Seasonal analysis of the tropane alkaloid ecgonine methyl ester and the occurrence of other highly-valued tropanes in the South African <Emphasis Type="Italic">Erythroxylum</Emphasis> trees

The coca family (Erythroxylaceae) consists of trees and shrubs sub-divided into four genera: Aneulophus, Nectaropetalum, Pinacopodium, and Erythroxylum, which include species with highly valuable medicinal compounds. E. delagoense, E. emarginatum, and E. pictum are endemic to southern Africa and have great pharmaceutical potential based on their traditional uses. Previous studies have shown certain inconsistencies in terms of the presence or absence of tropane alkaloids in these species, resulting in a need for further research and clarification. Therefore, the aim of this study was to determine the seasonal variation of the immediate biosynthetic precursor of cocaine, the tropane alkaloid, ecgonine methyl ester in the three South African Erythroxylum species by means of gas chromatography–mass spectrometry, as well as to conduct a phytochemical screening for observing the presence of other potential compounds and tropane alkaloids. We found significant differences in tropane concentrations from the seasonal variation study, explaining the discrepancies in previous reports on its presence/absence in these species. Furthermore, we report for the first time on the occurrence of selected highly valuable tropane alkaloids in E. emarginatum currently used in ‘blockbuster medicine’.     

Ultrastructure and large subunit rDNA sequences of Lepidodinium viride reveal a close relationship to Lepidodinium chlorophorum comb. nov. (=Gymnodinium chlorophorum)

The ultrastructure of the green dinoflagellate Lepididodinium viride M. M. Watanabe, S. Suda, I. Inouye Sawaguchi et Chihara was studied in detail. The nuclear envelope possessed numerous chambers each furnished with a nuclear pore, a similar arrangement to that found in other gymnodinioids. The flagellar apparatus was essentially identical to Gymnodinium chlorophorum Elbrächter et Schnepf, a species also containing chloroplasts of chlorophyte origin. Of particular interest was the connection of the flagellar apparatus to the nuclear envelope by means of both a fiber and a microtubular extension of the R3 flagellar root. This feature has not been found in other dinoflagellates and suggests a close relationship between these two species. This was confirmed by phylogenetic analysis based on partial sequences of the large subunit (LSU) rDNA gene of L. viride, G. chlorophorum and 16 other unarmoured dinoflagellates, including both the ‘type’ culture and a new Tasmanian isolate of G. chlorophorum. These two isolates had identical sequences and differed from L. viride by only 3.75% of their partial LSU sequences, considerably less than the difference between other Gymnodinium species. Therefore, based on ultrastructure, pigments and partial LSU rDNA sequences, the genus Lepidodinium was emended to encompass L. chlorophorum comb. nov.     

Adhesion of the probiotic strains <Emphasis Type="Italic">Enterococcus mundtii</Emphasis> ST4SA and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> 423 to Caco-2 cells under conditions simulating the intestinal tract,and in the presence of antibiotics and anti-inflammatory medicaments

Adhesion of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 (human carcinoma epithelial) cells was visualized by fluorescent staining. Both strains showed good adhesion compared to L. casei MB1, L. casei Shirota, L. johnsonii La1 and L. rhamnosus GG. No correlation was found between hydrophobicity, aggregation and adhesion to Caco-2 cells. Presence of antibiotics and anti-inflammatory medicaments reduced adhesion of bacterial strains to Caco-2 cells. Proteins sensitive to pepsin, trypsin and pronase are involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. Adhesion of Listeria monocytogenes ScottA to Caco-2 cells was not prevented by E. mundtii ST4SA and L. plantarum 423. Cell-free culture supernatants of strains ST4SA and 423, containing the antimicrobial peptides plantaricin 423 and peptide ST4SA, prevented the invasion of L. monocytogenes ScottA into Caco-2 cells.     

Bioprospecting for novel oxylipins in fungi: The presence of 3-hydroxy oxylipins in Pilobolus

As previously found in various members of the Mucorales, 3-hydroxy oxylipins in Mucor genevensis are associated with the sporangia, i.e. mainly the columella structure and between aggregating sporangiospores. To determine if this phenomenon is also true in distantly related members, the mucoralean fungus Pilobolus was examined. This fungus is characterized by relatively large sub sporangial-columella structures which actively eject sporangia in a sticky liquid for attachment onto herbage surrounding its growth medium – in this case horse dung. Strikingly, this fungus produced a novel oxylipin i.e. a 3-hydroxy monounsaturated fatty acid, possibly a nonenoic acid, which is mainly associated with the sub sporangial-columella structure and aggregating sporangiospores. The specificity of the antibody against 3-hydroxy oxylipins used in immunofluorescence mapping of the mucoralean fungi, was further confirmed in the yeast, Saccharomycopsis malanga which produces 3-hydroxy palmitate in crystal form. These crystals occur between aggregating yeast cells. On the basis of the available data, we hypothesize that 3-hydroxy oxylipins probably function as adhesives, attaching fungal cells to each other or to other surfaces through entropic based hydrophobic forces and/or hydrogen bonds.     

The presence of novel 3-hydroxy oxylipins on surfaces of hat-shaped ascospores of Ascoidea africana Batra & Francke-Grosmann

Immunofluorescence microscopy exposed the presence of novel 3-hydroxy oxylipins on the surfaces of aggregated hat-shaped ascospores of Ascoidea africana. These compounds were confirmed by gas chromatography - mass spectrometry analysis. Only the complete structure of a novel 3-hydroxy 10:1 could be determined.     

Oxylipin covered ascospores of Eremothecium coryli

Eremothecium coryli is known to produce intriguing spindle-shaped ascospores with long and thin whip-like appendages. Here, ultra structural studies using scanning electron microscopy, indicate that these appendages serve to coil around themselves and around ascospores causing spore aggregation. Furthermore, using immunofluorescence confocal laser scanning microscopy it was found that hydrophobic 3-hydroxy oxylipins cover the surfaces of these ascospores. Using gas chromatography–mass spectrometry, only the oxylipin 3-hydroxy 9:1 (a monounsaturated fatty acid containing a hydroxyl group on carbon 3) could be identified. Sequential digital imaging suggests that oxylipin-coated spindle-shaped ascospores are released from enclosed asci probably by protruding through an already disintegrating ascus wall.     

Acetylsalicylic acid as antifungal in <Emphasis Type="Italic">Eremothecium</Emphasis> and other yeasts

Interesting distribution patterns of acetylsalicylic acid (ASA, aspirin) sensitive 3-hydroxy (OH) oxylipins were previously reported in some representatives of the yeast genus Eremothecium—an important group of plant pathogens. Using immunofluorescence microscopy and 3-OH oxylipin specific antibodies in this study, we were able to map the presence of these compounds also in other Eremothecium species. In Eremothecium cymbalariae, these oxylipins were found to cover mostly the spiky tips of narrowly triangular ascospores while in Eremothecium gossypii, oxylipins covered the whole spindle-shaped ascospore with terminal appendages. The presence of these oxylipins was confirmed by chemical analysis. When ASA, a 3-OH oxylipin inhibitor, was added to these yeasts in increasing concentrations, the sexual stage was found to be the most sensitive. Our results suggest that 3-OH oxylipins, produced by mitochondria through incomplete β-oxidation, are associated with the development of the sexual stages in both yeasts. Strikingly, preliminary studies on yeast growth suggest that yeasts, characterized by mainly an aerobic respiration rather than a fermentative pathway, are more sensitive to ASA than yeasts characterized by both pathways. These data further support the role of mitochondria in sexual as well as asexual reproduction of yeasts and its role to serve as a target for ASA antifungal action.     

Isolation and characterization of NAD(P)H-dehydrogenases from seeds of the castor bean

Two pyridine nucleotide dehydrogenases have been isolated from castor bean seed extracts by a combination of ion exchange chromatography on DEAE-Sepharose and gel permeation chromatography on Sephadex G-200. The enzymes were designated D-I and D-II according to their elution position on DEAE-Sepharose. Both enzymes D-I and D-II are globular proteins which have MWs of 66 000 and 60 000, respectively. Dehydrogenation is observed with both NADH and NADPH as electron donors, while the electron acceptor specificity demonstrates that the enzymes are probably NAD(P)H: quinone oxidoreductases. Successful coupling of dehydrogenase activity with that of peroxidase indicates a possible role of the enzymes in seed germination.     

Factors Associated with Variations in Population HIV Prevalence across West Africa: Findings from an Ecological Analysis

Background

Population HIV prevalence across West Africa varies substantially. We assess the national epidemiological and behavioural factors associated with this.

Methods

National, urban and rural data on HIV prevalence, the percentage of younger (15–24) and older (25–49) women and men reporting multiple (2+) partners in the past year, HIV prevalence among female sex workers (FSWs), men who have bought sex in the past year (clients), and ART coverage, were compiled for 13 countries. An Ecological analysis using linear regression assessed which factors are associated with national variations in population female and male HIV prevalence, and with each other.

Findings

National population HIV prevalence varies between 0 4–2 9% for men and 0 4–5.6% for women. ART coverage ranges from 6–23%. National variations in HIV prevalence are not shown to be associated with variations in HIV prevalence among FSWs or clients. Instead they are associated with variations in the percentage of younger and older males and females reporting multiple partners. HIV prevalence is weakly negatively associated with ART coverage, implying it is not increased survival that is the cause of variations in HIV prevalence. FSWs and younger female HIV prevalence are associated with client population sizes, especially older men. Younger female HIV prevalence is strongly associated with older male and female HIV prevalence.

Interpretation

In West Africa, population HIV prevalence is not significantly higher in countries with high FSW HIV prevalence. Our analysis suggests, higher prevalence occurs where more men buy sex, and where a higher percentage of younger women, and older men and women have multiple partnerships. If a sexual network between clients and young females exists, clients may potentially bridge infection to younger females. HIV prevention should focus both on commercial sex and transmission between clients and younger females with multiple partners.