Global DNA methylation profiling in peripheral blood cells of South African women with gestational diabetes mellitus

Background/Objective: Recently, several studies have reported that DNA methylation changes in tissue are reflected in blood, sparking interest in the potential use of global DNA methylation as a biomarker for gestational diabetes mellitus (GDM). This study investigated whether global DNA methylation is associated with GDM in South African women.

Methods: Global DNA methylation was quantified in peripheral blood cells of women with (n?=?63) or without (n?=?138) GDM using the MDQ1 Imprint® DNA Quantification Kit.

Results: Global DNA methylation levels were not different between women with or without GDM and were not associated with fasting glucose or insulin concentrations. However, levels were 18% (p?=?0.012) higher in obese compared to non-obese pregnant women and inversely correlated with serum adiponectin concentrations (p?=?0.005).

Discussion: Contrary to our hypothesis, global DNA methylation was not associated with GDM in our population. These preliminary findings suggest that despite being a robust marker of overall genomic methylation that offers opportunities as a biomarker, global DNA methylation profiling may not offer the resolution required to detect methylation differences in the peripheral blood cells of women with GDM. Moreover, global DNA methylation in peripheral blood cells may not reflect changes in placental tissue. Further studies in a larger sample are required to explore the candidacy of a more targeted approach using gene-specific methylation as a biomarker for GDM in our population.     

Brief occlusion of the main pancreatic duct rapidly initiates signals which lead to increased duct cell proliferation in the rat

In the course of investigating the signals associated with pancreas regeneration, we have developed a method to initiate pancreatic duct cell proliferation by brief occlusion of the main pancreatic duct. The resulting duct cell proliferation, induced by temporary partial main duct occlusion, was compared to that induced by firmly tying a cellophane strip around the head of the pancreas for longer periods of time. Both methods stimulated a biphasic increase in duct cell proliferation, with proliferation maxima at 3 and 14 days post operation. The short duration of temporary main duct occlusion (60 s) that was needed to stimulate duct cell proliferation, and the similar duct cell proliferation profiles that were observed after both the temporary and the longer term main duct occlusion, led us to conclude that the signals which initiate proliferation occur rapidly at the beginning of each procedure.     

Isolation and structure elucidation of new radical oxidation products of 5-hydroxy steroids

Three new products have been isolated from the lead-tetraacetate version of the hypoiodite oxidation of 3beta,17beta-diacetoxy-5-hydroxy-5 alpha-androstane. Along with the expected 1(10)-unsaturated 5,10-seco steroidal 5-ketones, the fragmentation reaction gave two epimeric C-4 iodides. Their structural assignment was based on X-ray data of one of them ((4R,10S)-4-iodo-3beta,17beta-diacetoxy-5,10-secoandrostan-5-one). The third new product was found to be the 5 beta,6 beta-epoxide resulting from the dehydration of the tertiary alcohol followed by epoxidation of the intermediate Delta(5)-olefin.     

The effect of polysaccharide-degrading wine yeast transformants on the efficiency of wine processing and wine flavour

Commercial polysaccharase preparations are applied to winemaking to improve wine processing and quality. Expression of polysaccharase-encoding genes in Saccharomyces cerevisiae allows for the recombinant strains to degrade polysaccharides that traditional commercial yeast strains cannot. In this study, we constructed recombinant wine yeast strains that were able to degrade the problem-causing grape polysaccharides, glucan and xylan, by separately integrating the Trichoderma reesei XYN2 xylanase gene construct and the Butyrivibrio fibrisolvens END1 glucanase gene cassette into the genome of the commercial wine yeast strain S. cerevisiae VIN13. These genes were also combined in S. cerevisiae VIN13 under the control of different promoters. The strains that were constructed were compared under winemaking conditions with each other and with a recombinant wine yeast strain expressing the endo-beta-1,4-glucanase gene cassette (END1) from B. fibrisolvens and the endo-beta-1,4-xylanase gene cassette (XYN4) from Aspergillus niger, a recombinant strain expressing the pectate lyase gene cassette (PEL5) from Erwinia chrysanthemi and the polygalacturonase-encoding gene cassette (PEH1) from Erwinia carotovora. Wine was made with the recombinant strains using different grape cultivars. Fermentations with the recombinant VIN13 strains resulted in significant increases in free-flow wine when Ruby Cabernet must was fermented. After 6 months of bottle ageing significant differences in colour intensity and colour stability could be detected in Pinot Noir and Ruby Cabernet wines fermented with different recombinant strains. After this period the volatile composition of Muscat d'Alexandria, Ruby Cabernet and Pinot Noir wines fermented with different recombinant strains also showed significant differences. The Pinot Noir wines were also sensorial evaluated and the tasting panel preferred the wines fermented with the recombinant strains.     

Rationale for adjuvant fatty acid therapy to prevent radiotherapy failure and tumor recurrence during early laryngeal squamous cell carcinoma

Information from a preceding lipid study contributed to the pathobiological assessment of laryngeal squamous cell carcinoma (LSCC). Lipid-driven signaling pathways are responsible for laryngeal carcinogenesis and immunodeficiency. The construction of fatty acid (FA) profiles for LSCC allowed the identification of FA role players. The integration of lipid and clinicomolecular information encountered in the literature, in turn, allowed the identification of biological prognostic markers to distinguish between early (less aggressive) and advanced (more aggressive) LSCCs. High arachidonic acid (AA) and cyclooxygenase (COX-2) activities are criteria for less aggressive growth, whilst low AA and COX-2 activities occur during more aggressive growth. Excessive tobacco use and environmental smoke or human papillomavirus (HPV) infection and alcohol abuse can, respectively, elicit cumulative oxidative stress and an oxidative burst or interfere with signaling pathways during essential fatty acid (EFA) metabolism, all factors and events which may cause LSCC. Research revealed that enhanced COX-2 activity and Bcl-2 expression prevent apoptosis and, hence, LSCCs become resistant to radiotherapy. It was also observed that recurrent laryngeal cancers become more aggressive after radiotherapy failure. It is predicted that manipulation of AA activity and consequently a cascade of downstream factors that include COX-2 and Bcl-2 expression responsible for LSCC may have therapeutic potential to improve radiotherapy outcome during early LSCC. Adjuvant FA therapy to improve early LSCC management by counteracting radiotherapy failure and unwanted complications for further management is proposed. FA therapeutic strategies before and during radiotherapeutic courses need to be evaluated.     

Selective extraction of Cyclopia for enhanced in vitro phytoestrogenicity and benchmarking against commercial phytoestrogen extracts

Previous work established the phytoestrogenicity of "unfermented"Cyclopia (honeybush) extracts. The current study investigated the phytoestrogenicity of four Cyclopia harvestings (M6-9) for preparation of extracts with enhanced phytoestrogenicity for benchmarking against commercial preparations. Two extracts, from M6 (C. subternata) and M7 (C. genistoides), were identified as most phytoestrogenic using estrogen receptor binding, an estrogen receptor response element containing promoter reporter assay, alkaline phosphatase activity, and E-screen. M6 and M7 were sequentially and non-sequentially extracted with five solvents of differing polarities. Additionally, two extracts were prepared in the traditional way of preparing a cup of honeybush tea. The resultant 22 extracts were evaluated for estrogenicity. Select extracts were analysed by high-pressure liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS). The sequentially extracted M6 methanol extract (SM6Met) had the highest potency and the sequentially extracted M6 ethyl acetate extract (SM6EAc) had the highest efficacy of all the extracts. The HPLC results suggested enrichment of luteolin in SM6EAc and enrichment of an unidentified polyphenol in SM6Met. Benchmarking against four commercial phytoestrogenic preparations suggest that in terms of the assays used, Cyclopia extracts have comparable potency and efficacy to the commercial extracts and thus have potential as marketable phytoestrogenic nutraceuticals.     

Presence of a family of plasmids (29 to 65 kilobases) with a 26-kilobase common region in different strains of the sulfur-oxidizing bacterium Acidithiobacillus caldus

Three large cryptic plasmids from different isolates of Acidithiobacillus caldus were rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South African A. caldus strain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain "f," South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid "backbone" containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21 subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.     

The binding of snake venom cardiotoxins to heart cell membranes

Cobra venom cardiotoxins have the effect, inter alia, of causing systolic arrest of the heart. We have observed significant binding in vitro of ³⁵S-labelled cardiotoxins to mouse heart cell membranes. Part of the binding was saturable and could be displaced with homologous unlabelled cardiotoxins but not by neurotoxins or cardiotoxins inactivated by chemical modification. The specifically bound component represented more than 70% of total binding at saturation. Inclusion of Triton X-100 and NaCl in the phosphate-buffered incubation medium prevented nonspecific adsorption to centrifuge tube walls, and gave lower but more reproducible specific binding results, respectively. An apparent dissociation constant of 5·10^?7 M and a binding density of 500 pmol toxin/mg membrane protein were derived from the saturation isotherms.     

Electron Dense Artefactual Deposits in Tissue Sections: The Role of Ethanol, Uranyl Acetate and Phosphate Buffer

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.