Investigation of Multi-Factor Stress Corrosion Cracking Failure of Safe-End Feedwater Lines of Submarine Power System.

The corrosion process under the complex safe-end feedwater line conditions was investigated via experimental lab testing and numerical simulation. The corrosion of safe-end feedwater lines was controlled through the combination of galvanic corrosion, residual stress, and flow velocity. Firstly, galvanic corrosion occurred once the 20 steel was welded with 316L stainless steel. The pitting corrosion could be observed on the 20 steel side of the weld joint. Secondly, a vortex flow was detected around the welding bump and within the pits. The growth of the pits was accelerated in both the vertical and horizontal directions. Finally, under the residual stress condition, the stress intensity factor (K) at the bottom of the pits was easier to reach than the critical stress intensity factor (KISCC). Then, pitting was transformed into stress corrosion cracking which then propagated along the weld line. Therefore, the critical factor inducing the failure of safe-end feedwater lines was the combined action of galvanic corrosion, residual stress, and flow velocity.

Irradiation enhances the malignancy-promoting behaviors of cancer-associated fibroblasts.

Background: Cancer-associated fibroblasts (CAFs) are the important component of the tumor microenvironment (TME). Previous studies have found that some pro-malignant CAFs participate in the resistance to radiotherapy as well as the initiation and progression of tumor recurrence. However, the exact mechanism of how radiation affects CAFs remains unclear. This study aimed to explore the effect and possible mechanism of radiation-activated CAFs, and its influence on lung cancer. Methods: CAFs were isolated from surgical specimens in situ and irradiated with 8Gy x-rays. The changes in cell morphology and subcellular structure were observed. CAFs marker proteins such as FAP and α-SMA were detected by Western Blotting. Cell counting kit-8 (CCK8) assay, flow cytometry, wound healing assay, and transwell chamber assay was used to detect the activation of cell viability and migration ability. A nude mouse xenograft model was established to observe the tumorigenicity of irradiated CAFs in vivo. The genomic changes of CAFs after radiation activation were analyzed by transcriptome sequencing technology, and the possible mechanisms were analyzed. Results: The CAFs showed a disorderly growth pattern after X-ray irradiation. Subcellular observations suggested that metabolism-related organelles exhibited more activity. The expression level of CAFs-related signature molecules was also increased. The CAFs irradiated by 8Gy had good proliferative activity. In the (indirect) co-culture system, CAFs showed radiation protection and migration induction to lung cancer cell lines, and this influence was more obvious in radiation-activated CAFs. The radiation protection was decreased after exosome inhibitors were applied. Vivo study also showed that radiation-activated CAFs have stronger tumorigenesis. Transcriptome analysis showed that genes were enriched in several pro-cancer signaling pathways in radiation-activated CAFs. Conclusions: Our study confirmed that CAFs could be activated by ionizing radiation. Irradiation-activated CAFs could promote cancer cell proliferation, migration, radiotherapy tolerance, and tumorigenesis. These results suggested that irradiation-activated CAFs might participate in the recurrence of lung cancer after radiotherapy, and the inhibition of CAFs activation may be an important way to improve clinical radiotherapy efficacy.

Intracellular expression of arginine deiminase activates the mitochondrial apoptosis pathway by inhibiting cytosolic ferritin and inducing chromatin autophagy.

BACKGROUND: Based on its low toxicity, arginine starvation therapy has the potential to cure malignant tumors that cannot be treated surgically. The Arginine deiminase (ADI) gene has been identified to be an ideal cancer-suppressor gene. ADI expressed in the cytosol displays higher oncolytic efficiency than ADI-PEG20 (Pegylated Arginine Deiminase by PEG 20,000). However, it is still unknown whether cytosolic ADI has the same mechanism of action as ADI-PEG20 or other underlying cellular mechanisms. METHODS: The interactions of ADI with other protein factors were screened by yeast hybrids, and verified by co-immunoprecipitation and immunofluorescent staining. The effect of ADI inhibiting the ferritin light-chain domain (FTL) in mitochondrial damage was evaluated by site-directed mutation and flow cytometry. Control of the mitochondrial apoptosis pathway was analyzed by Western Blotting and real-time PCR experiments. The effect of p53 expression on cancer cells death was assessed by siTP53 transfection. Chromatin autophagy was explored by immunofluorescent staining and Western Blotting. RESULTS: ADI expressed in the cytosol inhibited the activity of cytosolic ferritin by interacting with FTL. The inactive mutant of ADI still induced apoptosis in certain cell lines of ASS- through mitochondrial damage. Arginine starvation also generated an increase in the expression of p53 and p53AIP1, which aggravated the cellular mitochondrial damage. Chromatin autophagy appeared at a later stage of arginine starvation. DNA damage occurred along with the entire arginine starvation process. Histone 3 (H3) was found in autophagosomes, which implies that cancer cells attempted to utilize the arginine present in histones to survive during arginine starvation. CONCLUSIONS: Mitochondrial damage is the major mechanism of cell death induced by cytosolic ADI. The process of chromatophagy does not only stimulate cancer cells to utilize histone arginine but also speeds up cancer cell death at a later stage of arginine starvation.

Acarbose, as a potential drug, effectively blocked the dynamic metastasis of EV71 from the intestine to the whole body.

Enterovirus 71 (EV71) is one of the main pathogens causing hand-foot-and-mouth disease (HFMD). The nose and mouth are usually the main infection entries of EV71 virus. However, its dynamic transport pathway from mouth to the whole body remains unknown. The reveal of this physiological mechanism in vivo will help to understand its transport direction, find its key proliferation nodes, and develop new preventive strategies. We trained a new strain of GFP-EV71 virus to be susceptible to mice brain by intracranial injection of mice. The adapted virus was oral-administrated to suckling mice. Then, the dynamic distributions of the virus in vivo were detected by living image system and fluorescence quantitation polymerase chain reaction (qPCR). We figured out the dynamic pathway of EV71 transport in vivo from intestine to peripheral tissue, then to the other organs. Small intestine was identified as a gateway for EV71 infection in vivo. Ileum was proved to be the main part of proliferation and transport of EV71 in small intestine of mice. EV71 was verified to enter small intestinal villus of mice through the infection of small intestinal epithelial cell. Acarbose displayed a good preventive effect on EV71 infection both in vivo and in vitro. Acarbose possibly decreased the intestinal infection of EV71 by blocking the receptor-binding sites on the surface of EV71 virion or by inhibiting various glycolic receptors on the cell surface. Thus, acarbose and its analogue may be the potential medicines to prevent EV71 infection.

Detecting EGFR mutations and ALK/ROS1 rearrangements in non-small cell lung cancer using malignant pleural effusion samples.

BACKGROUND: The study was conducted to evaluate the feasibility of using malignant pleural effusion (MPE) as a substitute specimen for genetic testing and to determine the significance of genetic profiling of MPE tumor cells to monitor non-small cell lung cancer (NSCLC) progression and therapeutic response. METHODS: We selected 168 NSCLC patients with MPE. We extracted MPE and enriched tumor cells using a custom-designed device. EGFR mutations and ALK/ROS1 fusions were then detected by quantitative real-time PCR, and the results were used to guide targeted therapy. We investigated drug responses through imaging. RESULTS: MPE tumor cells were detected in all patients. EGFR mutations and ALK/ROS1 rearrangements were detected in biopsy samples, treated MPE, and untreated MPE. We found that treated MPE had higher sensitivity and specificity than biopsy or untreated MPE. Among the 26 EGFR inhibitor patients, 13 showed a partial response, 7 had progressive disease, and 6 showed stable disease. Among the 16 patients that received ALK/ROS1 inhibitors, 8 had a partial response, 4 had progressive disease, and 4 showed stable disease. CONCLUSION: Our study provides a new, less invasive, and highly repeatable method of analyzing MPE tumor cells in NSCLC that facilitates precision medicine and genetic testing.

Mesenchymal stem cells: A double-edged sword in radiation-induced lung injury.

Radiation therapy is an important treatment modality for multiple thoracic malignancies. However, radiation-induced lung injury (RILI), which is the term generally used to describe damage to the lungs caused by exposure to ionizing radiation, remains a critical issue affecting both tumor control and patient quality of life. Despite tremendous effort, there is no current consensus regarding the optimal treatment approach for RILI. Because of a number of functional advantages, including self-proliferation, multi-differentiation, injury foci chemotaxis, anti-inflammation, and immunomodulation, mesenchymal stem cells (MSCs) have been a focus of research for many years. Accumulating evidence indicates the therapeutic potential of transplantation of MSCs derived from adipose tissue, umbilical cord blood, and bone marrow for inflammatory diseases, including RILI. However, reports have also shown that MSCs, including fibrocytes, lung hematopoietic progenitor cells, and ABCG2+ MSCs, actually enhance the progression of lung injuries. These contradictory results suggest that MSCs may have dual effects and that caution should be taken when using MSCs to treat RILI. In this review, we present and discuss recent evidence of the double-edged function of MSCs and provide comments on the prospects of these findings.

Arginine deiminase expressed in vivo, driven by human telomerase reverse transcriptase promoter, displays high hepatoma targeting and oncolytic efficiency.

Arginine starvation has the potential to selectively treat both primary tumor and (micro) metastatic tissue with very low side effects. Arginine deiminase (ADI; EC 3.5.3.6), an arginine-degrading enzyme, has been studied as a potential anti-tumor drug for the treatment of arginine-auxotrophic tumors. Though ADI-PEG20 (pegylated ADI by PEG 20,000) already passed the phase I/II clinical trials [1], it is just used as adjuvant therapy because of its low efficiency and less targeting. Then, this paper discussed the efficiency of arginine starvation mediated by ADI expressed in cytoplasm for liver cancers. In order to guarantee the tumor targeting, human telomerase reverse transcriptase (hTERT) promoter was used to drive the expression of ADI in vivo. To access the anti-tumor efficiency of ADI, p53 gene was used as the positive control. Thus, ADI displayed obvious cytotoxicity to BEL7402 and HUH7 cell lines in cytoplasm. The apoptosis rates rose from 15% to nearly 60% after changing the expression vectors from pcDNA4 plasmid to adenovirus. Compared with p53-adenovirus, ADI-adenovirus showed the higher oncolytic activity in the intratumoral injection model of mice. Tumor disappeared after the treatment of ADI-adenovirus for two weeks, and the mice pulled through all. Therefore, ADI is an ideal anti-tumor gene for caner targeting therapy with the help of hTERT promoter.

Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent.

Four residues of propeptide are essential for precursor folding of nattokinase.

Subtilisin propeptide functions as an intramolecular chaperone that guides precursor folding. Nattokinase, a member of subtilisin family, is synthesized as a precursor consisting of a signal peptide, a propeptide, and a subtilisin domain, and the mechanism of its folding remains to be understood. In this study, the essential residues of nattokinase propeptide which contribute to precursor folding were determined. Deletion analysis showed that the conserved regions in propeptide were important for precursor folding. Single-site and multi-site mutagenesis studies confirmed the role of Tyr10, Gly13, Gly34, and Gly35. During stage (i) and (ii) of precursor folding, Tyr10 and Gly13 would form the part of interface with subtilisin domain. While Gly34 and Gly35 connected with an α-helix that would stabilize the structure of propeptide. The quadruple Ala mutation, Y10A/G13A/G34A/G35A, resulted in a loss of the chaperone function for the propeptide. This work showed the essential residues of propeptide for precursor folding via secondary structure and kinetic parameter analyses.

Ritonavir binds to and downregulates estrogen receptors: molecular mechanism of promoting early atherosclerosis.

Estrogenic actions are closely related to cardiovascular disease. Ritonavir (RTV), a human immunodeficiency virus (HIV) protease inhibitor, induces atherosclerosis in an estrogen-related manner. However, how RTV induce pathological phenotypes through estrogen pathway remains unclear. In this study, we found that RTV increases thickness of coronary artery walls of Sprague Dawley rats and plasma free fatty acids (FFA) levels. In addition, RTV could induce foam cell formation, downregulate both estrogen receptor α (ERα) and ERß expression, upregulate G protein-coupled estrogen receptor (GPER) expression, and all of them could be partially blocked by 17ß-estradiol (E2), suggesting RTV acts as an antagonist for E2. Computational modeling shows a similar interaction with ERα between RTV and 2-aryl indoles, which are highly subtype-selective ligands for ERα. We also found that RTV directly bound to ERα and selectively inhibited the nuclear localization of ERα, and residue Leu536 in the hydrophobic core of ligand binding domain (LBD) was essential for the interaction with RTV. In addition, RTV did not change the secondary structure of ERα-LBD like E2, which explained how ERα lost the capacity of nuclear translocation under the treatment of RTV. All of the evidences suggest that ritonavir acts as an antagonist for 17ß-estradiol in regulating α subtype estrogen receptor function and early events of atherosclerosis.

Probing the cell death signaling pathway of HepG2 cell line induced by copper-1,10-phenanthroline complex.

Copper-1,10-phenanthroline (phen) complex [Cu(phen)2] has been typically known as DNA-cleaving agent. And now it becomes more important for developing multifunctional drugs with its improved cytotoxic properties. In our study, we probed the cytophysiological mechanism of Cu(phen)2. HepG2 cells were more sensitive to Cu(phen)2 with an IC50 of 4.03 µM than other three kinds of cell lines. After treated by Cu(phen)2, HepG2 cells had some typical morphological changes which happened to its nucleus. DNA ladder's occurence and Annexin V-positive increased cells indicated that Cu(phen)2 induced HepG2 cells into apoptosis. Further studies showed that Cu(phen)2 treatment resulted in significant G2/M phase arrest and collapse of mitochondrial membrane potential. Several cell cycle-related factors were down-regulated, including Cyclin A, Cyclin B1 and Cdc2. But p21 and p53 were up-regulated. DNA damage, microtubule disorganization and mitotic arrest through spindle assembly checkpoint activation were observed in Cu(phen)2-treated cells. The activation of caspase-3, 8 & 9 were checked out. The increased-expression ratio of Bax/Bcl-2 was detected. The expression levels of Bcl-xL and Bid were found to decrease. These meant that a mitochondrial-related apoptosis pathway was activated in treated HepG2 cells. Furthermore, some ER stress-associated signaling factors were found to be up-regulated, such as Grp78, XBP-1and CHOP. Ca(2+) was also found to be released from the ER lumen. Collectively, our findings demonstrate that Cu(phen)2 induces apoptosis in HepG2 cells via mitotic arrest and mitochondrial- and ER-stress-related signaling pathways.

Metallothionein 1 h tumour suppressor activity in prostate cancer is mediated by euchromatin methyltransferase 1.

Metallothioneins (MTs) are a group of metal binding proteins thought to play a role in the detoxification of heavy metals. Here we showed by microarray and validation analyses that MT1h, a member of MT, is down-regulated in many human malignancies. Low expression of MT1h was associated with poor clinical outcomes in both prostate and liver cancer. We found that the promoter region of MT1h was hypermethylated in cancer and that demethylation of the MT1h promoter reversed the suppression of MT1h expression. Forced expression of MT1h induced cell growth arrest, suppressed colony formation, retarded migration, and reduced invasion. SCID mice with tumour xenografts with inducible MT1h expression had lower tumour volumes as well as fewer metastases and deaths than uninduced controls. MT1h was found to interact with euchromatin histone methyltransferase 1 (EHMT1) and enhanced its methyltransferase activity on histone 3. Knocking down of EHMT1 or a mutation in MT1h that abrogates its interaction with EHMT1 abrogated MT1h tumour suppressor activity. This demonstrates tumour suppressor activity in a heavy metal binding protein that is dependent on activation of histone methylation.

Insights into the modulation of optimum pH by a single histidine residue in arginine deiminase from Pseudomonas aeruginosa.

Arginine deiminase (ADI) is a potential antitumor agent for the arginine deprivation treatment of L-arginine auxotrophic tumors. The optimum pH of ADI varies significantly, yet little is known about the origin of this variety. Here, Pseudomonas aeruginosa ADI (PaADI), an enzyme that functions only at acidic pH, was utilized as the model system. The results of UV-pH titration imply that the nucleophilic Cys406 thiol group is protonated in the resting state. The H405R single mutation resulted in an altered pH optimum (from pH 5.5 to 6.5), an increased k(cat) (from 9.8 s(-1) to 101.7 s(-1) at pH 6.5), and a shifted pH rate dependence (ascending limb pK(a) from 3.6 to 4.4). Other mutants were constructed to investigate the effects of hydrogen bonding, charge distribution, and hydrophobicity on the properties of the enzyme. The pH optima of His405 mutants were all shifted to a relatively neutral pH except for the H405E mutant. The results of kinetic characterizations and molecular dynamic simulations revealed that the active site hydrogen bonding network involving Asp280 and His405 plays an important role in controlling the dependence of PaADI activity on pH. Moreover, the H405R variant showed increased cytotoxicity towards arginine auxotrophic cancer cell lines.

Interaction of CSR1 with XIAP reverses inhibition of caspases and accelerates cell death.

Cellular Stress Response 1 (CSR1) is a tumor suppressor gene that is located at 8p21, a region that is frequently deleted in prostate cancer as well as a variety of human malignancies. Previous studies have indicated that the expression of CSR1 induces cell death. In this study, we found that CSR1 interacts with X-linked Inhibitor of Apoptosis Protein (XIAP), using yeast two-hybrid screening analyses. XIAP overexpression has been found in many human cancers, and forced expression of XIAP blocks apoptosis. Both in vitro and in vivo analyses indicated that the C-terminus of CSR1 binds XIAP with high affinity. Through a series of in vitro recombinant protein-binding analyses, the XIAP-binding motif in CSR1 was determined to include amino acids 513 to 572. Targeted knock-down of XIAP enhanced CSR1-induced cell death, while overexpression of XIAP antagonized CSR1 activity. The binding of CSR1 with XIAP enhanced caspase-9 and caspase-3 protease activities, and CSR1-induced cell death was dramatically reduced on expression of a mutant CSR1 that does not bind XIAP. However, a XIAP mutant that does not interact with caspase-9 had no impact on CSR1-induced cell death. These results suggest that cell death is induced when CSR1 binds XIAP, preventing the interaction of XIAP with caspases. Thus, this study may have elucidated a novel mechanism by which tumor suppressors induce cell death.

Gene deletions and amplifications in human hepatocellular carcinomas: correlation with hepatocyte growth regulation.

Tissues from 98 human hepatocellular carcinomas (HCCs) obtained from hepatic resections were subjected to somatic copy number variation (CNV) analysis. Most of these HCCs were discovered in livers resected for orthotopic transplantation, although in a few cases, the tumors themselves were the reason for the hepatectomies. Genomic analysis revealed deletions and amplifications in several genes, and clustering analysis based on CNV revealed five clusters. The LSP1 gene had the most cases with CNV (46 deletions and 5 amplifications). High frequencies of CNV were also seen in PTPRD (21/98), GNB1L (18/98), KIAA1217 (18/98), RP1-1777G6.2 (17/98), ETS1 (11/98), RSU1 (10/98), TBC1D22A (10/98), BAHCC1 (9/98), MAML2 (9/98), RAB1B (9/98), and YIF1A (9/98). The existing literature regarding hepatocytes or other cell types has connected many of these genes to regulation of cytoskeletal architecture, signaling cascades related to growth regulation, and transcription factors directly interacting with nuclear signaling complexes. Correlations with existing literature indicate that genomic lesions associated with HCC at the level of resolution of CNV occur on many genes associated directly or indirectly with signaling pathways operating in liver regeneration and hepatocyte growth regulation.

Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase.

Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to ß-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model.

Integrin alpha 7 interacts with high temperature requirement A2 (HtrA2) to induce prostate cancer cell death.

Integrins are a family of receptors for extracellular matrix proteins that have critical roles in human tissue development. Previous studies identified down-regulation and/or mutations of integrin alpha7 (ITGA7) in prostate cancer, liver cancer, soft tissue leiomyosarcoma, and glioblastoma multiforme. Here we report that expression of ITGA7 induced apoptosis in the human prostate cancer cell lines PC3 and DU145. Yeast two-hybrid analysis revealed that the C-terminus of ITGA7 interacts with high temperature requirement A2 (HtrA2), a serine protease with a critical role in apoptosis. Expression of ITGA7 increases the protease activity of HtrA2 both in vitro and in vivo. Deletion of the HtrA2 interaction domain abrogates the cell death activity of ITGA7, whereas down-regulation of HtrA2 dramatically reduced cell death mediated by ITGA7. In addition, site-directed protease-null mutant HtrA2S306A expression blocked apoptosis induced by ITGA7. Interestingly, interaction between ITGA7 and its ligand laminin 2 appears to protect against cell death, since depleting laminin beta2 with a small-interfering RNA significantly exacerbated apoptosis induced by ITGA7 expression. This report provides a novel insight into the mechanism by which ITGA7 acts as a tumor suppressor.

alpha-Bisabolol induces dose- and time-dependent apoptosis in HepG2 cells via a Fas- and mitochondrial-related pathway, involves p53 and NFkappaB.

In this study, the apoptotic effect of alpha-bisabolol, a sesquiterpene, against human liver carcinoma cell line HepG2 was investigated. MTT assay showed alpha-bisabolol could effectively induce cytotoxicity in several human cancer cell lines (PC-3, Hela, ECA-109 and HepG2). The results of nuclei morphology examination, DNA fragmentation detection, flow cytometry analysis and cleavage of poly(ADP-ribose) polymerase and caspases indicated alpha-bisabolol might induce dose- and time-dependent apoptosis in HepG2 cells. Western blot data also showed a cascade activation of caspases-8,-9,-3 and promoted expression of Fas, implying caspase-8 might function as an upstream regulator, and the Fas-related pathway might be involved in this process. Preparation of mitochondrial/cytosol fraction followed with immunoblot analysis showed the release of chromosome c from mitochondria, down-regulated expression of Bcl-2 and translocation of Bax, Bak and Bid, suggesting the mitochondrial-related pathway might be involved in alpha-bisabolol-induced apoptosis either. Detection of accumulation of nuclear wild-type p53 and up-regulated expression of NFkappaB indicated these two key regulator with transcriptional decision-making function in various signaling pathways might also play a role in alpha-bisabolol-induced apoptosis in HepG2 cells.

Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli.

Nattokinase (subtilisin NAT, NK) is a bacterial serine protease with strong fibrinolytic activity and it is a potent cardiovascular drug. In medical and commercial applications, however, it is susceptible to chemical oxidation, and subsequent inactivation or denaturation. Here we show that the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr(220) and Met(222), which were in the vicinity of the catalytic residue Ser(221) of the enzyme. Two nonoxidative amino acids (Ser and Ala) were introduced at these sites using site-directed mutagenesis. Active enzymes were successfully expressed in Escherichia coli with periplasmic secretion and enzymes were purified to homogeneity. The purified enzymes were analyzed with respect to oxidative stability, kinetic parameters, fibrinolytic activity and thermal stability. M222A mutant was found to have a greatly increased oxidative stability compared with wild-type enzyme and it was resistant to inactivation by more than 1 M H(2)O(2), whereas the wild-type enzyme was inactivated by 0.1 M H(2)O(2) (t(1/2) approximately 11.6 min). The other mutant (T220S) also showed an obvious increase in antioxidative ability. Molecular dynamic simulations on wild-type and T220S mutant proteins suggested that a hydrogen bond was formed between Ser(220) and Asn(155), and the spatial structure of Met(222) was changed compared with the wild-type. The present study demonstrates the feasibility of improving oxidative stability of NK by site-directed mutagenesis and shows successful protein engineering cases to improve stability of NK as a potent therapeutic agent.

Cloning, expression, characterization and phylogenetic analysis of arginine kinase from greasyback shrimp (Metapenaeus ensis).

Arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrate. The encoding AK gene from Shrimp Metapenaeus ensis (M. ensis) was cloned in prokaryotic expression plasmid pET-28a, and it was then expressed in Escherichia coil in dissoluble form. The recombinant protein was purified by following three chromatography steps in turn: CM-Cellulose cation-exchange, Sephacryl S-100HR gel filtrate and DEAE-Sepharose anion-exchange. The purified AK's apparent K(m) was 2.33+/-0.1 and 1.59+/-0.2 mM for ATP and l-arginine, respectively, while its optimum pH and temperature was 8.5 and 30 degrees C in the process of forward reaction, respectively. Phylogenetic analysis of cDNA-derived amino acid sequences for the AKs indicated a close affinity of M. ensis and another shrimp (Litopenaeus vannamei).