No abstract

Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the ‘repeated’ adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3‐α‐helix, raft‐like structure. Each pair of α‐helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel ‘tyrosine fork’ structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR.

Apocynin (4-hydroxy-3-methoxyacetophenone) is a major active ingredient from the rhizomes of Picrorhiza kurroa, a botanical plant used as an herbal medicine for treatment of a number of inflammatory diseases. Recently, apocynin is regarded as a specific inhibitor for NADPH oxidase in cell and animal models. In vitro studies indicated conversion of apocynin to diapocynin in the presence of peroxidases, e.g., myloperoxidase, posing the possibility that diapocynin also contributes to the anti-oxidative action of apocynin. The objectives of this study are to examine the bioavailability of apocynin to plasma, liver and brain tissue after intraperitoneal (i.p.) injection, and to examine whether apocynin is converted to diapocynin in vivo. Diapocynin was chemically synthetized and characterized by NMR and IR. Apocynin (5 mg/kg body wt) was injected i.p. to adult male Sprague-Dawley rats and plasma, liver and brain were collected at different times (30 min, 1 h and 2 h) after injection. Samples were treated with β-glucuronidase to hydrolyze the glycosyl linkage and analyzed by HPLC/MS. At 30 min and 1 h after injection, approximately 50% of apocynin was converted to its glycosyl derivative and was distributed in plasma, liver and brain. No diapocynin was detected in any samples. These results indicate rapid glycosylation of apocynin and its transport to blood and other organs but no apparent conversion to diapocynin in vivo.

Procedures based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and liquid chromatography-mass spectrometry (LC-MS) are described for analyzing diapocynin. Diapocynin was synthesized by oxidative coupling of two apocynin monomers, through the in situ generation of sulfate radicals. It was purified by washing 3 times each with boiling water, followed by boiling methanol. HPLC was used to determine the concentration of unreacted apocynin and other impurities and the purity of the diapocynin that had been synthesized. Negative-ion, atmospheric pressure chemical ionization (APCI) LC-MS was used to determine the molecular weights of impurities. The method using HPLC with UV detection provided a calibration curve that was linear from 0.16 to 24 microg/mL. The LC-MS method was linear from 0.005 to 2 microg/mL. It was found that diapocynin has low solubility in deionized water and corn oil but is soluble in dimethylsulfoxide (DMSO) and alkaline aqueous solutions. Also, diapocynin is 13 times more lipophilic than apocynin, even though both compounds have the same p K a of 7.4. The log of the octanol/water partition coefficient (log P) was 1.01 for apocynin and 1.82 for diapocynin. A solution of 5.5 mg/mL (16.7 mM) diapocynin in DMSO was found to be stable for at least 30 days when stored at room temperature.

Dietary supplements containing plant materials of Annonaceae species (Annona muricata L., A. squamosa L., A. mucosa JACQ., A. squamosa × cherimola Mabb.) were extracted by hot, pressurized ethyl acetate and analyzed for their effect in vitro on Lund human mesencephalic neurons. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell death was determined by lactate dehydrogenase levels. Three supplements strongly decreased the cell viability at extract concentrations of 1 µg/mL, of which 1 decreased cell viability at 0.1 µg/µL. Also, strong neuronal toxicities of these supplements were found. Cell death was observed at concentrations of 10 µg/mL. The degree of toxicity was comparable to the ones found in Annonaceous fruit extracts. Two fruit pulps of Annonaceae (A. muricata and A. squamosa) showed a reduction in cell viability at lower concentrations. The fruit pulp extract of A. muricata revealed the strongest neurotoxic effect, with 67% cell death at a concentration of 1 µg/mL. A high reduction in cell viability coupled with pronounced cell death was found at 0.1 µg/mL for an Annonaceous seed extract. These results demonstrate that the intake of dietary supplements containing plant material from Annonaceae may be hazardous to health in terms of neurotoxicity.