93 treatment at 10 h after incubation at 9 degrees C., Conclusion: Pscamk might play a role in germination and germ tube elongation of Pst urediospores. This study provides a basis for exploring pathogenesis of calcium signaling pathway during Pst infection.

Objective: To find catalytic center of MDV-1 UL13 and express it in vitro to investigate the function of UL13 kinase., Methods: UL13 gene was amplified by polymerase chain reaction (PCR) from MDV-1 CVI988/Rispens strain. The codon bias and antigenicity of UL13 in Escherichia coli was analyzed by online service GENEART (www.gcua.de)and DNAstar software respectively. Then the UL13 truncated fragments were expressed in Escherichia coli, and mice were immunized with the expressed Glutathione S Transferase fusion protein. The conserved domain was analyzed with protein blast and Cn3D 4.1 online software of National Center for Biotechnology Information., Results: UL13 gene was successfully amplified. The sequence analysis suggests that 259-400 and 431-513 amino acid residues are low abundance for rare codon and strong antigenicity in UL13. Result of conserved domain analysis demonstrated that 152-297 residue iskinase catalytic center of UL13. However, conserved glycin in kinase subdomain VII for most protein kinase was replaced by serine in UL13 and proline in kinase subdomain VIII replaced by cysteine. The serum from mice immunized with truncated fragment, 259-400 amino acids, could react with recombinant UL13 protein expressed in insect cells in immunofluorenscence assay., Conclusion: The 152-297 residue is kinase catalytic center of MDV-1 UL13; UL13 protein expressed in vitro induced specific antibodies against UL13.

Genomic DNA sequence analysis of phytochrome like photoreceptors in a number of bacteria revealed several open reading frames (ORFs) encoding proteins with amino acid sequences homologous to plant phytochromes. The phytochrome like photoreceptors, collectively called bacteriophytochromes, contain an N-terminal domain homologous to the chromophore-binding domain (CBD) of higher plants and a C-terminal domain of histidine kinase domain( HKD). Due to their simple structure, bacteriophytochromes broaden the view of phytochrome evolution and provide us with a simple model to investigate phytochrome-mediated light signal in higher plants. In this report, the bacteriophytochromes from Synechocystis sp. PCC6803 were investigated. The gene cph1 and its fragment cph1 (C-435) were isolated from the Synechocystis sp. PCC6803 genomic DNA by polymerase chain reaction(PCR) using specific primers. Then, the genes were cloned with the vector pBluescript, yielding plasmids pBlu-cphl and pBlu-cph1 ( C-435), before they are subcloned with the vector pET30, using the EcoRV and Xho I restriction sites. pBlu-cph1, pBlu-cph1 (N-435) were cleaved with Sma I and Xho I, and the released genes were ligated to the pET30a fragment. The E. coli [strain BL21 (DE3)] cells containing recombinant pET30a were grown in medium RB at 20 degrees C, and harvested 6 h later after induction with isopropyl thio-beta-D-galactoside (IPTG). Then, reconstitution systems were employed to study the characteristics of the genes. In the reconstitution system, autoassembly of aprotein of phytochrome with PCB was investigated. The chromophore addition was an autocatalytic process. Reconstitution products were red/infrared (R/FR) photochromic, which was similar to that of the phytoehrome in higher plants. How ever, the spectral change ratios (deltaAmax/deltaAmin) of the two fragments differed from each other. It was also shown that PCB was covalently bound to apo-protein via Zn2+ fluoresc ence SDS-PAGE. After irradiation by light of 700 nm, the maximum absorption spectrum o f holo-Cphl was 650nm. The absorption of it after denaturatior in the dark with ur ea in the presence of hydrochloric acid (pH = 2) was 660nm, which was similar with th at of cis-PCB. In addition, after irradiation by light of 650nm, the maximum absorption spectrum of holo-Cph1 was 700nm. The absorption of it after denaturation in the dark with urea in the presence of hydrochloric acid (pH = 2) was 600nm, which was similar with that of trans-PCB. The result showed that the photochromism of phytochrome resulted from the isomerizaation of chromophore (PCB in this report). The reconstitution of Cph1 (C-435) under the same condition supported the conclusion. Fluorescence emission spectrum of the products suggested that bacteriophytochrom e structure with cis-PCB was more stable than that with trans-PCB. The new reconstitution system in this report sets a base for the application of phytochrome as photochromic biomaterials in biosensors. In addition, phytochrome shows great potential in food, cosmetic and biological engineering, etc.

Objective: To isolate full length cDNA of a novel protein kinase and to deduce the protein kinase's classification position and functions., Methods: cDNA libraries gDNA library was screened with a partial cDNA clone which is homologous to human protein kinase DYRK2 as probe. FISH mapping was performed., Results: Two full length cDNAs of a novel protein kinase from human muscle cDNA library and human testis cDNA library were isolated. The full length cDNA from muscle has an open reading frame which is predicted to encode a protein of 588 amino acid residues and the cDNA from testis to encode a protein of 568 amino acid residues., Conclusion: Because the sequence from the 27th codon to the 3' end of the cDNA from muscle is identical to that from the 7th codon to the 3' end of the cDNA from testis, they should be different transcripts of the same gene. As the gene is highly homologous to human protein kinase DYRK2, the present authors termed the gene DYRK3. DYRK3 is homologous to many serine/threonine protein kinases such as yeast Yak1, human Clk1, human Mnb, drosophila melanogaster Mnb and Cdk2. DYRK3 should belong to the Clk family in CMGC group of serine/threonine protein kinase. DYRK3 has been mapped to chromosome 1q32 by FISH.