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Although enzyme catalysis is typified by high specificity, enzymes can catalyze various substrates (substrate promiscuity) and/or different reaction types (catalytic promiscuity) using a single active site. This interesting phenomenon is widely distributed in enzyme catalysis, with both fundamental and applied importance. To date, the mechanistic understanding of enzyme promiscuity is very limited. Herein, we report the structural mechanism underlying the substrate and catalytic promiscuity of Vibrio dual lipase/transferase (VDLT). Crystal structures of the VDLT from Vibrio alginolyticus (ValDLT) and its fatty acid complexes were solved, revealing prominent structural flexibility. In particular, the "Ser−His−Asp" catalytic triad machinery of ValDLT contains an intrinsically flexible oxyanion hole. Analysis of ligand-bound structures and mutagenesis showed that the flexible oxyanion hole and other binding residues can undergo distinct conformational changes to facilitate substrate and catalytic promiscuity. Our study reveals a previously unknown flexible form of the famous catalytic triad machinery and proposes a "catalytic site tuning" mechanism to expand the mechanistic paradigm of enzyme promiscuity. Vibrio dual lipases/transferases are virulence-related enzymes, with both substrate and catalytic promiscuity. Wang et al reveal their prominent structural flexibility, proposing a catalytic site tuning mechanism underlying enzyme promiscuity. [ABSTRACT FROM AUTHOR]

Thymidylate synthase (TS) is a key enzyme in the de novo biosynthesis of thymidine monophosphate, serving as a well-known drug target in chemotherapy against cancers and infectious diseases. Additional to its clinical value, TS is supposed to be a promising drug target in aquatic-disease control. To facilitate designing pathogen-specific TS inhibitors for shrimp-disease control, we report the crystal structures of TS from Litopenaeus vannamei (LvTS) in the apo form, LvTS-dUMP complex and LvTS-dUMP-raltitrexed complex at 2.27 Å, 1.54 Å, and 1.56 Å resolution, respectively. LvTS shares a similar fold with known TSs, existing as a dimer in the crystal. The apo LvTS and LvTS-dUMP take an open conformation, and raltitrexed binding induces structural changes into a closed conformation in LvTS-dUMP-raltitrexed. Compared to those in other known TS-dUMP-raltitrexed complexes with the closed conformation, the C-terminal loop in LvTS-dUMP-raltitrexed shifts its position away from the bound raltitrexed; the distance between C6 of dUMP and Sγ of the catalytic cysteine is obviously longer than that in the known TS structures with closed conformations, resembling that in the TS structures with open conformations. Other species-specific interactions with dUMP and raltitrexed are also observed. Therefore, LvTS-dUMP-raltitrexed adopts a loosely closed conformation with structural features intermediate between the closed and the open conformations that were reported in other TSs. Our study provides the first crustcean TS structure, and reveals species-specific interactions between TSs and the ligands, which would facilitate designing pathogen-specific TS inhibitors for shrimp-disease control. [ABSTRACT FROM AUTHOR]

Microbial diversity in the abyssal sediments beneath the seafloor of 30, 94, and 151 cm near the southern end of the Mariana Trench was analyzed in the Illumina HiSeq 2500 platform. Results show that the microbial populations were dominated by bacteria but merely no archaea were identified at the three depths. In the bacterial community, Proteobacteria and Firmicutes dominated the total taxon tags, followed by Bacteroidetes, Actinobacteria, Planctomycetes, Cyanobacteria, and Chloroflexi, which together account for over 99% of the total population. Similar to that in the seawater in the trench, the operational taxonomic units (OTUs) belonging to Gammaproteobacteria from the sediment samples showed high abundance. However, common bacterial OTUs in the water of the trench including Nitrospirae and Marinimicrobia were hardly found in the sediments from the southern Mariana Trench or the hadal region. Therefore, this study documented for the first time the compositions of microbial diversity in the trench sediments, revealed the difference in microbial diversity in water and sediment of the trench and will enrich the knowledge on the microbial diversity in the abyssal areas. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play vital roles in the regulation of cell cycle, cell growth, apoptosis, and tumorigenesis. Our previous studies showed that miR-526a positively regulated innate immune response by suppressing CYLD expression, however, the functional relevance of miR-526a expression and cell growth remains to be evaluated. In this study, miR-526a overexpression was found to promote cancer cell proliferation, migration, and anchor-independent colony formation. The molecular mechanism(s) of miR-526a-mediated growth stimulation is associated with rapid cell cycle progression and inhibition of cell apoptosis by targeting CYLD. Taken together, these results provide evidence to show the stimulatory role of miR-526a in tumor migration and invasion through modulation of the canonical NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Hypoxia inducible factor (HIF)-2α is an important transcription factor that contributes to tumor proliferation and progression. Beclin 1 is a key mediator of autophagy, and dysfunction of Beclin 1 is implicated in tumorigenicity. This study was designed to investigate the expression patterns of HIF-2α and Beclin 1 and to clarify their clinical significance in chondrosarcoma. Methods: The mRNA and protein levels of HIF-2α and Beclin 1 in chondrosarcoma and the corresponding nontumor tissues were analyzed by real-time polymerase chain reaction and Western blot, respectively. The protein expression of HIF-2α and Beclin 1 was investigated by immunohistochemistry, and their associations with clinicopathological factors and overall survival were evaluated. Results: HIF-2α was remarkably elevated, whereas Beclin 1 was significantly reduced in chondrosarcoma compared with the corresponding nontumor tissues. High HIF-2α level and negative Beclin 1 expression were 52.9% and 58.8% in chondrosarcoma specimens, respectively. HIF-2α and Beclin 1 were associated with histological grade and Musculoskeletal Tumor Society stage. There was a significant inverse relationship between HIF-2α and Beclin 1. HIF-2α and Beclin 1 had significant impacts on the prognosis of chondrosarcoma patients. Multivariate analysis revealed that Beclin 1 was an independent prognostic factor for overall survival of patients with chondrosarcoma. Conclusions: Elevated HIF-2α levels assocaited with low Beclin 1 expression play a role in the development of chondrosarcoma. Beclin 1 can serve as a novel biomarker to predict survival of chondrosarcoma patients, and may represent a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Clinical management of chondrosarcoma remains a challenging problem, largely due to the toxicity and resistance of this tumor to conventional chemotherapy. Programmed Cell Death 5 (PDCD5) is a protein that accelerates apoptosis in different cell types in response to various stimuli, and has been shown to be down-regulated in many cancer tissues. In this study, mRNA and protein levels of PDCD5 were found to be up-regulated in cisplatin-treated SW1353 chondrosarcoma cells compared with untreated cells. Recombinant human PDCD5 (rhPDCD5) was also shown to sensitize chondrosarcoma cells to cisplatin-based chemotherapy, with inhibition of cell growth and apoptosis detected both in vitro and in vivo. Increased expression of Bax and decreased expression of Bcl-2 were also observed, along with release of cytochrome c from mitochondria into the cytosol. Additionally, cleavage of caspase-9 and caspase-3, as well as the cleavage of poly (ADP-ribose) polymerase (PARP), were detected, suggesting that sensitization of chondrosarcoma cells involves the intrinsic mitochondrial apoptosis pathway. In vivo, the treatment of a xenograft model of chondrosarcoma with rhPDCD5 and cisplatin significantly inhibited tumor cell proliferation and induced apoptosis compared to treatment with cisplatin alone. Overall, these data provide a theoretical basis for the administration of rhPDCD5 and cisplatin for the treatment of patients with chondrosarcoma. [ABSTRACT FROM AUTHOR]

Spinal cord metastatic malignancies and locally invasive paraspinal carcinomas were severe pain and disabling complications. The treatment modality was unclear. We retrospectively evaluated the safety and efficacy of (125)I seed implantation for spinal metastatic and paraspinal malignancies in our center. A total of 19 patients and 22 procedures were included in this study. Eight patients received a seed implant in an operation, and 11 patients were implanted percutaneously under CT-guidance. The number of (125)I seeds implanted ranged from 6 to 100, with a median of 26. The specific activity of (125)I seeds ranged from 0.45 to 0.80 mCi per seed, with a median of 0.5 mCi. The minimal peripheral doses of (125)I seed implantation were 90-140 Gy, with median of 120 Gy. Follow-up ranged from 3 to 69 months with a median of 22 months. All patients tolerated seed implantation well. The 1, 2, 3, and 5 year local control rates were 63%, 47%, 31%, and 3%, respectively, with a median of 22 months (3-61 months). The 1, 2, 3, and 5 year survival rates were 74%, 56%, 43%, and 43%, respectively, with a median of 33 months. Ten patients (52.6%) developed distant metastases. Two (10.5%) patients showed recurrence at 13 and 39 months. Seven (36.8%) patients were still alive, 84% (16/19) of patients had either normal or improved ambulation following seed implant. No myelopathies were encountered. Our study suggests a promising local therapy for a selected population of patients with spinal cord metastatic or paraspinal carcinoma. [ABSTRACT FROM AUTHOR]

The tyrosine kinase, c-Abl, plays important roles in many aspects of cellular function. The activity of c-Abl is tightly controlled, but the underlying mechanism is unclear. Recent studies suggest that c-Abl function is regulated by distinct lipids in different cell types. In the present study, we show that the DNA replication factor, proliferating cell nuclear antigen (PCNA), interacts with c-Abl and destabilizes c-Abl by promoting its polyubiquitination and degradation. Moreover, deletion of a domain in c-Abl, the PIP box, disrupts its interaction with PCNA, abolishes the PCNA-induced degradation of nuclear c-Abl, and substantially increases the nuclear c-Abl apoptotic function. These findings indicate that PCNA negatively regulates the stability of c-Abl and thereby inhibits apoptosis in the response to DNA damage. [ABSTRACT FROM AUTHOR]

Simvastatin has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which simvastatin exerts its effects is still unclear. We previously reported that simvastatin promotes bone morphogenetic protein 2 (BMP-2) expression, induces osteoblastic differentiation, and inhibits adipocytic differentiation in mouse bone marrow stromal cells (BMSCs), and that this occurs, at least in part, via a BMP-2-dependent pathway. The aim of this study was to investigate further the mechanisms by which simvastatin stimulates osteogenesis in mouse BMSCs. To determine whether simvastatin-mediated osteogenesis was dependent on BMP-2, mouse BMSCs were treated with nonimmune normal mouse IgG or BMP-2 neutralizing antibodies combined with different concentrations of simvastatin. Surprisingly, the stimulatory effect of simvastatin on alkaline phosphatase (ALP) activity was not completely blocked by neutralizing BMP-2 monoclonal antibody treatment. Interestingly, we found that estrogen receptor-alpha (ER-alpha) protein levels increased after mouse BMSCs were treated with simvastatin for 72 h in a concentration-dependent manner. Moreover, the stimulatory effect of simvastatin on ALP activity in BMSCs was blocked by the estrogen receptor agonist ICI 182,780, and cotreatment with 17-beta-estradiol and simvastatin increased ALP activities by two-to threefold in the BMSCs compared with treatment with simvastatin alone. These results suggest that simvastatin-induced in vitro osteogenesis in mouse BMSCs is mediated, at least in part, by induction of ER-alpha and not by BMP-2 alone. These results provide new insight into the mechanisms of simvastatin-induced bone formation in BMSCs. [ABSTRACT FROM AUTHOR]

Homogeneous charge compression ignition (HCCI) has challenges in ignition timing control, combustion rate control, and operating range extension. In this paper, HCCI combustion was studied in a two-cylinder gasoline direct injection (GDI) engine with negative valve overlap (NVO). A two-stage gasoline direct injection strategy combined with negative valve overlap was used to control mixture formation and combustion. The gasoline engine could be operated in HCCI combustion mode at a speed range of 800–2 200 r/min and load, indicated mean effective pressure (IMEP) range of 0.1–0.53 MPa. The engine fuel consumption is below 240 g/(kW−1·h−1), and the NO x emission is below 4 × 10−5 without soot emission. The effect of different injection strategies on HCCI combustion was studied. The experimental results indicated that the coefficient of variation of the engine cycle decreased by using NVO with two-stage direct injection; the ignition timing and combustion rate could be controlled; and the operational range of HCCI combustion could be extended. [ABSTRACT FROM AUTHOR]

The c-Abl and Arg nonreceptor tyrosine kinases are activated in the response of cells to oxidative stress. The present studies demonstrate that treatment of cells with 0.1?mM H2O2 is associated with increased tyrosine phosphorylation of Arg and little effect on Arg levels. By contrast, exposure to 1.0?mM H2O2 decreased Arg phosphorylation. Treatment with 1.0?mM H2O2 was also associated with ubiquitination and degradation of Arg. The results show that Arg is stabilized in response to 0.1?mM H2O2 by autophosphorylation of Y-261, consistent with involvement of the Arg kinase function in regulating Arg levels. The results further demonstrate that c-Abl-mediated phosphorylation of Arg on Y-261 similarly confers Arg stabilization. In concert with these results, phosphorylation of Arg on Y-261 blocked H2O2-induced ubiquitination and thereby Arg degradation and inactivation. These findings demonstrate that Arg phosphorylation and degradation are differentially regulated by the degree of oxidative stress, and that Arg stability is conferred by phosphorylation of Y-261.Oncogene (2005) 24, 2433-2440. doi:10.1038/sj.onc.1208454 Published online 7 February 2005 [ABSTRACT FROM AUTHOR]

A random mutagenesis library of gp120-801 (a large segment of the envelope protein gene of HIV-1) was constructed using error-prone PCR and DNA shuffling methods, and one mutant of gp120-801 was selected from this library using a green fluorescent protein (GFP) fusion vector. After being cloned into pEX31b and expressed in E. coli, the expressed fusion protein reached to 15% of total bacterial proteins for the mutant but was just 1–2% for the wild type. [ABSTRACT FROM AUTHOR]