Kinetics of sono-photooxidative degradation of poly(alkyl methacrylate)s

The mechano-chemical degradation of poly(methyl methacrylate) (PMMA), poly(ethyl methacrylate) (PEMA) and poly(n-butyl methacrylate) (PBMA) using ultrasound (US), ultraviolet (UV) radiation and a photoinitiator (benzoin) has been investigated. The degradation of the polymers was monitored using the reduction in number average molecular weight (M-n) and polydispersity (PDI). A degradation mechanism that included the decomposition of the initiator, generation of polymer radicals by the hydrogen abstraction of initiator radicals, reversible chain transfer between stable polymer and polymer radicals was proposed. The mechanism assumed mid-point chain scission due to US and random scission due to UV radiation. A series of experiments with different initial M-n of the polymers were performed and the results indicated that, irrespective of the initial PDI, the PDI during the sono-photooxidative degradation evolved to a steady state value of 1.6 +/- 0.05 for all the polymers. This steady state evolution of PDI was successfully predicted by the continuous distribution kinetics model. The rate coefficients of polymer scission due to US and UV exhibited a linear increase and decrease with the size of the alkyl group of the poly(alkyl methacrylate)s, respectively. (C) 2010 Elsevier B.V. All rights reserved.

Vinyl Monomer Based Polyperoxides as Potential Initiators for Radical Polymerization: An Exploratory Investigation with Poly(.alpha.-methylstyrene peroxide)

We describe the use of poly(alpha-methylstyrene peroxide) (P alpha MSP), an alternating copolymer of alpha-methylstyrene and oxygen, as initiator for the radical polymerization of vinyl monomers. Thermal decomposition of P alpha MSP in 1,4-dioxane follows first-order kinetics with an activation energy (E(a)) of 34.6 kcal/mol. Polymerization of methyl methacrylate (MMA) and styrene using P alpha MSP as an initiator was carried out in the temperature range 60-90 degrees C. The kinetic order with respect to the initiator and the monomer was close to 0.5 and 1.0, respectively, for both monomers. The E(a) for the polymerization was 20.6 and 22.9 kcal/mol for MMA and styrene, respectively. The efficiency of P alpha MSP was found to be in the range 0.02-0.04. The low efficiency of P alpha MSP was explained in terms of the unimolecular decomposition of the alkoxy radicals which competes with primary radical initiation. The presence of peroxy segments in the main chain of PMMA and polystyrene was confirmed from spectroscopic and DSC studies. R(i)'/2I values for P alpha MSP compared to that of BPO at 80 degrees C indicate that P alpha MSP can be used as an effective high-temperature initiator.

A novel (main chain) (side chain) polymeric peroxide: Synthesis and initiating characteristics of poly(alpha-(tert-butylperoxymethyl)styrene peroxide)

A novel (main chain)-(side chain) vinyl polyperoxide, poly(alpha-(tert-butylperoxymethyl)styrene peroxide) (MCSCPP), an alternating copolymer of alpha-(tert-butylperoxymethyl)styrene (TPMS) and oxygen, has been synthesized by the oxidative polymerization of TPMS. The MCSCPP was characterized by H-1 NMR, C-13 NMR, IR, DSC, EI-MS, and GC-MS studies. The overall activation energy (E(a)) for the degradation of MCSCPP was found to be 27 kcal/mol. Formaldehyde and alpha-(tert-butylperoxy)acetophenone (TPAP) were identified as the primary degradation products of MCSCPP; TPAP was found to undergo further degradation. The side chain peroxy groups were found to be thermally more stable than those in the main chain. Polymerization of styrene in the presence of MCSCPP as initiator, at 80 degrees C, follows classical kinetics. The presence of peroxy segments in the polystyrene chain was confirmed by both H-1 NMR and thermal decomposition studies. Interestingly, unlike other vinyl polyperoxides, the MCSCPP initiator shows an increase in molecular weight with conversion.

Important role of the amino acid attached to tRNA in formylation and in initiation of protein synthesis in Escherichia coli

In attempts to convert an elongator tRNA to an initiator tRNA, we previously generated a mutant elongator methionine tRNA carrying an anticodon sequence change from CAU to CUA along with the two features important for activity of Escherichia coli initiator tRNA in initiation. This mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only when aminoacylated with methionine by overproduction of methionyl-tRNA synthetase. Here we show that the Mi:2 tRNA is normally aminoacylated in vivo with lysine and that the tRNA aminoacylated with lysine is a very poor substrate for formylation compared with the same tRNA aminoacylated with methionine. By introducing further changes at base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in the E. coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tRNA transformylase results in partial formylation of the mutant tRNA and activity of the formyllysyl-tRNAs in initiation of protein synthesis. Thus, the G4:C69 base pair contributes toward formylation of the tRNA and protein synthesis in E. coli can be initiated with formyllysine. We also discuss the implications of these and other results on recognition of tRNAs by E. coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases.

Studies of moisture induced crosslinking in a novel vinyl ether maleic anhydride copolymer

A novel vinyl ether, 2,2-dimethyl-4-vinyioxymethyl-1,3-dioxol (DMVMD), that has a dimethyl ketal protected vicinal diol functionality was synthesizied from readily available starting materials, such as glycerol, acetone and acetylene. Copolymerisation of DMVMD with maleic anhydride (MAH) in various molar ratios was carried out using a free radical initiator. The composition of the copolymer was established by conductometric titration, and was found to be 1:1 irrespective of the monomer feed composition thus establishing its alternating nature. The copolymer formed clear free standing films upon solvent casting which became insoluble upon prolonged exposure to ambeint atmosphere. The insolubility is ascribed to moisture induced crosslinking. A plausible mechanism for the crosslinking involves the hydrolysis of some of the anhydride groups, followed by acid catalysed deketalization, and then by the reaction of the alcoholic groups, thus generated, with the residual anhydride to give ester crosslinks. This hypothesis was confirmed both by model reactions and insitu FT-IR studies.

Solution grafting of maleic anhydride onto polyethylene and compatibilization of polyetylene-nylon blends

Maleic anhydride (MAH) has been grafted onto high density polyethylene (HDPE) with benzoyl peroxide (BOP) initiator in toluene solution. Maximum degree of grafting (12%) without crosslinking has been obtained using MAH/HDPE and BOP/HDPE weight ratios of 1.0 and 0.15 respectively, at 110 degrees C. The HDPE-g-MAH compatibilizer is found to drastically reduce the dispersed phase size and also to produce homogeneous blends for relatively low concentrations of dispersed phase in HDPE/nylon blends. Addition of this compatibilizer results in increase of tensile strength and modulus with increasing nylon content of HDPE/nylon blends, while the opposite is found for the blends without any added compatibilizer.

Grafting of m-isopropenyl-alpha, alpha-dimethylbenzyl-isocyanate (m-TMI) onto isotactic polypropylene: Synthesis and characterization

A novel vinyl monomer with an isocyanate functional group, m-isopropenyl-alpha,alpha-dimethylbenzyl-isocyanate (m-TMI), was grafted onto isotactic polypropylene (i-PP) using dicumyl peroxide (DCP) as the initiator. This would open up the possibility of using the grafted polymer with the reactive isocyanate group as compatibilizer for blending carbohydrates such as cellulose with. polypropylene. The grafting was carried out in a Brabender Plasticoder at 180degreesC. The effects of monomer and initiator concentrations on the yield of grafting were investigated by performing statistical analysis. While the grafting yield increased with the concentration of DCP at any given concentration of m-TMI, the variation of the grafting yield with m-TMI concentration, for a given concentration of DCP, went through a maximum, the optimum yield of 7.8% (w/w) being obtained at 10 wt.% concentration of both DCP and m-TMI. The grafting reaction is. accompanied by considerable chain scission of I-PP, resulting in a decrease in the molecular weight of the grafted polymer. While the molecular weight drops sharply even at a low concentration of DCP, there occurs no further significant change in the molecular weight even at much higher concentrations of the initiator.

Synthetic Triterpenoid Cyano Enone of Methyl Boswellate Activates Intrinsic, Extrinsic, and Endoplasmic Reticulum Stress Cell Death Pathways in Tumor Cell Lines

We explored the effect of a novel synthetic triterpenoid compound cyano enone of methyl boswellates (CEMB) on various prostate cancer and glioma cancer cell lines. CEMB displayed concentration-dependent cytotoxic activity with submicromolar lethal dose 50% (LD(50)) values in 10 of 10 tumor cell lines tested. CEMB-induced cytotoxicity is accompanied by activation of downstream effector caspases (caspases 3 and 7) and by upstream initiator caspases involved in both the extrinsic (caspase 8) and intrinsic (caspase 9) apoptotic pathways. By using short interfering RNAs (siRNA), we show evidence that knockdown of caspase 8, DR4, Apaf-1, and Bid impairs CEMB-induced cell death. Similar to other proapoptotic synthetic triterpenoid compounds, CEMB-induced apoptosis involved endoplasmic reticulum stress, as shown by partial rescue of tumor cells by siRNA-mediated knockdown of expression of genes involved in the unfolded protein response such as IRE1 alpha, PERK, and ATF6. Altogether, our results suggest that CEMB stimulates several apoptotic pathways in cancer cells, suggesting that this compound should be evaluated further as a potential agent for cancer therapy. Mol Cancer Ther; 10(9); 1635-43. (C)2011 AACR.

Cryptic AUG is important for 48S ribosomal assembly during internal initiation of translation of coxsackievirus B3 RNA

We have investigated the possible role of a conserved cis-acting element, the cryptic AUG, present in the 5' UTR of coxsackievirus B3 (CVB3) RNA. CVB3 5' UTR contains multiple AUG codons upstream of the initiator AUG, which are not used for the initiation of translation. The 48S ribosomal assembly takes place upstream of the cryptic AUG. We show here that mutation in the cryptic AUG results in reduced efficiency of translation mediated by the CVB3 IRES; mutation also reduces the interaction of mutant IRES with a well characterized IRES trans-acting factor, the human La protein. Furthermore, partial silencing of the La gene showed a decrease in IRES activity in the case of both the wild-type and mutant. We have demonstrated here that the interaction of the 48S ribosomal complex with mutant RNA was weaker compared with wild-type RNA by ribosome assembly analysis. We have also investigated by chemical and enzymic modifications the possible alteration in secondary structure in the mutant RNA. Results suggest that the secondary structure of mutant RNA was only marginally altered. Additionally, we have demonstrated by generating compensatory and non-specific mutations the specific function of the cryptic AUG in internal initiation. Results suggest that the effect of the cryptic AUG is specific and translation could not be rescued. However, a possibility of tertiary interaction of the cryptic AUG with other cis-acting elements cannot be ruled out. Taken together, it appears that the integrity of the cryptic AUG is important for efficient translation initiation by the CVB3 IRES RNA.

Inhibition of the Interaction Between NS3 Protease and HCV IRES With a Small Peptide: A Novel Therapeutic Strategy

Recently, we have demonstrated that the protease domain of NS3 alone can bind specifically to hepatitis C virus (HCV) internal ribosome entry site (IRES) near the initiator AUG, dislodges human La protein and inhibits translation in favor of viral RNA replication. Here, by using a computational approach, the contact points of the protease on the HCV IRES were putatively mapped. A 30-mer NS3 peptide was designed from the predicted RNA-binding region that retained RNA-binding ability and also inhibited IRES-mediated translation. This peptide was truncated to 15 mer and this also demonstrated ability to inhibit HCV RNA-directed translation as well as replication. More importantly, its activity was tested in an in vivo mouse model by encapsulating the peptide in Sendai virus virosomes followed by intravenous delivery. The study demonstrates for the first time that the HCV NS3-IRES RNA interaction can be selectively inhibited using a small peptide and reports a strategy to deliver the peptide into the liver.

Distinctive contributions of the ribosomal P-site elements m(2)G966, m(5)C967 and the C-terminal tail of the S9 protein in the fidelity of initiation of translation in Escherichia coli

The accuracy of pairing of the anticodon of the initiator tRNA (tRNA(fMet)) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m(2)G966 (methylated by RsmD), m(5)C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNA(fMet). We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA(CAU)(fmet) (tRNA(fMet) with CAU anticodon); CAC and CAU with tRNA(GUG)(fme); UAG with tRNA(GAU)(fMet) using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA(CAU)(fMet)lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

Initiation with Elongator tRNAs

In all domains of life, initiator tRNA functions exclusively at the first step of protein synthesis while elongator tRNAs extend the polypeptide chain. Unique features of initiator tRNA enable it to preferentially bind the ribosomal P site and initiate translation. Recently, we showed that the abundance of initiator tRNA also contributes to its specialized role. This motivates the question, can a cell also use elongator tRNA to initiate translation under certain conditions? To address this, we introduced non-AUG initiation codons CCC (Pro), GAG (Glu), GGU (Gly), UCU (Ser), UGU (Cys), ACG (Thr), AAU (Asn), and AGA (Arg) into the uracil DNA glycosylase gene (ung) used as a reporter gene. Enzyme assays from log-phase cells revealed initiation from non-AUG codons when intracellular initiator tRNA levels were reduced. The activity increased significantly in stationary phase. Further increases in initiation from non-AUG codons occurred in both growth phases upon introduction of plasmid-borne genes of cognate elongator tRNAs. Since purine-rich Shine-Dalgarno sequences occur frequently on mRNAs (in places other than the canonical AUG codon initiation contexts), initiation with elongator tRNAs from the alternate contexts may generate proteome diversity under stress without compromising genomic integrity. Thus, by changing the relative amounts of initiator and elongator tRNAs within the cell, we have blurred the distinction between the two classes of tRNAs thought to be frozen through years of evolution.

Specific Sequence of a Beta Turn in Human La Protein May Contribute to Species Specificity of Hepatitis C Virus

Human La protein is known to be an essential host factor for translation and replication of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that residues responsible for interaction of human La protein with the HCV internal ribosomal entry site (IRES) around the initiator AUG within stem-loop IV form a beta-turn in the RNA recognition motif (RRM) structure. In this study, sequence alignment and mutagenesis suggest that the HCV RNA-interacting beta-turn is conserved only in humans and chimpanzees, the species primarily known to be infected by HCV. A 7-mer peptide corresponding to the HCV RNA-interacting region of human La inhibits HCV translation, whereas another peptide corresponding to the mouse La sequence was unable to do so. Furthermore, IRES-mediated translation was found to be significantly high in the presence of recombinant human La protein in vitro in rabbit reticulocyte lysate. We observed enhanced replication with HCV subgenomic and full-length replicons upon overexpression of either human La protein or a chimeric mouse La protein harboring a human La beta-turn sequence in mouse cells. Taken together, our results raise the possibility of creating an immunocompetent HCV mouse model using human-specific cell entry factors and a humanized form of La protein.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved.