Pieter Swart

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.

Mechanism for the stabilization in vivo of the aziridine precursor 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride by serum proteins

Oral and intraperitoneal administration of 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (Compound A), an analogue of phenyl aziridine precursors that occur in the shrub Salsola tuberculatiformis Botsch, had a contraceptive effect on female Wistar rats with a concomitant decrease in total body, uterus, and every mass and an increase in abronal mass. Compound A elicited a Type II difference spectrum and inhibited the Type I deoxycorticosterone (DOC) induced difference spectrum of sheep adrenal cytochrome P450c11 in a manner similar to that of S2, a biologically active fraction isolated from S. tuberculatiformis. The effects of Compound A on the spectral properties of P450c11 were diminished with time in PBS. Electrospray mass spectrometry (ES-MS) indicated that the rate of cyclization of Compound A to the corresponding aziridine followed a time course similar to the attenuation of cytochrome P450c11 inhibition. It was concluded that the aziridine precursor. Compound A, rather than aziridine itself, was the inhibiting agent of sheep adrenal P450c11. Addition of sheep and rat plasma prevented the attenuation of the effect of Compound A on the spectral properties of cytochrome P450c11. Subsequent ES-MS analysis indicated that Compound A was stabilized in plasma by sex hormone binding globulin and corticosteroid binding globulin. These results suggest a mechanism whereby natural plant products, which are highly reactive and unstable in vitro, can be stabilized by binding to plasma proteins, and so remain biologically active in vivo.

11β-Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: A putative role in castration resistant prostate cancer?

Adrenal C19 steroids, dehydroepiandrostenedione (DHEA(S)) and androstenedione (A4), play a critical role in castration resistant prostate cancer (CRPC) as they are metabolised to dihydrotestosterone (DHT), via testosterone (T), or via the alternate 5α-dione pathway, bypassing T. Adrenal 11OHA4 metabolism in CRPC is, however, unknown. We present a novel pathway for 11OHA4 metabolism in CRPC leading to the production of 11ketoT (11KT) and novel 5α-reduced C19 steroids - 11OH-5α-androstanedione, 11keto-5α-androstanedione, 11OHDHT and 11ketoDHT (11KDHT). The pathway was validated in the androgen-dependent prostate cancer cell line, LNCaP. Androgen receptor (AR) transactivation studies showed that while 11KT and 11OHDHT act as a partial AR agonists, 11KDHT is a full AR agonist exhibiting similar activity to DHT at 1nM. Our data demonstrates that, while 11OHA4 has negligible androgenic activity, its metabolism to 11KT and 11KDHT yields androgenic compounds which may be implicated, together with A4 and DHEA(S), in driving CRPC in the absence of testicular T.

Enzymatic cleaning of ultrafiltration membranes fouled by abattoir effluent

Proteins and lipids are the major membrane foulants present in abattoir process effluent. The successful use of ultrafiltration (UF) to treat such effluent streams depends on the effective removal of these foulants, which are mostly hydrophobic fouling species, from the membrane surface. Lipases and proteases were used in this study to clean flat-sheet polysulphone membranes (PSMs) fouled in abattoir effluent. The lipases from Candida cylindracea, Pseudomonas mendocina and Aspergillus oryzea were used alone, as well as in combination with the proteases from Bacillus licheniformis, Protease A (a protein engineered protease specific for low temperature wash conditions) and Aspergillus oryzea. Enzyme assays were first performed to identify active bacterial proteases and lipases. The parameters used to determine the cleaning efficiencies of the enzymes used were: (i) lipid content adsorbed onto the membranes; (ii) protein content adsorbed onto the membranes; and (iii) pure-water flux (PWF) after enzymatic cleaning. The results show the ability of enzymes to remove adsorptive fouling, and indicate their usefulness in enzyme-based cleaning regimes for membranes operating on abattoir effluents.

The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: Quantification of steroid intermediates and end products in H295R cells

The steroid hormone output of the adrenal gland is crucial in the maintenance of hormonal homeostasis, with hormonal imbalances being associated with numerous clinical conditions which include, amongst others, hypertension, metabolic syndrome, cardiovascular disease, insulin resistance and type 2 diabetes. Aspalathus linearis (Rooibos), which has been reported to aid stress-related symptoms linked to metabolic diseases, contains a wide spectrum of bioactive phenolic compounds of which aspalathin is unique. In this study the inhibitory effects of Rooibos and the dihydrochalcones, aspalathin and nothofagin, were investigated on adrenal steroidogenesis. The activities of both cytochrome P450 17α-hydroxylase/17,20 lyase and cytochrome P450 21-hydroxylase were significantly inhibited in COS-1 cells. In order to study the effect of these compounds in H295R cells, a human adrenal carcinoma cell line, a novel UPLC-MS/MS method was developed for the detection and quantification of twenty-one steroid metabolites using a single chromatographic separation. Under both basal and forskolin-stimulated conditions, the total amount of steroids produced in H295R cells significantly decreased in the presence of Rooibos, aspalathin and nothofagin. Under stimulated conditions, Rooibos decreased the total steroid output 4-fold and resulted in a significant reduction of aldosterone and cortisol precursors. Dehydroepiandrosterone-sulfate levels were unchanged, while the levels of androstenedione (A4) and 11β-hydroxyandrostenedione (11βOH-A4) were inhibited 5.5 and 2.3-fold, respectively. Quantification of 11βOH-A4 showed this metabolite to be a major product of steroidogenesis in H295R cells and we confirm, for the first time, that this steroid metabolite is the product of the hydroxylation of A4 by human cytochrome P450 11β-hydroxylase. Taken together our results demonstrate that Rooibos, aspalathin and nothofagin influence steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid and androgen pathways, thus possibly contributing to the alleviation of negative effects arising from elevated glucocorticoid levels.

Pharmacokinetics and In Vivo Gene Transfer of Plasmid DNA Complexed with Mannosylated Poly(L-Lysine) in Mice

Abstract To achieve mannose receptor-mediated, cell-specific, in vivo gene transfer by intravenous injection of plasmid DNA, mannosylated poly(L-lysine) (Man-PLL) was synthesized as a carrier molecule, and mixed with a plasmid DNA encoding chloramphenicol acetyl-transferase (CAT) gene to form DNA/Man-PLL complex. The particle size and zeta potential of DNA/Man-PLL (prepared at 1:0.7 on a weight basis) were determined to be 220 nm and +12 mV, respectively. The pharmacokinetics of the DNA/Man-PLL complex was assessed in mice using 32P-labeled DNA ([32P]DNA). After intravenous injection of [32P]DNA/Man-PLL, the radioactivity in plasma fell rapidly and was recovered mainly in the liver nonparenchymal cells. The amount in the liver reached more than 80% of the dose. Radioactivity observed in kidney, lung, and spleen was very low compared to that in the liver. Then, the in vivo gene expression after intravenous injection of DNA/Man-PLL was examined by a CAT assay. Highest CAT activity was detected in the liver, but no activity was detected in the lung, kidney, and spleen. These results clearly indicate that a cell-specific gene delivery system can be developed by regulating the biodistribution of DNA/carrier complex through the control of its physicochemical properties.

Charge modification of plasma and milk proteins results in antiviral active compounds

Previous studies have shown that acylated plasma and milk proteins with increased negative charge, derived from various animal and human sources, are potent anti‐HIV compounds. The antiviral effects seemed to correlate positively with the number of negative charges introduced into the various polypeptides: proteins with a high content of basic amino acids in which all of the available εNH2 groups were anionized yielded the most potent anti‐HIV compounds. It remained unclear however whether the total net negative charge of the various derivatized proteins, or rather the charge density on the protein backbone, is essential for the observed anti‐HIV activity. Earlier studies have shown that acylated albumins preferentially block the process of HIV/cell fusion through binding to the HIV envelope proteins gp120 and gp41 as well as to the cell surface of the HIV target cells. Some of these polyanionic proteins have been shown to interfere also with the gp120–CD4 mediated virus/cell binding. The relative contribution of these effects to the anti‐HIV activity may depend both on the total negative charge introduced as well as the hydrophobicity of the acylating reagent added to the particular proteins. In this study we show that the higher the charge density of the derivatized proteins, the more potent their HIV replication inhibiting effects are. In contrast, the addition of positive charge to the studied plasma and milk proteins through amination resulted in a reduced anti‐HIV activity but a clearly increased anti‐HCMV activity, with IC50 values in the low micromolar concentration range. Interestingly, native lactoferrin (Lf) was antivirally active against both HIV and HCMV. Acylation or amination of Lf increased the anti‐HIV and anti‐HCMV activity, respectively. The N‐terminal portion of Lf appeared essential for its anti‐HCMV effect: N‐terminal deletion variants of human Lf were less active against HCMV. Circular dichroism of the modified proteins showed that the secondary structure of the tested proteins was only moderately influenced by acylation and/or covalent attachment of drugs, making these (derivatized) proteins useful candidates as antiviral agents and/or intrinsically active drug carriers. The relatively simple chemical derivatization as well as the abundant sources of blood plasma and milk proteins provides attractive opportunities for the preparation of potent and relatively cheap antiviral agents for systemic or local applications. Copyright

Mechanism of anti-HIV activity of negatively charged albumins: Biomolecular interaction with the HIV-1 envelope protein gp120

A novel class of polyanionic proteins with potent anti-human immunodeficiency virus type 1 activity, the negatively charged albumins (NCAs), have been reported previously. In vitro antiviral assays established that these compounds preferentially inhibit virus-cell fusion and syncytium formation and that virus-cell binding is less affected. Here the interaction of the NCAs with synthetic peptides composed of 15-36 amino acids and corresponding to different parts of the gp120 envelope protein is described. Among the gp120 peptides tested, binding of the NCAs was observed only with the s0-called V3 loop (amino acids 296-330) and the C-terminal part of gp120. A higher number of negatively charged residues in the albumins resulted in higher binding affinities. NCAs in which, in addition to negative charges, up to 7 or 14 lactose or mannose groups were introduced, respectively did not exhibit increasing binding affinity. In contrast, mannosylated albumin containing about 14 mannose groups showed an increased binding compared with native albumin. Binding of the NCAs to the V3 and C-terminal oligopeptide was competitively inhibited by sulfated polysaccharide heparin and dextran sulfate. This finding indicates that the binding between the gp120 peptides and the NCAs is likely caused by electrostatic interactions. However, the fact that the dissociation constants of dextran sulfate and heparin are orders of magnitude larger compared with the NCAs indicates that the spatial structure of the proteins and/or hydrophobic interactions between the NCAs and the envelope protein may also be involved.

Developmental Trauma is Associated with Behavioral Hyperarousal, Altered HPA Axis Activity, and Decreased Hippocampal Neurotrophin Expression in the Adult Rat

Abstract:  Effects of early‐life trauma on adult behavioral responses, corticosterone (CORT) concentration, and levels of nerve growth factor (NGF), brain‐derived neutrophic factor (BDNF), and neurotrophin‐3 (NT‐3) in hippocampus and frontal cortex were investigated. Traumatized animals showed an increase in rearing in both the elevated plus maze and open field after adult restress, higher basal levels of CORT, lower levels of BDNF in dorsal hippocampus, and lower levels of NT‐3 in dorsal and ventral hippocampus. Trauma‐related behavioral hyperarousal and altered hypothalamic‐pituitary‐adrenal (HPA) axis activity may be mediated by decreases in hippocampal neurotrophin expression.

Viral Load in Breast Milk Correlates with Transmission of Human Cytomegalovirus to Preterm Neonates, but Lactoferrin Concentrations Do Not

ABSTRACT In vitro, lactoferrin (LF) strongly inhibits human cytomegalovirus (HCMV), which led us to hypothesize that in vivo HCMV might also be inhibited in secretions with high LF concentrations. In breast milk, high viral loads observed as high viral DNA titers tended to coincide with higher LF levels. However, the LF levels did not correlate to virus transmission to preterm infants. The viral load in the transmitting group was highest compared to the nontransmitting group. We conclude that viral load in breast milk is an important factor for transmission of the virus.