Francois H. van der Westhuizen

Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells

The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and apoptosis. HeLa cells were titrated with rotenone, resulting in dose-dependent decrease in complex I activity and elevated ROS production at activities lower than 33%. Expression of MT2A (MT isoform 2A), but not MT1A or MT1B RNA, was significantly inducible by rotenone (up to 7-fold), t-BHP (t-butyl hydroperoxide; 5-fold) and CdCl2 (50-fold), but not ZnCl2. Myxothiazol treatment did not elevate either ROS or MT2A levels, which supports a ROS-related mechanism for rotenone-induced MT2A expression. To evaluate the role of MT2A expression, MT2A and MT1B were overexpressed in HeLa cells and treated with rotenone. Compared with control and MT1B-overexpressing cells, ROS production was significantly lower and cell viability higher in MT2A-overexpressing HeLa cells when ROS production was enhanced by treatment with t-BHP. Mitochondrial membrane potential was noticeably less reduced in both MT-overexpressing cell lines. MT2A overexpression in rotenone-treated cells also significantly reduced or delayed apoptosis induction, as measured by caspase 3/7 activity and cytosolic nucleosome enrichment. We conclude that MT2A offers significant protection against the main death-causing consequences of rotenone-induced complex I deficiency in HeLa cells. Our results are in support of the protective role against oxidative stress ascribed to MTs and provide evidence that MT2A expression may be a beneficial downstream adaptive response in complex I-deficient cells.

Chemoprotective properties of rooibos (Aspalathus linearis), honeybush (Cyclopia intermedia) herbal and green and black (Camellia sinensis) teas against cancer promotion induced by fumonisin B1 in rat liver.

The chemoprotective properties of unfermented and fermented rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) herbal teas, and green and black teas (Camellia sinensis) were investigated against fumonisin B1 (FB1) promotion in rat liver utilizing diethylnitrosamine (DEN) as cancer initiator. The various teas differently affected the clinical chemical parameters associated with liver and kidney damage associated with FB1 suggesting specific FB1/iron/polyphenolic interactions. Green tea enhanced (P<0.05) the FB1-induced reduction of the oxygen radical absorbance capacity, while fermented herbal teas and unfermented honeybush significantly (P<0.05) decreased FB1-induced lipid peroxidation in the liver. The teas exhibited varying effects on FB1-induced changes in the activities of catalase, glutathione peroxidase (GPx) glutathione reductase (GR) as well as the glutathione (GSH) status. Unfermented rooibos and honeybush significantly (P<0.05) to marginally (P<0.1) reduced the total number of foci (>10microm), respectively, while all the teas reduced the relative amount of the larger foci. Fermentation seems to reduce the protective effect of the herbal teas. Differences in the major polyphenolic components and certain FB1/polyphenolic/tissue interactions may explain the varying effects of the different teas on the oxidative parameters, hepatotoxic effects and cancer promotion in rat liver.

OXPHOS gene expression and control in mitochondrial disorders

The cellular consequences of deficiencies of the mitochondrial OXPHOS system include a variety of direct and secondary changes in metabolite homeostasis, such as ROS, Ca(2+), ADP/ATP, and NAD/NADH. The adaptive responses to these changes include the transcriptional responses of nuclear and mitochondrial genes that are mediated by these metabolites, control of the mitochondria permeability transition pore, and a great variety of secondary signalling elements. Among the transcriptional responses reported over more than a decade using material harboring mtDNA mutations, deletions, or depletions, nuclear and mitochondrial DNA OXPHOS genes have mostly been up-regulated. However, it is evident from the limited data in a variety of disease models that expression responses are highly diverse and inconsistent. In this article, the mechanisms and controlling elements of these transcriptional responses are reviewed. In addition, the elements that need to be evaluated, in order to gain an improved perspective of the manner in which OXPHOS genes respond and impact on mitochondrial disease expression, are highlighted.

Glycaemic control improves fibrin network characteristics in type 2 diabetes – A purified fibrinogen model

Diabetic subjects have been shown to have altered fibrin network structures. One proposed mechanism for this is non-enzymatic glycation of fibrinogen due to high blood glucose. We investigated whether glycaemic control would result in altered fibrin network structures due to decreased fibrinogen glycation. Twenty uncontrolled type 2 diabetic subjects were treated with insulin in order to achieve glycaemic control. Twenty age- and body mass index (BMI)-matched non-diabetic subjects were included as a reference group. Purified fibrinogen, isolated from plasma samples was used for analysis. There was a significant decrease in fibrinogen glycation (6.81 to 5.02 mol glucose/mol fibrinogen) with a corresponding decrease in rate of lateral aggregation (5.86 to 4.62) and increased permeability (2.45 to 2.85 x 10(-8) cm(2)) and lysis rate (3.08 to 3.27 microm/min) in the diabetic subjects after glycaemic control. These variables correlated with markers of glycaemic control. Fibrin clots of non-diabetic subjects had a significantly higher ratio of inelastic to elastic deformation than the diabetic subjects (0.10 vs. 0.09). Although there was no difference in median fiber diameter between diabetic and non-diabetic subjects, there was a small increase in the proportion of thicker fibers in the diabetic samples after glycaemic control. Results from SDS-PAGE indicated no detectable difference in factor XIIIa-crosslinking of fibrin clots between uncontrolled and controlled diabetic samples. Diabetic subjects may have altered fibrin network formation kinetics which contributes to decreased pore size and lysis rate of fibrin clots. Achievement of glycaemic control and decreased fibrinogen glycation level improves permeability and lysis rates in a purified fibrinogen model.

Phytochemical contents and antioxidant capacities of two Aloe greatheadii var. davyana extracts.

Aloe greatheadii var. davyana (Asphodelaceae) is used among rural South African communities to treat arthritis, skin cancer, burns, eczema, psoriasis, digestive problems, high blood pressure and diabetes, despite very little supporting scientific evidence. Due to increased interest by both the scientific community and industry regarding the medicinal uses of this plant species, we identified, quantified and compared the phytochemical contents and antioxidant capacities of two extracts of A. greatheadii; a leaf gel extract (LGE) and a 95 % aqueous ethanol leaf gel extract (ELGE), using various modified extraction procedures, GC-MS and spectrophotometry. Apart from extensively characterizing this medicinal plant with regards to its organic acid, polyphenols/phenolic acid, alcohol, aldehyde, ketone, alkane, pyrimidine, indole, alkaloid, phytosterol, fatty acid and dicarboxylic acid contents and antioxidant capacities, we describe a modified extraction procedure for the purpose of general phytochemical characterization, and compare this to a 95 % aqueous ethanol extraction technique. From the results it is clear that A. greatheadii contains a variety of compounds with confirmed antioxidant capacity and other putative health benefits (such as blood glucose, cholesterol and cortisol lowering properties) relating to the prevention or treatment of diabetes, cardiovascular disease, cancer and hypertension. The results also indicate that separate ethyl acetate/diethyl ether and hexane extractions of the LGE, better serve for general phytochemical characterization purposes, and 95 % aqueous ethanol extraction for concentrating selective groups of health related compounds, hence justifying its use for biological in vivo efficacy studies.

Blood glutathione and subclinical atherosclerosis in African men: the SABPA study

BACKGROUND Sub-Saharan Africans face an increasing burden of hypertension and related cardiac and cerebrovascular morbidity and mortality, making the identification of factors leading to early vascular abnormalities imperative. METHODS We investigated the possible influence of the antioxidant glutathione (GSH) on early subclinical atherosclerosis in 63 hypertensive (aged 45.2 years) and 34 normotensive (aged 38.9 years; P < 0.001) nondiabetic African men. We measured ambulatory daytime systolic and diastolic blood pressure (SBP, DBP) as well as daytime mean arterial pressure (MAP), carotid intima-media thickness (CIMT), and calculated the cross-sectional wall area. We determined the reduced form of GSH in whole blood and blood glucose in serum. RESULTS Blood glucose (110 vs. 92 mg/dl; P < 0.001) and CIMT (0.75 vs. 0.61 mm; P < 0.001) were higher in hypertensives compared to normotensives. No significant difference existed for GSH. Associations in normotensives suggested the hypotensive effect of GSH after single (SBP: r = -0.35, P < or = 0.05; DBP: r = -0.37, P < or = 0.05; MAP: r = -0.38, P < or = 0.05) and multiple (SBP: B = -0.015, P < 0.05; DBP: B = -0.011, P < 0.05; MAP: B = -0.012, P < 0.05) regression analyses. In hypertensives, CIMT (B = -0.00027, P < 0.01) and cross-sectional wall area (CSWA) (B = -0.0066, P < 0.05) correlated negatively with GSH. These findings were consistent after excluding 10 human immunodeficiency virus (HIV)-positive hypertensive subjects. CONCLUSIONS In hypertensive African men, CIMT is negatively associated with GSH, suggesting a possible contributory role of attenuated GSH levels in the development of subclinical atherosclerosis.

The effect of glycaemic control on fibrin network structure of type 2 diabetic subjects

Diabetic subjects have been shown to have altered fibrin network structures. One possible cause may be fibrinogen glycation resulting in altered structure/function properties. We investigated the effect of glucose control on fibrinogen glycation and fibrin network structure in type 2 diabetes. Blood samples were taken from twenty uncontrolled diabetic subjects at baseline to determine the levels of fibrinogen glycation and fibrin network structures. The subjects were then treated with insulin until blood glucose control was achieved before end blood samples were taken. Twenty age- and BMI-matched non-diabetic subjects were included as a reference group. The diabetic subjects had significantly higher mean fibrinogen glycation at baseline than the non-diabetic subjects (7.84 vs. 3.89 mol glucose / mol fibrinogen; p < 0.001). This was significantly reduced during the intervention (7.84 to 5.24 mol glucose / mol fibrinogen; p < 0.0002) in the diabetic group. Both groups had high mean fibrinogen concentrations (4.25 and 4.02 g/l, diabetic and non-diabetic subjects respectively). There was no difference in fibrinogen concentration, porosity, compaction and kinetics of clot formation between the diabetic subjects and non-diabetic subjects at baseline, nor were there any changes during the intervention despite the reduced fibrinogen glycation. Fibrin network characteristics correlated well with fibrinogen but not with any markers of glycaemic control. Improved glycaemic control resulted in decreased fibrinogen glycation but not fibrinogen concentration. It seems as though porosity, compaction and kinetics of clot formation are more related to fibrinogen concentration than fibrinogen glycation in this model.

A significant decline in IGF-I may predispose young Africans to subsequent cardiometabolic vulnerability

OBJECTIVES Low serum IGF-I is an independent risk factor for diabetes and cardiovascular disease. These noncommunicable diseases are extremely common in urban black South Africans, but their IGF-I concentration is unknown. We aimed to compare serum IGF-I concentrations of African and Caucasian people, investigate their age-related IGF-I decline, and determine whether IGF-I could account, at least in part, for the high prevalence of noncommunicable diseases in black Africans. RESEARCH DESIGN AND METHODS This cross-sectional study involved 211 African and 316 Caucasian men and women (aged 20-70 yr). Fasting glucose, insulin, lipids, albumin, creatinine, liver enzymes, cotinine, high-sensitivity C-reactive protein, reactive oxygen species, IGF-I, blood pressure (BP), and pulse wave velocity were determined. RESULTS IGF-I was lower in Africans (P < 0.001) and in both ethnicities declined significantly by age quartiles (P < 0.001). In African men and women, IGF-I declined significantly from age quartile 1 to 2 (r = -0.65, P < 0.001), not seen in young Caucasian men and women (r = -0.08, P = 0.45; r = -0.10, P = 0.34). This was confirmed after adjustment for BP, insulin resistance, high-sensitivity C-reactive protein, cotinine, gamma-glutamyl transferase, and reactive oxygen species. Only young Africans showed significant negative correlations of IGF-I with BP, pulse wave velocity, and high-density lipoprotein cholesterol. CONCLUSIONS Africans presented lower IGF-I levels than Caucasians due to an accelerated decline in serum IGF-I concentration prior to 40 yr of age. Strong associations of low serum IGF-I with blood pressure and arterial stiffness in young Africans suggest that the loss of cardiometabolic protection by IGF-I could predispose them to earlier disease onset.

Combined Tarsal and Carpal Tunnel Syndrome in Mucolipidosis Type III

Mucolipidosis type III (MLIII) (MIM# 252600) is an uncommon autosomal recessive disorder that results from uridine 5′‐diphosphate‐N‐acetylglucosamine: lysosomal hydrolase N‐acetyl‐1‐phosphotransferase or UDP‐GlcNAc 1‐phophotransferase deficiency. Clinical manifestations include developmental delay, short stature and other structural abnormalities. Less common clinical features, such as carpal tunnel syndrome, claw hand deformities, trigger fingers, and claw toes have previously been reported, but no specific association with tarsal tunnel syndrome has been reported in the literature. Tarsal tunnel syndrome is caused by entrapment of the posterior tibialis nerve in the tunnel formed by the medial malleolus of the ankle and the flexor retinaculum. It causes pain in the heel and sole of the foot as well as abnormal sensation in the distribution area of nervus tibialis posterior. In adults, the most common cause described is a ganglion. The phenomenon is rare in children and the published series are small. This case report portrays the presentation of a young girl with breath‐holding spells secondary to painful bilateral tarsal tunnel syndrome and trigger fingers subsequently diagnosed with MLIII.

Characterisation of the influence of genetic variations on the enzyme activity of a recombinant human glycine N-acyltransferase

Human glycine N-acyltransferase (human GLYAT) detoxifies a wide range of endogenous and xenobiotic metabolites, including benzoate and salicylate. Significant inter-individual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. To investigate the influence of single nucleotide polymorphisms (SNPs) in the GLYAT coding sequence on enzyme activity, we expressed and characterised a recombinant human GLYAT. Site-directed mutagenesis was used to generate six non-synonymous SNP variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. We also generated an E227Q mutant, which lacks the catalytic residue proposed by Badenhorst et al. (2012). This mutant was inactive compared to the wild-type recombinant human GLYAT. A molecular model of human GLYAT containing coenzyme A (CoA) was generated which revealed that the inactivity of the R199C variant could be due to the substitution of the highly conserved Arg(199) and destabilisation of an α-loop-α motif which is important for substrate binding in the GNAT superfamily. The finding that SNP variations in the human GLYAT gene influence the kinetic properties of the enzyme may explain some of the inter-individual variation in glycine conjugation capacity, which is relevant to the metabolism of xenobiotics such as aspirin and the industrial solvent xylene, and to the treatment of some metabolic disorders.