In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis.
Anti-Bacterial Agents ; chemistry ; metabolism ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Genetic Engineering ; Micromonosporaceae ; chemistry ; enzymology ; genetics ; metabolism ; Mutation ; Polyketide Synthases ; chemistry ; genetics ; metabolism ; Protein Domains ; Sirolimus ; analogs & derivatives ; metabolism

PURPOSE: This study sought to determine the 1-year clinical effectiveness and safety of a biodegradable, polymer-containing Biolimus A9™-eluting stent (BES) in Korean patients with acute coronary syndrome (ACS). MATERIALS AND METHODS: A total of 1000 ACS patients with 1251 lesions who underwent implantation of BESs at 22 centers in Korea were enrolled between May 2011 and July 2013. We assessed major adverse cardiac events (MACE) defined as the composite of cardiac death, non-fatal myocardial infarction (MI), and clinical-driven target vessel revascularization at 12 months. RESULTS: Patient mean age was 62.6±11.4 years. 72.8% of the patients were male, 28.5% had diabetes, 32.8% had multi-vessel disease (MVD), and 47.9% presented with acute MI (AMI). The mean global registry of acute coronary events risk score of all patients was 103.0±27.6. The number of stents per patient was 1.3±0.6. The incidences of MACE and definite stent thrombosis at 12 months were 3.9% and 0.2%, respectively. On multivariate Cox-regression analysis, age ≥65 years was identified as an independent predictors of 1-year MACE (hazard ratio=2.474; 95% confidence interval=1.202−5.091). Subgroup analyses revealed no significant differences in the incidence of MACE between patients with and without diabetes (4.3% vs. 3.7%, p=0.667), between those who presented with and without AMI (4.4% vs. 3.4%, p=0.403), and between those with and without MVD (4.6% vs. 3.5%, p=0.387). CONCLUSION: Our study demonstrated excellent 1-year clinical outcomes of BES implantation in patients at low-risk for ACS.
Acute Coronary Syndrome/drug therapy ; Aged ; Drug-Eluting Stents/adverse effects ; Female ; Humans ; Incidence ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Multivariate Analysis ; Proportional Hazards Models ; Republic of Korea ; Sirolimus/adverse effects ; Sirolimus/analogs & derivatives ; Sirolimus/therapeutic use ; Time Factors ; Treatment Outcome

BACKGROUNDStudies have suggested that use of prolonged dual antiplatelet therapy (DAPT) following new generation drug-eluting stent implantation may increase costs and potential bleeding events. This study aimed to investigate the association of DAPT status with clinical safety in patients undergoing everolimus-eluting stent (EES) implantation in the SEEDS study (A Registry to Evaluate Safety and Effectiveness of Everolimus Drug-eluting Stent for Coronary Revascularization) at 2-year follow-up.

METHODSThe SEEDS study is a prospective, multicenter study, where patients (n = 1900) with small vessel, long lesion, or multi-vessel diseases underwent EES implantation. Detailed DAPT status was collected at baseline, 6-month, 1- and 2-year. DAPT interruption was defined as any interruption of aspirin and/or clopidogrel more than 14 days. The net adverse clinical events (NACE, a composite endpoint of all-cause death, all myocardial infarction (MI), stroke, definite/probable stent thrombosis (ST), and major bleeding (Bleeding Academic Research Consortium II-V)) were investigated according to the DAPT status at 2-year follow-up.

RESULTSDAPT was used in 97.8% of patients at 6 months, 69.5% at 12 months and 35.4% at 2 years. It was observed that the incidence of NACE was low (8.1%) at 2 years follow-up, especially its components of all-cause death (0.9%), stroke (1.1%), and definite/probable ST (0.7%). DAPT was not an independent predictor of composite endpoint of all-cause death/MI/stroke (hazard ratio [HR]: 0.693, 95% confidence interval [CI]: 0.096-4.980, P = 0.715) and NACE (HR: 1.041, 95% CI: 0.145-7.454, P = 0.968). Of 73 patients who had DAPT interruption, no patient had ST at 12-month, and only 1 patient experienced ST between 1- and 2-year (1.4%). There was a high frequency of major bleeding events (53/65, 82.5%) occurred in patients receiving DAPT treatment.

CONCLUSIONSProlonged DAPT use was not associated with improved clinical safety. The study emphasized that duration of DAPT needs to be shortened in Chinese patients following EES implantation (ClinicalTrials.gov identifier: NCT 01157455).

Adolescent ; Adult ; Aged ; Aspirin ; therapeutic use ; Drug-Eluting Stents ; Everolimus ; Female ; Humans ; Male ; Middle Aged ; Platelet Aggregation Inhibitors ; therapeutic use ; Prospective Studies ; Sirolimus ; analogs & derivatives ; therapeutic use ; Thrombosis ; drug therapy ; Ticlopidine ; analogs & derivatives ; therapeutic use ; Treatment Outcome ; Young Adult

Amino Acid Substitution ; Exocytosis ; HEK293 Cells ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Membrane Proteins ; chemistry ; metabolism ; Microscopy, Confocal ; Protein Binding ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; Sirolimus ; analogs & derivatives ; chemistry ; metabolism ; Tacrolimus Binding Proteins ; chemistry ; genetics ; metabolism

The aim of this study was to illustrate the mechanism of inhibiting the cell growth in diffuse large B-cell lymphoma by histone deacetylase inhibitor valproic acid (VPA) combined with mTOR inhibitor temsirolimus (TEM). MTT assay and Wright's stain were used to assess cell growth inhibition and to detect the cell morphological changes respectively. The cell apoptosis, cell cycle and cell autophagy were determined by flow cytometry. Ultrastructure changes were confirmed by electron microscopy. Protein changes were detected by Western blot. The results showed that both VPA and TEM alone inhibited cell proliferation and the effect was more obvious in the combination group. VPA combined with TEM induced cell arrest in G0/G1 phase and upregulated the expression of autophagy-related protein LC3, without cell apoptosis. Moreover, typical autophagosomes were observed, further confirming the presence of autophagy. Western blot showed the changes of proteins involved in autophagy signaling pathway. VPA decreased HDAC1 and HDAC3 expression and increased histone acetylation, suggesting that VPA also affected lymphoma cell proliferation through epigenetic modification. It is concluded that the combined treatment of VPA and TEM induces cell cycle arrest and cell autophagy, which provides a new clue for their clinical application in diffuse large B-cell lymphoma.
Autophagy ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Epigenesis, Genetic ; Histone Deacetylase 1 ; metabolism ; Histone Deacetylases ; metabolism ; Humans ; Lymphoma, Large B-Cell, Diffuse ; pathology ; Microtubule-Associated Proteins ; metabolism ; Sirolimus ; administration & dosage ; analogs & derivatives ; pharmacology ; Valproic Acid ; administration & dosage ; pharmacology

BACKGROUNDSirolimus-eluting stents (SES) are reported to be associated with reduced late lumen loss (LLL), resulting in less frequent restenosis when compared to bare-metal stent. The current study aimed to assess the difference in LLL between SES with biodegradable and with permanent polymer.

METHODSFrom March 2010 to June 2011, 300 consecutive patients having only biodegradable polymers or permanent polymer SES for all diseased vessels were included. Serial quantitative coronary analysis was performed on both the "in-stent" and "segment" area, including the stented segment, as well as both five mm margins proximal and distal to the stent. The primary endpoint was the LLL defined as the minimal lumen diameter (MLD) post-stenting minus the MLD at nine-month after the indexed procedure.

RESULTSLLL was comparable between the two stents. Importantly, LLL for the distal segment (median 0.05 mm, interquartile 0 to 0.09 mm) was less severe compared with in-stent (median 0.13 mm, interquartile 0.08 to 0.18 mm) and proximal segment LLL (median 0.12 mm, interquartile 0.06 to 0.14 mm, all P < 0.001). In general, the LLL was associated with the post-procedure MLD (b = 0.28, P = 0.002), hyperlipidemia (b = 0.14, P = 0.021), and calcified lesions (b = 0.58, P = 0.001). The R(2) and Radj of the multiple regression model were 0.651 and 0.625, respectively.

CONCLUSIONSSES with either biodegradable or permanent polymer had lower value of LLL. The small amount of LLL at the distal segment possibly contributed to the less distal edge stenosis.

Aged ; Aspirin ; therapeutic use ; Coronary Restenosis ; prevention & control ; Drug-Eluting Stents ; Female ; Humans ; Male ; Middle Aged ; Polymers ; chemistry ; Regression Analysis ; Sirolimus ; therapeutic use ; Ticlopidine ; analogs & derivatives ; therapeutic use

As the first nationwide Korean prospective multicenter data collection registry, the Korea Acute Myocardial Infarction Registry (KAMIR) launched in November 2005. Through a number of innovative approaches, KAMIR suggested new horizons about acute myocardial infarction (AMI) which contains unique features of Asian patients from baseline characteristics to treatment strategy. Obesity paradox was existed in Korean AMI patients, whereas no gender differences among them. KAMIR score suggested new risk stratifying method with increased convenience and an enhanced accuracy for the prediction of adverse outcomes. Standard loading dose of clopidogrel was enough for Asian AMI patients. Triple antiplatelet therapy with aspirin, clopidogrel and cilostazol could improve clinical outcomes than dual antiplatelet therapy with aspirin and clopidogrel. Statin improved clinical outcomes even in AMI patients with very low LDL-C levels. The rate of percutaneous coronary intervention was higher and door-to-balloon time was shorter than the previous reports. Zotarolimus eluting stents as the 2nd generation drug-eluting stent (DES) was not superior to the 1st generation DES, in contrast to the western AMI studies. KAMIR made a cornerstone in the study of Korean AMI and expected to be new standards of care for AMI with the renewal of KAMIR design to overcome its pitfalls.
Acute Disease ; Drug-Eluting Stents ; Humans ; Myocardial Infarction/*diagnosis/drug therapy ; Platelet Aggregation Inhibitors/therapeutic use ; Registries ; Republic of Korea ; Risk Factors ; Severity of Illness Index ; Sirolimus/analogs & derivatives/therapeutic use

No abstract available.
Alveolitis, Extrinsic Allergic/*chemically induced/diagnosis/drug therapy ; Biopsy ; Drug Hypersensitivity/diagnosis/drug therapy/*etiology ; Drug-Eluting Stents/*adverse effects ; Humans ; Male ; Middle Aged ; Percutaneous Coronary Intervention/*adverse effects/instrumentation ; Sirolimus/adverse effects/*analogs & derivatives ; Steroids/therapeutic use ; Tomography, X-Ray Computed ; Treatment Outcome

We aimed comparing two-year clinical outcomes of the Everolimus-Eluting Promus and Paclitaxel-Eluting TAXUS Liberte stents used in routine clinical practice. Patients with objective evidence of ischemia and coronary artery disease eligible for PCI were prospectively randomized to everolimus-eluting stent (EES) or paclitaxel-eluting stent (PES) groups. The primary end-point was ischemia-driven target vessel revascularization (TVR) at 2 yr after intervention, and the secondary end-point was a major adverse cardiac event (MACE), such as death, myocardial infarction (MI), target lesion revascularization (TLR), TVR or stent thrombosis. A total of 850 patients with 1,039 lesions was randomized to the EES (n=425) and PES (n=425) groups. Ischemic-driven TVR at 2 yr was 3.8% in the PES and 1.2% in the EES group (P for non-inferiority=0.021). MACE rates were significantly different; 5.6% in PES and 2.5% in EES (P = 0.027). Rates of MI (0.8% in PES vs 0.2% in EES, P = 0.308), all deaths (1.5% in PES vs 1.2% in EES, P = 0.739) and stent thrombosis (0.3% in PES vs 0.7% in EES, P = 0.325) were similar. The clinical outcomes of EES are superior to PES, mainly due to a reduction in the rate of ischemia-driven TVR.
Antineoplastic Agents, Phytogenic/administration & dosage/therapeutic use ; Coronary Artery Disease/*drug therapy/mortality ; Coronary Restenosis/prevention & control ; *Drug-Eluting Stents ; Female ; Humans ; Immunosuppressive Agents/administration & dosage/therapeutic use ; Male ; Middle Aged ; Paclitaxel/administration & dosage/*therapeutic use ; Percutaneous Coronary Intervention/*methods ; Prospective Studies ; Sirolimus/administration & dosage/*analogs & derivatives/therapeutic use ; Thrombosis ; Treatment Outcome

Autophagy is a conserved and programmed catabolic process that degrades damaged proteins and organelles. But the underlying mechanism and functions of autophagy in the ischemia-reperfusion (IR)-induced injury are unknown. In this study, we employed simulated IR of N2a cells as an in vitro model of IR injury to the neurons and monitored autophagic processes. It was found that the levels of Beclin-1 (a key molecule of autophay complex, Beclin-1/class III PI3K) and LC-3II (an autophagy marker) were remarkably increased with time during the process of ischemia and the process of reperfusion after 90 min of ischemia, while the protein kinases p70S6K and mTOR which are involved in autophagy regulation showed delayed inactivation after reperfusion. Administration of 3-methyladenine (3MA), an inhibitor of class III PI3K, abolished autophagy during reperfusion, while employment of rapamycin, an inhibitor of mTORC1 (normally inducing autophagy), surprisingly weakened the induction of autophagy during reperfusion. Analyses of mitochondria function by relative cell viability demonstrated that autophagy inhibition by 3-MA attenuated the decline of mitochondria function during reperfusion. Our data demonstrated that there were two distinct dynamic patterns of autophagy during IR-induced N2a injury, Beclin-1/class III PI3K complex-dependent and mTORC1-dependent. Inhibition of over-autophagy improved cell survival. These suggest that targeting autophagy therapy will be a novel strategy to control IR-induced neuronal damage.
Adenine ; analogs & derivatives ; pharmacology ; Animals ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Autophagy ; Beclin-1 ; Cell Line, Tumor ; Cell Survival ; Mechanistic Target of Rapamycin Complex 1 ; Mice ; Mitochondria ; metabolism ; Multiprotein Complexes ; antagonists & inhibitors ; metabolism ; Neurons ; drug effects ; metabolism ; Neuroprotective Agents ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Reperfusion Injury ; metabolism ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism