Anthraquinones from Hedyotis capitellata

Four new furanoanthraquinones, 2-hydroxymethyl-3,4-[2'-(1-hydroxy-1-methylethyl)-dihydrofurano]-8-hydroxyanthraquinone, 2-hydroxymethyl-3,4-[1'-hydroxy-2'-(1-hydroxy-1-methylethyl)-dihydrofurano]-8-hydroxyanthraquinone, 2-hydroxymethyl-3,4-[2'-1-hydroxy-1-methylethyl)-dihydrofurano]anthraquinone and 2-methyl-3,4-[2'-(1-hydroxy-1-methylethyl)-dihydrofurano] anthraquinone or capitellataquinone A-D and four known anthraquinones, rubiadin, anthragallol 2-methyl ether, alizarin 1-methyl ether and digiferruginol, together with scopoletin were isolated from the stems of Hedyotis capitellata Wall (Rubiaceae). Lucidin-3-O-beta-glucoside was isolated from the roots of the plant. Characterization of the new compounds was carried out by extensive NMR studies using FGCOSY, FGHMQC, FGHMBC and DEPT-135 in addition to other spectroscopic methods.     

Flexibility in HIV-1 assembly subunits: solution structure of the monomeric C-terminal domain of the capsid protein

The protein CA forms the mature capsid of human immunodeficiency virus. Hexamerization of the N-terminal domain and dimerization of the C-terminal domain, CAC, occur during capsid assembly, and both domains constitute potential targets for anti-HIV inhibitors. CAC homodimerization occurs mainly through its second helix, and is abolished when its sole tryptophan is mutated to alanine. Previous thermodynamic data obtained with the dimeric and monomeric forms of CAC indicate that the structure of the mutant resembles that of a monomeric intermediate found in the folding and association reactions of CAC. We have solved the three-dimensional structure in aqueous solution of the monomeric mutant. The structure is similar to that of the subunits in the dimeric, nonmutated CAC, except the segment corresponding to the second helix, which is highly dynamic. At the end of this region, the polypeptide chain is bent to bury several hydrophobic residues and, as a consequence, the last two helices are rotated 90 degrees when compared to their position in dimeric CAC. The previously obtained thermodynamic data are consistent with the determined structure of the monomeric mutant. This extraordinary ability of CAC to change its structure may contribute to the different modes of association of CA during HIV assembly, and should be taken into account in the design of new drugs against this virus.     

Synthesis of amides from (E)-3-(1-chloro-3,4-dihydronaphthalen-2-yl)acrylic acid and substituted amino acid esters as NorA efflux pump inhibitors of Staphylococcus aureus

Inhibitors for NorA efflux pump of Staphylococcus aureus have attracted the attention of many researchers towards the discovery and development of novel efflux pump inhibitors (EPIs). In an attempt to find specific potent inhibitors of NorA efflux pump of S. aureus, a total of 15 amino acid conjugates of 3-(1-chloro-3,4-dihydronaphthalen-2-yl)acrylic acid (4–18) were synthesized using a simple convenient synthetic approach and bioevaluated against NorA efflux pump. Two compounds 7 and 8 (each having MEC of 1.56?µg/mL) were found to restore the activity of ciprofloxacin through reduction of the MIC elucidated by comparing the ethidium bromide efflux in dose dependent manner in addition to ethidium bromide efflux inhibition and accumulation study using NorA overexpressing strain SA-1199B. Most potent compounds among these were able to restore the antibacterial activity of ciprofloxacin completely against SA-1199B. Structure activity relationship (SAR) studies and docking study of potent compounds 7 and 8 could elucidate the structural requirements necessary for interaction with the NorA efflux pumps. On the whole, compounds 7 and 8 have ability to reverse the NorA efflux mediated resistance and could be further optimized for development of potent efflux pump inhibitors.     

Substrate specificity and products of side-reactions catalyzed by jasmonate:amino acid synthetase (JAR1)

Jasmonate:amino acid synthetase (JAR1) is involved in the function of jasmonic acid (JA) as a plant hormone. It catalyzes the synthesis of several JA-amido conjugates, the most important of which appears to be JA-Ile. Structurally, JAR1 is a member of the firefly luciferase superfamily that comprises enzymes that adenylate various organic acids. This study analyzed the substrate specificity of recombinant JAR1 and determined whether it catalyzes the synthesis of mono- and dinucleoside polyphosphates, which are side-reaction products of many enzymes forming acyl approximately adenylates. Among different oxylipins tested as mixed stereoisomers for substrate activity with JAR1, the highest rate of conversion to Ile-conjugates was observed for (+/-)-JA and 9,10-dihydro-JA, while the rate of conjugation with 12-hydroxy-JA and OPC-4 (3-oxo-2-(2Z-pentenyl)cyclopentane-1-butyric acid) was only about 1-2% that for (+/-)-JA. Of the two stereoisomers of JA, (-)-JA and (+)-JA, rate of synthesis of the former was about 100-fold faster than for (+)-JA. Finally, we have demonstrated that (1) in the presence of ATP, Mg(2+), (-)-JA and tripolyphosphate the ligase produces adenosine 5'-tetraphosphate (p(4)A); (2) addition of isoleucine to that mixture halts the p(4)A synthesis; (3) the enzyme produces neither diadenosine triphosphate (Ap(3)A) nor diadenosine tetraphosphate (Ap(4)A) and (4) Ap(4)A cannot substitute ATP as a source of adenylate in the complete reaction that yields JA-Ile.     

Residue specific partitioning of KL4 into phospholipid bilayers

KL₄, which has demonstrated success in the treatment of respiratory distress, is a synthetic helical, amphipathic peptide mimetic of lung surfactant protein B. The unusual periodicity of charged residues within KL₄ and its relatively high hydrophobicity distinguish it from canonical amphipathic helical peptides. Here we utilized site specific spin labeling of both lipids and the peptide coupled with EPR spectroscopy to discern the effects of KL₄ on lipid dynamics, the residue specific dynamics of hydrophobic regions within KL₄, and the partitioning depths of specific KL₄ residues into the DPPC/POPG and POPC/POPG lipid bilayers under physiologically relevant conditions. KL₄ induces alterations in acyl chain dynamics in a lipid-dependent manner, with the peptide partitioning more deeply into DPPC-rich bilayers. Combined with an earlier NMR study of changes in lipid dynamics on addition of KL₄ (V.C. Antharam et al., 2009), we are able to distinguish how KL₄ affects both collective bilayer motions and intramolecular acyl chain dynamics in a lipid-dependent manner. EPR power saturation results for spin labeled lipids demonstrate that KL₄ also alters the accessibility profiles of paramagnetic colliders in a lipid-dependent manner. Measurements of dynamics and depth parameters for individual spin-labeled residues within KL₄ are consistent with a model where the peptide partitions deeply into the lipid bilayers but lies parallel to the bilayer interface in both lipid environments; the depth of partitioning is dependent on the degree of lipid acyl chain saturation within the bilayer.     

FTIR studies of the redox partner interaction in cytochrome P450: The Pdx–P450cam couple

Recently we have developed a new approach to study protein–protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam–Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP–FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in β-sheets and α-helix content, a decrease in the population of random coil/3₁₀-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam–Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx–P450cam complex.     

The important role of stratum corneum lipids for the cutaneous barrier function

The skin protects the body from unwanted influences from the environment as well as excessive water loss. The barrier function of the skin is located in the stratum corneum (SC). The SC consists of corneocytes embedded in a lipid matrix. This lipid matrix is crucial for the lipid skin barrier function. This paper provides an overview of the reported SC lipid composition and organization mainly focusing on healthy and diseased human skin. In addition, an overview is provided on the data describing the relation between lipid modulations and the impaired skin barrier function. Finally, the use of in vitro lipid models for a better understanding of the relation between the lipid composition, lipid organization and skin lipid barrier is discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Isolation,identification, and quantification of Pentylcurcumene from Geophila repens: A new class of cholinesterase inhibitor for Alzheimer’s disease

The aerial part of Geophila repens (L.) I.M. Johnst (Rubiaceae) has been used in India to improve intelligence and memory for a long time. As part of our ongoing efforts in discovering potential bioactive compounds from G. repens, we have studied the isolation, identification, and quantification of a new class of cholinesterase inhibitor from G. repens for Alzheimer’s disease (AD). Terpene was isolated from hydroalcohol extract of G. repens (GRHA) and its structure was identified “Pentylcurcumene” by spectroscopic data. HPTLC fingerprint analysis was performed and good separation was achieved in mobile phase (benzene:methanol; 7.5:2.5, v/v, 254 and 366 nm; R_f 0.51). The method was validated using ICH guidelines in terms of linearity, specificity, sensitivity, accuracy, precision, robustness and stability. In cellular antioxidant studies e.g. DPPH, oxygen-radical-absorbance-capacity (ORAC) and cell-based-antioxidant-protection-in-erythrocytes (CAP-e) assays showed that, Pentylcurcumene showed remarkably different degrees of antioxidant activities in dose-dependent manner. Pentylcurcumene demonstrated anticholinesterase activities e.g. IC₅₀ of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition were 73.12 ± 0.56 and 97.65 ± 0.46 μg/ml, respectively. To better understand enzyme kinetics, Lineweaver-Burk plot of Pentylcurcumene displayed the highest affinity with competitive inhibition (reversible) towards both AChE (V_max 0.8) and BChE (V_max 0.6). An improved and advanced HPTLC tool of bioautography detection of Pentylcurcumene has been successfully demonstrated its anticholinesterase activities. Molecular docking simulations of Pentylcurcumene (ligand) and enzymes (proteins) exhibited the binding of ligand at active sites of AChE (human/rat) and BChE (human/homology) efficiently and also predicted the hydrophobic interaction of drug towards different amino acid residue within proteins. As per the results of antioxidant study and with the support of molecular docking analysis, it is concluded that Pentylcurcumene could be a potential first-line cholinesterase-inhibitor for AD.