Potential role of DNA-dependent protein kinase in cellular resistance to ionizing radiation

In this paper, we study the ability of DNA-PK-deficient (M059J) and -proficient (M059K) cells to undergo the rate of cellular proliferation, cell cycle distribution and apoptosis after 10 Gy X-ray irradiation, and the role of DNA-PK in radiosensitivity. The results showed that M059J cells exhibited hyper-radiosensitivity compared with M059K cells. A strong G2 phase arrest was observed in M059J cells post irradiation. Significant accumulation in the G2 phase in M059J cells was accompanied by apoptosis at 12 h. Altogether, the data suggested that DNA-PK may have two roles in mammalian cells after DNA damage, a role in DNA DSB repair and a second role in DNA-damaged cells to traverse a G2 checkpoint, by which DNA-PK may affect cellular sensitivity to ionizing radiation. 地址: [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Chinese Acad Sci, Inst Modern Phys, Lanzhou 730000, Peoples R China; [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Key Lab Heavy Ion Radiat Med Gansu Prov, Lanzhou 730000, Peoples R China; [Li Ning; Wang Yanling] Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China; [Wang Xiaohu] Gansu Tumor Hosp, Dept Radiotherapy, Lanzhou 730050, Peoples R China

Microarray-based Raman spectroscopic assay for kinase inhibition by gold nanoparticle probes

In this paper, a microarray-based surface-enhanced Raman spectroscopic (SERS) assay for detection of kinase functionality and inhibition has been reported. Biotinylated anti-phosphoserinen antibodies mark the phosphorylation and inhibition events and gold nanoparticles are attached to the antibodies by standard avidin-biotin chemistry, followed by silver deposition for SERS signal enhancement. The avidin conjugated fluorescein is used as SERS probe. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA), its well known substrate, kemptide, and three inhibitors, H89, HA1077, and KN62 have been chosen here to establish the SERS assay. As expected, highly selective inhibition of PKA is demonstrated with the inhibitor H89 and the inhibition assay enable to detect kinase inhibition as well as derive IC50 (half maximal inhibitory concentration) plots.

Microarray-based kinase inhibition assay by gold nanoparticle probes

We report on the development of a new class of kinase microarray for the detection of kinase inhibition based on marking peptide phosphorylation/biotinylation events by attachment of gold nanoparticles followed by silver deposition for signal enhancement. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate-dependent protein kinase (PKA), and its well-known substrate, kemptide, were used for the purpose of monitoring phosphorylation and inhibition. As expected, highly selective inhibition of PKA is demonstrated with the four inhibitors: H89, HA1077, mallotoxin, and KN62. Furthermore, an inhibition assay demonstrates the ability to detect kinase inhibition as well as derive IC50 (half-maximal inhibitory concentration) plots.

Involvement of p90 ribosomal S6 kinase in termination of cell cycle arrest during development of Artemia-encysted embryos

Artemia has evolved a unique developmental pattern of encysted embryos to cope with various environmental threats. Cell divisions totally cease during the preemergence developmental stage from gastrula to prenauplius. The molecular mechanism of this, however, remains unknown. Our study focuses on the involvement of p90 ribosomal S6 kinase (RSK), a family of serine/threonine kinase-mediating signal transduction downstream of mitogen-activated protein kinase cascades, in the termination of cell cycle arrest during the post-embryonic development of Artemia-encysted gastrula. With immunochemistry, morphology, and cell cycle analysis, the identified Artemia RSK was established to be specifically activated during the post-embryonic and early larval developmental stages when arrested cells of encysted embryos resumed mitoses. In vivo knockdown of RSK activity by RNA interference, kinase inhibition, and antibody neutralization consistently induced defective larvae with distinct gaps between the exoskeleton and internal tissues. In these abnormal individuals, mitoses were detected to be largely inhibited in the affected regions. These results display the requirement of RSK activity during Artemia development and suggest its role in termination of cell cycle (G(2)/M phase) arrest and promotion of mitogenesis. Our findings may, thus, provide insights into the regulation of cell division during Artemia post-embryonic development and reveal further aspects of RSK functions.

Arginine kinase from Litopenaeus vannamei: Cloning, expression and catalytic properties

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. in this paper, the full-length cDNA of AI( was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LIPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Eschetichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular cloning and response to laminarin stimulation of arginine kinase in haemolymph in Chinese shrimp, Fenneropenaeus chinensis

Arginine kinase (AK) was previously reported as a phosphagen-ATP phosphotransferase found in invertebrates. In this study, an 1184 bp cDNA was cloned and sequenced. It contained an open reading frame of 1068 bp that coded for 356 deduced amino acids of AK in Fenneropenaeus chinensis. The calculated molecular mass of AK is 40129.73 Da and pI is 5.92. The predicted protein showed a high level of identity to known AK in invertebrates and creatine kinase from vertebrates, which belong to a conserved family of ATP:guanidino phospho-transferases. In addition, AK protein in plasma of F. chinensis was identified using two-dimensional electrophoresis (2DE) and electrospray ionization mass spectrometry (ESI-MS) according to the calculated molecular mass and pI. AK was significantly decreased in the plasma of F. chinensis at 45 min and recovered at 3 It after laminarin injection as confirmed by 2DE and ESI-MS. The results showed that AK was one of the most significantly changed proteins on two-dimensional gel in the plasma proteins of F. chinensis at 45 min and 3 It after simulation. (c) 2005 Elsevier Ltd. All rights reserved.

Characterization of DYRK2 (dual-specificity tyrosine-phosphorylation-regulated kinase 2) from Zebrafish (Dario rerio)

Proteins of the DYRK (dual-specificity tyrosine-phosphorylation-regulated kinase) family are characterized by the presence of a conserved kinase domain and N-terminal DH box. DYRK2 is involved in regulating key developmental and cellular processes, such as neurogenesis, cell proliferation, cytokinesis, and cellular differentiation. Herein, we report that the ortholog of DYRK2 found in zebrafish shares about 70% identity with that of human, mouse, and chick. RT-PCR showed that DYRK2 is expressed maternally and zygotically. In-situ hybridization results show that DYRK2 is expressed in somite cells that will develop into muscles. Our results provide preliminary evidence for investigating the in-vivo function of DYRK2 in zebrafish muscle development.

Heterogeneous asymmetric hydrogenation of ethyl pyruvate on chirally modified Pt/Al2O3 catalyst with fixed-bed reactor

The enantioselective hydrogenation of ethyl pyruvate on the cinchonidine modified Pt/Al2O3 catalyst was investigated using a high-pressure reaction system with a fixed-bed reactor for the purpose to produce the,chiral product without separating the catalyst from the reaction system. The reaction was also investigated in a batch reactor for comparison. About 60% e. e. and 90% e. e. were obtained with the fixed-bed reactor and the batch reactor respectively, demonstrating the possibility for the heterogeneous asymmetric hydrogenation in the fixed-bed reactor. Some adsorbed chiral modifier, cinchonidine, can be slowly removed from the surface of Pt/Al2O3 under the continuous flow reaction, as a result, the e, e, values drops with the reaction time in the fixed-bed reactor. The enantio-selectivity is higher in the fixed-bed reactor, but lower in the batch reactor when ethanol was used as solvent than that when acetic acid as solvent. CO was used as molecular probe to characterize the adsorption of cinchonidine an the catalyst surface by IR spectroscopy, A red shift observed in IR spectra of coadsorbed CO with cinchonidine suggests that the cinchonidine adsorption is mainly through the pi -interaction with platinum surface and donating electron to the platinum surface.