Angiotensin converting enzyme gene polymorphism in Korean patients with primary knee osteoarthritis

Angiotensin converting enzyme (ACE) plays an important role in the physiology of vasculature, blood pressure and inflammation. ACE gene, known to have insertion/deletion (I/D) polymorphism, has been widely investigated in its relation with cardiovascular and neurodegenerative diseases and longevity. ACE gene polymorphism in an inflammation associated osteoarthritis (OA) patients is not known. Here we have investigated ACE gene polymorphism in 142 Korean primary knee OA patients and 135 healthy volunteers to establish any clinical correlates between ACE polymorphism and knee osteoarthritis. Clinical parameters such as disease onset age, Kellgren-Lawrence grade and Lequesne's functional index provided additional analysis of the relationship of ACE polymorphism and clinical features of OA. Early onset OA showed significantly higher allele frequency and carriage rate of I than late onset OA. Radiographically severe and functionally poor OA showed higher carriage rate of I allele than radiographically mild and functionally good OA, respectively. This study first reports ACE gene polymorphism to be a risk factor for early onset, severe form primary knee OA.     

NF-κB-dependent miR-31/155 biogenesis is essential for TNF-α-induced impairment of endothelial progenitor cell function

Endothelial progenitor cell (EPC) dysfunction impairs vascular function and remodeling in inflammation-associated diseases, including preeclampsia. However, the underlying mechanism of this inflammation-induced dysfunction remains unclear. In the present study, we found increases in TNF-α and miR-31/155 levels and reduced numbers of circulating EPCs in patients with preeclampsia. Patient-derived mononuclear cells (MNCs) cultured in autologous serum had decreased endothelial nitric oxide synthase (eNOS) expression, nitric oxide production, and differentiation into EPCs with angiogenic potential, and these effects were inhibited by a TNF-α-neutralizing antibody and miR-31/155 inhibitors. Moreover, TNF-α treatment of normal MNCs increased miR-31/155 biogenesis, decreased eNOS expression, reduced EPC differentiation, and impaired angiogenic potential. The TNF-α-induced impairment of EPC differentiation and function was rescued by NF-κB p65 knockdown or miR-31/155 inhibitors. In addition, treatment of MNCs with synthetic miR-31/155 or an eNOS inhibitor mimicked the inhibitory effects of TNF-α on eNOS expression and EPC functions. Moreover, transplantation of EPCs that had been differentiated from TNF-α-treated MNCs decreased neovascularization and blood perfusion in ischemic mouse hindlimbs compared with those of normally differentiated EPCs. These findings suggest that NF-κB activation is required for TNF-α-induced impairment of EPC mobilization, differentiation, and function via miR-31/155 biogenesis and eNOS downregulation. Our data provide a new role for NF-κB-dependent miR-31/155 in EPC dysfunction under the pathogenic conditions of inflammation-associated vascular diseases, including preeclampsia.Subject terms: Differentiation, Cell biology     

Quantitative and qualitative measure of intralaboratory two-dimensional protein gel reproducibility and the effects of sample preparation, sample load, and image analysis

We investigate one approach to assess the quantitative variability in two-dimensional gel electrophoresis (2-DE) separations based on gel-to-gel variability, sample preparation variability, sample load differences, and the effect of automation on image analysis. We observe that 95% of spots present in three out of four replicate gels exhibit less than a 0.52 coefficient of variation (CV) in fluorescent stain intensity (% volume) for a single sample run on multiple gels. When four parallel sample preparations are performed, this value increases to 0.57. We do not observe any significant change in quantitative value for an increase or decrease in sample load of 30% when using appropriate image analysis variables. Increasing use of automation, while necessary in modern 2-DE experiments, does change the observed level of quantitative and qualitative variability among replicate gels. The number of spots that change qualitatively for a single sample run in parallel varies from a CV = 0.03 for fully manual analysis to CV = 0.20 for a fully automated analysis. We present a systematic method by which a single laboratory can measure gel-to-gel variability using only three gel runs.     

Reliability of estimation of recombination energies of molecular radical cations by charge exchange and a test of the exoergicity rule

Charge exchange occurring in a collision cell of a mass spectrometer was used previously to find the very long-lived excited electronic states of molecular cations generated by electron ionization. The underlying criterion in the method, called the exoergicity rule, was that only electronically exoergic and resonant processes have large cross sections at low collision energy. The validity of this rule has been checked in this work by estimating the recombination energies of some molecular ions based on the presence/absence of charge-exchange signals from gases with different ionization energies, and comparing these estimates with the literature values. It is concluded that a recombination energy can be estimated to within an upper limit uncertainty of 0.1 eV with this technique, which validates the exoergicity rule proposed previously.     

Melatonin prevents doxorubicin-induced cardiotoxicity through suppression of AMPKα2-dependent mitochondrial damage

The clinical application of doxorubicin, one of the most effective anticancer drugs, has been limited due to its adverse effects, including cardiotoxicity. One of the hallmarks of doxorubicin-induced cytotoxicity is mitochondrial dysfunction. Despite intensive research over recent decades, there are no effective approaches for alleviating doxorubicin-induced cytotoxicity. Melatonin, a natural hormone that is primarily secreted by the pineal gland, is emerging as a promising adjuvant that protects against doxorubicin-induced cytotoxicity owing to its pharmaceutical effect of preserving mitochondrial integrity. However, the underlying mechanisms are far from completely understood. Here, we provide novel evidence that treatment of H9c2 cardiomyoblasts with doxorubicin strongly induced AMP-activated protein kinase α2 (AMPKα2), which translocated to mitochondria and interfered with their function and integrity, ultimately leading to cellular apoptosis. These phenomena were significantly blocked by melatonin treatment. The levels of AMPKα2 in murine hearts were tightly associated with cardiotoxicity in the context of doxorubicin and melatonin treatment. Therefore, our study suggests that the maintenance of mitochondrial integrity is a key factor in reducing doxorubicin-induced cytotoxicity and indicates that AMPKα2 may serve as a novel target in the design of cytoprotective combination therapies that include doxorubicin.Subject terms: Biochemistry, Diseases     

20-O-acylcamptothecin derivatives: evidence for lactone stabilization

Convincing UV and NMR spectrophotometric evidence is presented which demonstrates that at physiological pH, 7.4, 20-O-acyl derivatives of camptothecin (CPT) are substantially more stable in the lactone form than the 20-OH parent. Additionally, it was determined by HPLC analysis that the lactone ring of a 20-O-ether derivative of CPT underwent endocyclic ring opening at pH > or =8.5, while the lactone ring of 20-O-acyl CPT derivatives remained unaffected. PEG (and other smaller alkyl) 20-O-acyl-CPT derivatives released native CPT at pH > 9.5, which arises from exocyclic cleavage, thus precluding isolation of any open CPT acyl PEG (or alkyl) carboxylate forms.     

Influence of film preparation procedures on the crystallinity, morphology and mechanical properties of LLDPE films

Influence of various film preparation procedures on the crystallinity, morphology and mechanical properties of pure linear low-density polyethylene and its calcite filled composite films has been studied using differential scanning calorimeter (DSC), wide-angle X-ray diffractometer (WAXRD), atomic force microscope (AFM) and ultimate tensile testing machine (UTM). The film preparation procedures include variation in cooling rates such as quenching, force (fan) and natural cooling and in techniques such as extrusion followed by melt squeezing and compression molding. The heat of fusion (from DSC), the degree of crystallinity (from WAXRD) and the crystallite size (from WAXRD and AFM) are found to be the highest for naturally cooled specimen, followed by fan cooled and quenched ones. The AFM images of surface topology exhibit stacked lamellar morphology for forcefully cooled (fan cooled and quenching) samples and spherulitic ‘lozenges’ for naturally cooled ones. The Young’s modulus and yield stress (from UTM) are the highest for naturally cooled samples, followed by fan cooled and quenched ones. Amongst the calcite filled composites, the ‘base film’, which is prepared by extrusion followed by melt squeezing and natural cooling, exhibits the lowest heat of fusion, degree of crystallinity and Young’s modulus, but the highest yield stress, elongation at break and tensile strength compared to the compression molded ones.     

A comparison of three commercially available isoelectric focusing units for proteome analysis: the multiphor, the IPGphor and the protean IEF cell

We tested and compared three different commercially available instruments for isoelectric focusing for proteome analysis by two-dimensional protein electrophoresis. These instruments, the Multiphor, the IPGphor, and the Protean IEF cell, were used with 18 cm immobilized pH gradient strips and run under various conditions. The total number of spots and features was obtained by Melanie software (Bio-Rad Laboratories) and separately by visual inspection. The Multiphor consistently resulted in the highest number of spots detected per gel independent of sample type, immobilized pH gradient (IPG) and method to calculate the number of spots. The Protean IEF cell had the next highest number of spots detected per gel. In the experiments performed, the IPGphor afforded good reproducibility in the total number of Melanie-detected spots from gel to gel while the Protean IEF cell offered better reproducibility in the total number of manually detected spots from gel to gel. Among gels run with the different instruments, differences in the quality of the ammoniacal silver stain were also observed. A measure of quantitative reproducibility suggests that the Protean IEF cell, which was the easiest instrument to use, performs better than the other instruments, although all three instruments had demonstrated good quantitative reproducibility in the experiments performed.     

A catalytic approach of blending CO2-activating MOF struts for cycloaddition reaction in a helically interlaced Cu(II) amino acid imidazolate framework: DFT-corroborated investigation

In CO₂ transformation catalysis, the synthesis of cyclic carbonates using two classes of MOF catalysts viz., zeolitic imidazolate frameworks (ZIF) and MOFs with carboxylate-capped SBUs have gained large attention. Herein we propose the strategy of employing a unified multifunctional framework formed in the metal-centered assembly of imidazole and amino-carboxylates for CO₂ transformation, such as propylene carbonate (PC) by the cycloaddition of CO₂ with propylene oxide. The framework {[Cu(L-asp)(1,4-bix)_0.5]·3H₂O}_n (CuAspBix) comprises of the amino acid building units, L-aspartic acid (L-Asp) and the flexible ligand, 1,4-bis(imidazole-1-yl methyl)benzene [1,4-Bix]. The 1,4-Bix ligand with imidazole terminals renders elongated M-M distances and flexibility in comparison with pristine ZIF materials. The cumbersome synthesis procedure poor phase purity of the bulk catalyst in solvothermal conditions were improved by a microwave-assisted synthesis, preserving the structural and physicochemical properties. Minimal energy input or room temperatures for the catalysis occurred via the synergistic participation of CuAspBix and quaternary ammonium bromide salt, demonstrated by density-functional theory computational studies to propose mechanistic pathway of the reaction. Reaction conditions were optimized by altering the parameters. The heterogeneous catalyst was reused four times without a significant change in activity.

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