Role of Spm–Cer‐S1P signalling pathway in MMP‐2 mediated U46619‐induced proliferation of pulmonary artery smooth muscle cells: protective role of epigallocatechin‐3‐gallate

During remodelling of pulmonary artery, marked proliferation of pulmonary artery smooth muscle cells (PASMCs) occur s , which contributes to pulmonary hypertension. Thromboxane A₂ (TxA₂) has been shown to produce pulmonary hypertension. The present study investigates the inhibitory effect of epigallocatechin‐3‐gallate (EGCG) on the TxA₂ mimetic, U46619‐induced proliferation of PASMCs. U46619 at a concentration of 10 nM induces maximum proliferation of bovine PASMCs. Both pharmacological and genetic inhibitors of p³⁸MAPK, NF‐κB and MMP‐2 significantly inhibit U46619‐induced cell proliferation. EGCG markedly abrogate U46619‐induced p³⁸MAPK phosphorylation, NF‐κB activation, proMMP‐2 expression and activation, and also the cell proliferation. U46619 causes an increase in the activation of sphingomyelinase (SMase) and sphingosine kinase (SPHK) and also increase sphingosine 1 phosphate (S1P) level. U46619 also induces phosphorylation of ERK1/2, which phosphorylates SPHK leading to an increase in S1P level. Both pharmacological and genetic inhibitors of SMase and SPHK markedly inhibit U46619‐induced cell proliferation. Additionally, pharmacological and genetic inhibitors of MMP‐2 markedly abrogate U46619‐induced SMase activity and S1P level. EGCG markedly inhibit U46619‐induced SMase activity, ERK1/2 and SPHK phosphorylation and S1P level in the cells. Overall, Sphingomyeline–Ceramide–Sphingosine‐1‐phosphate (Spm–Cer–S1P) signalling axis plays an important role in MMP‐2 mediated U46619‐induced proliferation of PASMCs. Importantly, EGCG inhibits U46619 induced increase in MMP‐2 activation by modulating p³⁸MAPK–NFκB pathway and subsequently prevents the cell proliferation. Copyright © 2015 John Wiley & Sons, Ltd.     

Systemic Negligence: Why It Is Morally Important for Developing World Bioethics

In the context of clinical and non‐clinical biomedical practices, negligence is usually understood as a lapse of a specific professional duty by a healthcare worker or by a medical facility. This paper tries to delineate systemic negligence as another kind of negligence in the context of health systems, particularly in developing countries, that needs to be recognized and addressed. Systemic negligence is not just a mere collection of stray incidences of medical errors and system failures in a health system, but is proposed in this paper as a more pervasive kind of neglect. Several non‐medical factors, such as lack of social and political will, also contribute to it and hence is more difficult to address in a health system. This paper argues that recognizing systemic negligence and including it research agenda have special moral importance for researchers in developing world bioethics, public health ethics and for health activists in the developing world. For, it can be a potent health system barrier, and can seriously impair efforts to ensure patient safety, particularly in the weaker health systems. As it erodes accountability in a health system, addressing it is also important for the twin goals of ensuring patient safety and improving health system performance. Above all, it needs to be addressed because the tolerance of its persistence in a health system seems to undervalue health as a social good.     

ABCG9, ABCG11 and ABCG14 ABC transporters are required for vascular development in Arabidopsis

In order to obtain insights into the regulatory pathways controlling phloem development, we characterized three genes encoding membrane proteins from the G sub‐family of ABC transporters (ABCG9, ABCG11 and ABCG14), whose expression in the phloem has been confirmed. Mutations in the genes encoding these dimerizing ‘half transporters’ are semi‐dominant and result in vascular patterning defects in cotyledons and the floral stem. Co‐immunoprecipitation and bimolecular fluorescence complementation experiments demonstrated that these proteins dimerize, either by flexible pairing (ABCG11 and ABCG9) or by forming strict heterodimers (ABCG14). In addition, metabolome analyses and measurement of sterol ester contents in the mutants suggested that ABCG9, ABCG11 and ABCG14 are involved in lipid/sterol homeostasis regulation. Our results show that these three ABCG genes are required for proper vascular development in Arabidopsis thaliana.     

Identification,purification and characterization of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle plasma membrane

Bovine pulmonary artery smooth muscle tissue possesses matrix metalloproteinase-2 (72 kDa gelatinase: MMP-2; E.C. 3.4.24.24) as revealed by immunoblot studies of its plasma membrane suspension with polyclonal MMP-2 antibody. In this report, we described the purification and partial characterization of MMP-2 in the plasma membrane fraction of the smooth muscle. MMP-2 has been purified from plasma membrane fraction of bovine pulmonary artery smooth muscle to homogeneity using a combination of purification steps. Heparin sepharose purified preparation of 72 kDa progelatinase is composed of two distinct population of zymogens: a 72 kDa progelatinase tightly complexed with TIMP-2 (an ambient tissue inhibitor of metalloprotease in the smooth muscle plasma membrane), and a native 72 kDa progelatinase free of any detectable TIMP-2. The homogeneity of the native 72 kDa progelatinase form is demonstrated by SDS-PAGE under non-reducing condition, non-denaturing native gel electrophoresis. The purified TIMP-2 free proenzyme electrophoresed as a single band of 72 kDa which could be activated by APMA with the formation of 62 and 45 kDa active species. The proenzyme is activated poorly by trypsin but not by plasmin. The purified 72 kDa progelatinase is stable at aqueous solution and does not spontaneously autoactivate. The purified 72 kDa gelatinase exhibited properties that are typical of MMP-2 obtained from other sources. These are: (i) its activity is dependent on the divalent cation, Ca+2, and is inhibited by EDTA, EGTA and 1:1 0-phenanthroline; (ii) it was inhibited by a, macroglobulin but not by the inhibitors of serine, cysteine, thiol, aspartic proteinases and calpains; (iii) it was found to be inhibited by TIMP-2, the specific inhibitor of MMP-2; (iv) like MMP-2, obtained from other sources, its major substrates were found to be collagens (type IV and V) and gelatins (type I, IV and V). Additionally, the purified MMP-2 degrades Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH (dinitrophenyl labelled peptide), a well known synthetic substrate for the MMP-2.     

Role of matrix metalloprotease-2 in oxidant activation of Ca<Superscript>2+</Superscript>ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane

Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H₂O₂ (1 mM) stimulated Ca²⁺ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca²⁺ATPase activity by H₂O₂ in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca²⁺-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA, 1: 10-phenanthroline, a2-macroglobulin and tissue inhibitor of metalloprotease-2 (TIMP-2) indicating that the Ca²⁺-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding tissue inhibitor of metalloprotease in the membrane. In addition to increasing the Ca²⁺ATPase activity, H₂O₂ also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability to degrade¹⁴C-gelatin. The protease activity and the Ca²⁺ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H₂O₂ was due to reactive oxidant species(es). Both basal and H₂O₂ stimulated MMP-2 activity and Ca²⁺ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1: 10-phenanthroline, α₂-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H₂O₂ increased MMP-2 activity and that subsequently stimulated Ca²⁺ATPase activity in the plasma membrane. This was further confirmed by the following observations: (i) adding low doses of MMP-2 or H₂O₂ to the smooth muscle membrane suspension caused submaximal increase in Ca²⁺ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity; (ii) combined treatment of the membrane with low doses of MMP-2 and H₂O₂ augments further the Ca²⁺ATPase activity caused by the respective low doses of either H₂O₂ or MMP-2; and (iii) pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity in the membrane caused by the combined treatment of MMP-2 and H₂O₂.     

Thermostable amylolytic enzymes from a cellulolytic fungusMyceliophthora thermophila D14 (ATCC 48 104)

Summary The production of amylolytic enzymes by a thermophilic cellulolytic fungus,Myceliophthora thermophila D14 was investigated by batch cultivation in Czapek-Dox medium at 45° C. Among various nitrogenous compounds used, NaNO₃ and KNO₃ were found to be the best for amylase production. Starch, cellobiose and maltose induced the synthesis of amylase while glucose, fructose, galactose, lactose, arabinose, xylose, sorbitol, mesoinositol and sucrose did not. Calcium ions had the most stimulating effect on enzyme formation amongst many ions investigated. The synthesis of amylolytic enzymes was dependent on growth and occurred predominantly in the mid-stationary phase. The enzyme was active in a broad temperature range (50° C–60° C) and displayed activity optima at 60° C and pH 5.6.     

Characterization of Salmonella typhi OmpC and OmpF porins engineered with HIV‐gp41 epitope on the surface loops

Porins form trimers in the outer membrane and help transport nutrients and waste products across the bacterial cell membrane. Porin loops are suitable candidates as display systems due to their high immunogenicity and presentation at the bacterial cell surface. In this study, Salmonella typhi porins (OmpC and OmpF) engineered with the Kennedy peptide from gp41 of HIV were characterised. The chimeric OmpC carrying the Kennedy peptide in loop7 did not trimerise, whereas the chimeric OmpF with the epitope in loop5 formed trimers and also was recognised by the antibodies in the HIV patient serum. The results suggest that chimeric S. typhi OmpF may be taken further as a potential candidate to develop as an epitope display system. Proteins 2017; 85:657–664. © 2016 Wiley Periodicals, Inc.