Quaternary salts of 4,3' and 4,4' bis-pyridinium monooximes: synthesis and biological activity

Six unsymmetrical bis-quaternary monooximes viz. dibromides of 1-(4-hydroxyiminomethyl pyridinium)-3-(3/4-carbamoyl pyridinium)propane, 1-(4-hydroxyiminomethyl pyridinium)-4-(3/4-carbamoyl pyridinium) butane, 1-(4-hydroxyiminomethyl pyridinium)-5-(3/4-carbamoyl pyridinium)pentane were synthesized and characterized by spectral data. Their ability to reactivate tetraethyl pyrophosphate inhibited mouse total brain cholinesterase was investigated and compared with 2-pyridine aldoxime chloride (2-PAM). All the compounds were found to be more effective acetylcholinesterase reactivators when compared with the conventional oxime, 2-PAM, except the compound (5a) with pentylene bridge and carbamoyl group present at fourth position. The bis-pyridinium monooximes with 3-carbamoyl group were more potent reactivators than the corresponding 4-carbamoyl compounds and bis-oximes tested.     

A new triterpenoid from the fern Adiantum lunulatum and evaluation of antibacterial activity

A new triterpenoid, 22,29xi-epoxy-30-norhopane-13beta-ol (1) was isolated together with six known compounds viz., fern-9(11)-en-6alpha-ol. fern-9(11)-ene, fern-9(11)-en-25-oic acid, fern-9(11)-en-28-ol, filicenol-B, adiantone and oxidation product of fern-9(11)-en-6alpha-ol obtained as 6-oxofern-9(11)-ene from the whole plant of Adiantum lunulatum, and their structures were elucidated by means of spectroscopic analysis and antibacterial evaluation of these compounds were conducted.     

Synthesis and biological activity of isoamoenylin,a metabolite of Dendrobium amoenum

Isoamoenylin (6), a dihydrostilbene from Dendrobium amoenum, was synthesised from 3,4,5-trimethoxybenzaldehyde (1) in four steps with an overall yield of 60%. The spectral data for synthetic 6 are in good agreement with those of the natural product. Isoamoenylin showed moderate antioxidative and weak antibacterial activities.     

Metabolites and analogs of 1alpha,25-dihydroxyvitamin D(3): evaluation of actions in bone

Analogs of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] activate both genomic mechanisms via the nuclear vitamin D(3) receptor (nVDR) and nongenomic pathways via the plasma membrane vitamin D(3) receptor (pmVDR). Both of these pathways are normally activated by 1alpha,25(OH)(2)D(3), but as a result of synthesis of numerous analogs of 1alpha,25(OH)(2)D(3) these pathways can be distinguished. We used increasing doses of vitamin D(3) analogs to determine their potencies of action on these two distinct pathways, measuring calcium channel potentiation as an indicator of the nongenomic action and measuring increases in osteocalcin mRNA and protein release and bone resorption as indicators of genomic action. We found that both 25(OH)-16,23E-diene-D(3) (R) and 1alpha,25(OH)(2)-16,23E-diene-D(3) (A) are 10-fold more potent than 1alpha,25(OH)(2)D(3) for activation of the nongenomic pathway because double bonds in the side chain and the D ring increase the affinity for calcium channel potentiation. While the C-1alpha-hydroxyl group is not necessary for potentiation of calcium channels, methyl groups at this position can alter the affinity for calcium channel potentiation. On the other hand, 1000 fold higher concentrations of nongenomic analogs were needed compared to 1alpha,25(OH)(2)D(3) to increase osteocalcin mRNA or protein release. 1alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorovitamin D(3), (E) is an agent that is 10 fold more potent than 1alpha,25(OH)(2)D(3) at increasing osteocalcin mRNA and protein release, whereas 1alpha,25(OH)(2)-3-epi-D(3) increases osteocalcin mRNA and protein with a potency over 10 fold lower than 1alpha,25(OH)(2)D(3). These results suggest that double bonds in the side chain and the D ring stabilize action on the nongenomic pathway whereas F(6) on the terminal portion of the side chain increases potency for nVDR. On the other hand, while the C-1alpha-hydroxyl group is necessary for activation of genomic events via nVDR, the activation of nongenomic events occurs in the absence of this group.     

Generation of a complete, soluble, and catalytically active sterol 14 alpha-demethylase-reductase complex.

Sterol 14 alpha-demethylation is one of the key steps of sterol biosynthesis in eukaryotes and is catalyzed by cytochrome P450 sterol 14 alpha-demethylase (other names being CYP51 and P45014DM) encoded by ERG11. This enzyme activity is supported by an associated NAPDH-dependent reductase encoded by NCPR1 (NCP1), which is also associated with the endoplasmic reticulum. A diglycine linker recognition site (Gly-Gly-Ile-Glu-Gly-Arg-Gly-Gly) for the protease factor Xa, also containing a thrombin recognition site, was inserted just beyond the N-terminal hydrophobic segment of Candida albicans Erg11p. This modified enzyme was heterologously expressed at a level of 2.5 nmol of Erg11p/mg of protein as an integral endoplasmic reticulum protein. Following purification, treatment of the modified protein with factor Xa or thrombin resulted in sequence-specific cleavage and production of a soluble N-terminal truncated Erg11p which exhibited spectral characteristics identical to those of the purified full-length, wild-type form. Furthermore, reconstitution of the soluble enzyme with soluble yeast Ncpr1p, expressed and purified as an N-terminal deletion of 33 amino acids encompassing its membrane anchor, resulted in a fully functional and soluble eukaryotic Erg11p system. The complex was disrupted by high-salt concentration, reflecting the importance of electrostatic forces in the protein-protein interaction. The results demonstrate the membrane anchor serves to localize Erg11p to the ER where the substrate is located, but is not essential in either Ncpr1p or Erg11p activity. The possibility of cocrystallization of an active soluble eukaryotic 14 alpha-demethylase can be envisaged.     

Molecular Characterisation of Small Molecule Agonists Effect on the Human Glucagon Like Peptide-1 Receptor Internalisation

The glucagon-like peptide receptor (GLP-1R), which is a G-protein coupled receptor (GPCR), signals through both Gαs and Gαq coupled pathways and ERK phosphorylation to stimulate insulin secretion. The aim of this study was to determine molecular details of the effect of small molecule agonists, compounds 2 and B, on GLP-1R mediated cAMP production, intracellular Ca²⁺ accumulation, ERK phosphorylation and its internalisation. In human GLP-1R (hGLP-1R) expressing cells, compounds 2 and B induced cAMP production but caused no intracellular Ca²⁺ accumulation, ERK phosphorylation or hGLP-1R internalisation. GLP-1 antagonists Ex(9–39) and JANT-4 and the orthosteric binding site mutation (V36A) in hGLP-1R failed to inhibit compounds 2 and B induced cAMP production, confirming that their binding site distinct from the GLP-1 binding site on GLP-1R. However, K334A mutation of hGLP-1R, which affects Gα_s coupling, inhibited GLP-1 as well as compounds 2 and B induced cAMP production, indicating that GLP-1, compounds 2 and B binding induce similar conformational changes in the GLP-1R for Gα_s coupling. Additionally, compound 2 or B binding to the hGLP-1R had significantly reduced GLP-1 induced intracellular Ca²⁺ accumulation, ERK phosphorylation and hGLP-1R internalisation. This study illustrates pharmacology of differential activation of GLP-1R by GLP-1 and compounds 2 and B.     

Determination of total fluorine in serum and other biological materials by oxygen bomb and reverse extraction techniques.

A procedure is described to determine submicrogram amounts of total fluorine in biological materials by the oxygen bomb technique. The fluoride in the bomb washings is determined with the fluoride ion electrode, when necessay, following reverse extraction of fluoride. This procedure averts the hazards of loss of fluorine and contamination with extraneous fluoride encountered in the open ashing procedures.     

Subcellular fractionation enhances proteome coverage of pancreatic duct cells

ObjectivesSubcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increase proteome coverage.MethodsWe used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared – via mass spectrometry-based analysis – the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates.ResultsWe identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle.ConclusionsSubcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease.     

Requirement of Nse1, a subunit of the Smc5-Smc6 complex, for Rad52-dependent postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, postreplication repair (PRR) of UV-damaged DNA occurs by a Rad6-Rad18- and an Mms2-Ubc13-Rad5-dependent pathway or by a Rad52-dependent pathway. The Rad5 DNA helicase activity is specialized for promoting replication fork regression and template switching; previously, we suggested a role for the Rad5-dependent PRR pathway when the lesion is located on the leading strand and a role for the Rad52 pathway when the lesion is located on the lagging strand. In this study, we present evidence for the requirement of Nse1, a subunit of the Smc5-Smc6 complex, in Rad52-dependent PRR, and our genetic analyses suggest a role for the Nse1 and Mms21 E3 ligase activities associated with this complex in this repair mode. We discuss the possible ways by which the Smc5-Smc6 complex, including its associated ubiquitin ligase and SUMO ligase activities, might contribute to the Rad52-dependent nonrecombinational and recombinational modes of PRR.     

Synthesis, structural revision, and antioxidant activities of antimutagenic homoisoflavonoids from Hoffmanosseggia intricata

Intricatinol and intricatin, the two homoisoflavonoids isolated from Hoffmanosseggia intricata, and two analogs have been synthesized from pyrogallol in three steps. The spectral data of synthetic intricatinol are in good agreement with those of natural metabolite, but the spectral data of intricatin are not corroborative with those of the natural product. The structure of intricatin has been thus revised to 8-methoxybonducellin, a compound isolated from Caesalpinia pulcherrima. The antioxidant activity of all the four homoisoflavonoids was determined by superoxide (NBT) and DPPH free radical scavenging methods. The synthetic analog 7,8-dihydroxy-3-[(3,4-dihydroxyphenyl)methylene]chroman-4-one displayed excellent activity in both methods.