Theoretical insights into thermal cyclophanediene to dihydropyrene electrocyclic reactions; a comparative study of Woodward Hoffmann allowed and forbidden reactions

The thermally allowed electrocyclic reaction syn-cyclophanediene (CPD) to dihydropyrene (DHP) was compared with the disallowed thermal electrocyclic reaction in anti CPD through density functional theory (DFT) calculations at the B3LYP/6-31?+?G(d) level. Moreover, the results were also compared with the electrocyclization of 1,3,5 hexatriene to 1,3-cyclohexadiene . The Woodward-Hoffmann (W-H) allowed thermal reaction in syn CPD 11 has a calculated activation barrier of 6.23 kcal mol^?1, compared with 29 kcal mol^?1 for the electrocyclization of 1,3,5 hexatriene to 1,3-cyclohexadiene. The enhanced acceleration of electrocyclization is believed to arise from geometrically enforced spatially aligned termini of the hexatriene. Substituents at the electrocyclic terminus of cyclophanediene significantly affected (up to three fold) the activation barriers. Mono-substitution of CPD has substituent dependent acceleration or deceleration whereas di-substitution always increased the activation barrier. The activation barrier for electrocyclization in 33 is 4.44 kcal mol^?1, which is the lowest activation barrier for any thermal electrocyclic reaction. Cyclophanedienes (CPDs) substituted with electron-rich substituents cyclized with high activation barriers and vice versa, a phenomenon significantly different from electrocyclic reaction of 1,3,5-hexatriene where no such trend is traceable. Comparison of W-H allowed and forbidden electrocyclization in syn and anti CPDs, respectively, revealed quite similar electronic demand, although the transition states are different in nature. The transition state for a W-H forbidden reaction is biradicaloid, with most of the spin density at the electrocyclic termini; however, the transition state for a W-H allowed reaction has no such contribution. We also believe that this is the first study of its type, where W-H allowed and forbidden reactions are compared on a similar set of molecules, and compared for electronic effect through substituents.     

Lipophilic cations: a group of model substrates for the multidrug-resistance transporter.

The possibility that simple lipophilic cations such as tetraphenylphosphonium (TPA+), triphenylmethylphosphonium (TPMP+), and diphenyldimethylphosphonium (DDP+) are substrates for the multidrug-resistance transport protein, P-glycoprotein, was tested. Hamster cells transfected with and overexpressing mouse mdr1 or mouse mdr3 exhibit high levels of resistance to TPP+ and TPA+ (20-fold) and somewhat lower levels of resistance to TPMP+ and DDP+ (3-12-fold). Transfected cell clones expressing mdr1 or mdr3 mutants with decreased activity against drugs of the MDR spectrum (e.g., Vinca alkaloids and anthracyclines) also show reduced resistance to lipophilic cations. Studies with radiolabeled TPP+ and TPA+ demonstrate that increased resistance to cytotoxic concentrations of these lipophilic cations is correlated quantitatively with a decrease in intracellular accumulation in mdr1- and mdr3-transfected cells. This decreased intracellular accumulation is shown to be strictly dependent on intact intracellular nucleotide triphosphate pools and is reversed by verapamil, a known competitive inhibitor of P-glycoprotein. Taken together, these results demonstrate that lipophilic cations are a new class of substrates for P-glycoprotein and can be used to study its mechanism of action in homologous and heterologous systems.     

Peroxisomal Acyl-CoA oxidase 4 activity differs between Arabidopsis accessions

In plants, peroxisomes are the primary site of fatty acid β-oxidation. Following substrate activation, fatty acids are oxidized by Acyl-CoA Oxidase (ACX) enzymes. Arabidopsis has six ACX genes, although ACX6 is not expressed. Biochemical characterization has revealed that each ACX enzyme acts on specific chain-length targets, but in a partially overlapping manner, indicating a degree of functional redundancy. Genetic analysis of acx single and double mutants in the Columbia (Col-0) accession revealed only minor phenotypes, but an acx3acx4 double mutant from Wassileskija (Ws) is embryo lethal. In this study, we show that acx3acx4(Col) and acx1acx3acx4(Col) mutants are viable and that enzyme activity in these mutants is significantly reduced on a range of substrates compared to wild type. However, the triple mutant displays only minor defects in seed-storage mobilization, seedling development, and adult growth. Although the triple mutant is defective in the three most active and highly-expressed ACX proteins, increases in ACX2 expression may support partial β-oxidation activity. Comparison of acx mutant alleles in the Col-0 and Ws accessions reveals independent phenotypes; the Ws acx4 mutant uniquely shows increased sensitivity to propionate, whereas the Col-0 acx4 allele has sucrose-dependent growth in the light. To dissect the issues between Col-0 and Ws, we generated mixed background mutants. Although alleles with the Col-0 acx4 mutant were viable, we were unable to isolate an acx3acx4 line using the Ws acx4 allele. Reducing ACX4 expression in several Arabidopsis backgrounds showed a split response, suggesting that the ACX4 gene and/or protein functions differently in Arabidopsis accessions.     

In vitro antituberculosis activities of the constituents isolated from Haloxylon salicornicum

In vitro antituberculosis activities of fractions and pure compounds (1–20) including seven triterpenes, two alkaloids, two cycloheximide derivatives, two coumarins six sterol derivatives and a long chain alcohol, respectively, isolated from Haloxylon salicornicum were determined against Mycobecterium tuberculosis H37Rv. Actively growing cultures were tested by rapid colorimetric method while the stationary phase cultures were tested by drug exposure methods for bactericidal activity. The MIC values were found to be 50 μg/ml for compounds 15, 19 and 20 where as rest of the compounds invariably showed MIC value of 100 μg/ml against the logarithmic phase culture. These were compare to Isoniazid as a control drug. The compounds exhibited no activity against the stationary phase culture of M. tuberculosis H37Rv up to 200 μg/ml. Further studies are required to investigate the in vivo efficacies and activities of the compounds in combination with antimicrobials that are already being used for TB therapy.     

A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA

The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter, MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26). In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance. In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations. These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA. We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed.     

Determinants of substrate recognition by the Escherichia coli multidrug transporter MdfA identified on both sides of the membrane

The Escherichia coli multidrug transporter MdfA contains a membrane-embedded charged residue (Glu-26) that was shown to play an important role in substrate recognition. To identify additional determinants of multidrug recognition we isolated 58 intragenic second-site mutations that restored the function of inactive MdfA E26X mutants. In addition, two single-site mutations that enhanced the activity of wild-type MdfA were identified. Most of the mutations were found in two regions, the cytoplasmic half of transmembrane segments (TMs) 4, 5, and 6 (cluster 1) and the periplasmic half of TM 1 and 2 (cluster 2). The identified residues were mutated to cysteines in the background of a functional cysteine-less MdfA, and substrate protection against alkylation was analyzed. The results support the suggestion that the two clusters are involved in substrate recognition. Using inverted membrane vesicles we observed that a proton electrochemical gradient (Deltamicro(H(+)), inside positive and acidic) enhanced the substrate-protective effect in the cytoplasmic region, whereas it largely reduced this effect in the periplasmic side of MdfA. Therefore, we propose that substrates interact with two sites in MdfA, one in the cytoplasmic leaflet of the membrane and the other in the periplasmic leaflet. Theoretically, these domains could constitute a large part of the multidrug pathway through MdfA.     

Wound dressing based on PVA nanofiber containing silk fibroin modified with GO/ZnO nanoparticles for superficial wound healing: In vitro and in vivo evaluations

Silk fibroin (SF), extracted from Bombyx mori, has unique physicochemical properties to achieve an efficient wound dressing. In this study, reduced graphene oxide (RGO)/ZnO NPs/silk fibroin nanocomposite was made, and an innovative nanofiber of SF/polyvinyl alcohol (PVA)/RGO/ZnO NPs was ready with the electrospinning technique and successfully characterized. The results of MIC and OD analyses were used to investigate the synthesized materials' antibacterial effects and displayed that the synthesized materials could inhibit growth against Staphylococcus aureus and Escherichia coli bacteria. However, both in vitro cytotoxicity (MTT) and scratch wound studies have shown that RGO/ZnO NPs and SF/PVA/RGO/ZnO NPs are not only non-toxic to NIH 3T3 fibroblasts, but also can cause cell viability, cell proliferation, and cell migration. Furthermore, improving the synthesized nanofiber's structural properties in the presence of RGO and ZnO NPs has been confirmed by performing tensile strength, contact angle, and biodegradation analyses. Also, in a cell attachment analysis, fibroblast cells had migrated and expanded well in the nanofibrous structures. Moreover, in vivo assay, SF/PVA/RGO/ZnO NPs nanofiber treated rats and has been shown significant healing activity and tissue regeneration compared with other treated groups. Therefore, this study suggests that SF/PVA/RGO/ZnO NPs nanofiber is a hopeful wound dressing for preventing bacteria growth and improving superficial wound repair.     

Nucleoside transport in mammalian cell membranes

Summary Transport of the nucleoside analog cytosine-arabinoside (CAR) in transformed hamster cells in culture has been studied in conditions of minimal metabolic conversion. Uptake (zero-trans in) properties at 20°C over a limited range of CAR concentrations were characterized by aK _m of 350 m and a maximal velocity (V) of 780 m·min^–1 (V/K _m =2.28 min^–1). Equilibrium exchange at 20°C over a wider range of concentrations was best described by a saturable component with aK _m of 500 m and av of 1230 m·min^–1 (V/K _m =2.26 min^–1) and either a saturable component of highK _m or a nonsaturable component ofk=0.3 min^–1. For the saturable component, thev/K _m values were similar in both procedures.CAR transport was inhibited by various metabolizable nucleosides. Uptake of some of these nucleosides was inhibited by CAR. CAR transport and uridine uptake were inhibited in a reversible but partially competitive fashion by high affinity probes like S-(p-nitrobenzyl-6-mercaptoinosine (NBMI) (K _i <0.5nm) and in an irreversible fashion by SH reagents such as N-ethylmaleiimide (NEM). The organomercurialp-hydroxymercuribenzene sulfonate (pMBS) markedly stimulated transport of these nucleosides, but also markedly potentiated the inhibitory effects of either NBMI or NEM. These effects are interpreted either in terms of models which invoke allosteric properties or in terms of two transport systems which display distinct chemical susceptibilities to externally added probes.