Alpha-glucosidase inhibitory and antiplasmodial properties of terpenoids from the leaves of Buddleja saligna Willd

In our continuing search for biologically active natural product(s) of plant origin, Buddleja saligna, a South African medicinal plant, was screened in line with its traditional use for antidiabetic (yeast alpha glucosidase inhibitory) and antiplasmodial (against a chloroquine sensitive strain of Plasmodium falciparum (NF54)) activities. The hexane fraction showed the most promising activity with regards to its antidiabetic (IC₅₀?=?260?±?0.112?µg/ml) and antiplasmodial (IC₅₀?=?8.5?±?1.6?µg/ml) activities. Using activity guided fractionation three known terpenoids (betulonic acid, betulone and spinasterol) were isolated from this species for the first time. The compounds displayed varying levels of biological activities (antidiabetic: 27.31?µg/ml?≥?IC₅₀?≥?5.6?µg/ml; antiplasmodial: 14?µg/ml?≥?IC₅₀?≥?2?µg/ml) with very minimal toxicity.     

Vibrational and electronic circular dichroism study of the interactions of cationic porphyrins with (dG-dC)10 and (dA-dT)10

The interactions of two different porphyrins, without axial ligands-5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin-Cu(II) tetrachloride (Cu(II)TMPyP) and with bulky meso substituents-5,10,15,20-tetrakis(N,N,N-trimethylanilinium-4-yl)porphyrin tetrachloride (TMAP), with (dG-dC)10 and (dA-dT)10 were studied by combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopy at different [oligonucleotide]/[porphyrin] ratios, where [oligonucleotide] and [porphyrin] are the concentrations of oligonucleotide per base-pair and porphyrin, respectively. The combination of VCD and ECD spectroscopy enables us to identify the types of interactions, and to specify the sites of interactions: The intercalative binding mode of Cu(II)TMPyP with (dG-dC)(10), which has been well described, was characterized by a new VCD "marker" and it was shown that the interaction of Cu(II)TMPyP with (dA-dT)10 via external binding to the phosphate backbone and major groove binding caused transition from the B to the non-B conformer. TMAP interacted with the major groove of (dG-dC)10, was semi-intercalated into (dA-dT)10, and caused significant variation in the structure of both oligonucleotides at the higher concentration of porphyrin. The spectroscopic techniques used in this study revealed that porphyrin binding with AT sequences caused substantial variation of the DNA structure. It was shown that VCD spectroscopy is an effective tool for the conformational studies of nucleic acid-porphyrin complexes in solution.     

Impact of an extruded nucleotide on cleavage activity and dynamic catalytic core conformation of the hepatitis delta virus ribozyme

The self-cleaving hepatitis delta virus (HDV) ribozyme is essential for the replication of HDV, a liver disease causing pathogen in humans. The catalytically critical nucleotide C75 of the ribozyme is buttressed by a trefoil turn pivoting around an extruded G76. In all available crystal structures, the conformation of G76 is restricted by stacking with G76 of a neighboring molecule. To test whether this crystal contact introduces a structural perturbation into the catalytic core, we have analyzed approximately 200 ns of molecular dynamics (MD) simulations. In the absence of crystal packing, the simulated G76 fluctuates between several conformations, including one wherein G76 establishes a perpendicular base quadruplet in the major groove of the adjacent P1 stem. Second-site mutagenesis experiments suggest that the identity of the nucleotide in position 76 (N76) indeed contributes to the catalytic activity of a trans-acting HDV ribozyme through its capacity for hydrogen bonding with P1. By contrast, in the cis-cleaving genomic ribozyme the functional relevance of N76 is less pronounced and not correlated with the P1 sequence. Terbium(III) footprinting and additional MD show that the activity differences between N76 mutants of this ribozyme are related instead to changes in average conformation and modified cross-correlations in the trefoil turn.     

B3LYP/6-311++G* * study of structure and spin-spin coupling constant in heparin disaccharide

Structures of heparin disaccharide have been analyzed by DFT using the B3LYP/6-311++G( * *) method. The optimized geometries of two forms of this disaccharide, differing in the conformation ((1)C(4) and (2)S(0)) of the IdoA2S residue, confirmed considerable influences of the sulfate and the carboxylate groups upon the pyranose ring geometries. The computed energies showed that disaccharide having the (1)C(4) form of the IdoA2S residue is more stable than that with the (2)S(0) form. Interatomic distances, bond and torsion angles showed that interconversion of the IdoA2S residue results in geometry changes in the GlcN,6S residue as well. Three-bond proton-proton and proton-carbon spin-spin coupling constants computed for both forms agree with the experimental data and indicate that only two chair forms contribute to the conformational equilibrium in disaccharide. Influences of the charged groups upon the magnitudes of spin-spin coupling constants are also discussed.     

Genetic analysis of autosomal and X-linked markers across a mouse hybrid zone

In this paper, we present results of the first comprehensive study of the introgression of both autosomal and sex-chromosome markers across the central European portion of the hybrid zone between two house mouse subspecies, Mus musculus musculus and M. m. domesticus. More than 1800 individuals sampled from 105 sites were analyzed with a set of allozyme loci (hopefully representing neutral or nearly neutral markers) and X-linked loci (which are assumed to be under selection). The zone center is best modeled as a single straight line independent of fine-scale local geographic or climatic conditions, being maintained by a balance between dispersal and selection against hybrids. The width (w) of the multilocus autosomal cline was estimated as 9.6 km whereas the estimate for the compound X-chromosome cline was about 4.6 km only. As the former estimate is comparable to that of the Danish portion of the zone (assumed to be much younger than the central European one), zone width does not appear to be related to its age. The strength (B) of the central barrier was estimated as about 20 km; with dispersal (sigma) of about 1 km/gen(1/2), this means effective selection (s*) is approximately 0.06-0.09 for autosomal loci and about 0.25 for X-linked loci. The number of loci under selection was estimated as N= 56-99 for autosomes and about 380 for X-linked loci. Finally, we highlight some potential pitfalls in hybrid zone analyses and in comparisons of different transects. We suggest that conclusions about parts of the mouse genome involved in reproductive isolation and speciation should be drawn with caution and that analytical approaches always providing some estimates should not be used without due care regarding the support or confidence of such estimates, especially if conclusions are based on the difference between these estimates. Finally, we recommend that analysis in two-dimensional space, dense sampling, and rigorous treatment of data, including inspection of likelihood profiles, are essential for hybrid zone studies.     

An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells

Recent studies demonstrated the importance of the mitochondrial ATP in the regulation of a novel long-chain fatty acid generation/export system in mitochondria of diabetic rat heart. In steroidogenic systems, mitochondrial ATP and intramitochondrial arachidonic acid (AA) generation are important for steroidogenesis. Here, we report that mitochondrial ATP is necessary for the generation and export of AA, steroid production and steroidogenic acute regulatory protein induction supported by cyclic 3'-5'-adenosine monophosphate in steroidogenic cells. These results demonstrate that ATP depletion affects AA export and provide new evidence of the existence of the fatty acid generation and export system involved in mitochondrial cholesterol transport.     

Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.     

Genetic basis for the biosynthesis of methylglucose lipopolysaccharides in Mycobacterium tuberculosis

Mycobacteria produce two unusual polymethylated polysaccharides, the 6-O-methylglucosyl-containing lipopolysaccharides (MGLP) and the 3-O-methylmannose polysaccharides, which have been shown to regulate fatty acid biosynthesis in vitro. A cluster of genes dedicated to the synthesis of MGLP was identified in Mycobacterium tuberculosis and Mycobacterium smegmatis. Overexpression of the putative glycosyltransferase gene Rv3032 in M. smegmatis greatly stimulated MGLP production, whereas the targeted disruption of Rv3032 in M. tuberculosis and that of the putative methyltransferase gene MSMEG2349 in M. smegmatis resulted in a dramatic reduction in the amounts of MGLP synthesized and in the accumulation of precursors of these molecules. Disruption of Rv3032 also led to a significant decrease in the glycogen content of the tubercle bacillus, indicating that the product of this gene is likely to be involved in the elongation of more than one alpha-(1-->4)-glucan in this bacterium. Results thus suggest that Rv3032 encodes the alpha-(1-->4)-glucosyltransferase responsible for the elongation of MGLP, whereas MSMEG2349 encodes the O-methyltransferase required for the 6-O-methylation of these compounds.     

Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi

The mode of action of xylanase A from a phytopathogenic bacterium, Erwinia chrysanthemi, classified in glycoside hydrolase family 5, was investigated on xylooligosaccharides and polysaccharides using TLC, MALDI-TOF MS and enzyme treatment with exoglycosidases. The hydrolytic action of xylanase A was found to be absolutely dependent on the presence of 4-O-methyl-D-glucuronosyl (MeGlcA) side residues in both oligosaccharides and polysaccharides. Neutral linear beta-1,4-xylooligosaccharides and esterified aldouronic acids were resistant towards enzymatic action. Aldouronic acids of the structure MeGlcA(3)Xyl(3) (aldotetraouronic acid), MeGlcA(3)Xyl(4) (aldopentaouronic acid) and MeGlcA(3)Xyl(5) (aldohexaouronic acid) were cleaved with the enzyme to give xylose from the reducing end and products shorter by one xylopyranosyl residue: MeGlcA(2)Xyl(2), MeGlcA(2)Xyl(3) and MeGlcA(2)Xyl(4). As a rule, the enzyme attacked the second glycosidic linkage following the MeGlcA branch towards the reducing end. Depending on the distribution of MeGlcA residues on the glucuronoxylan main chain, the enzyme generated series of shorter and longer aldouronic acids of backbone polymerization degree 3-14, in which the MeGlcA is linked exclusively to the second xylopyranosyl residue from the reducing end. Upon incubation with beta-xylosidase, all acidic hydrolysis products of acidic oligosaccharides and hardwood glucuronoxylans were converted to aldotriouronic acid, MeGlcA(2)Xyl(2). In agreement with this mode of action, xylose and unsubstituted oligosaccharides were essentially absent in the hydrolysates. The E. chrysanthemi xylanase A thus appears to be an excellent biocatalyst for the production of large acidic oligosaccharides from glucuronoxylans as well as an invaluable tool for determination of the distribution of MeGlcA residues along the main chain of this major plant hemicellulose.