Vaughan Oosthuizen

The 3.2-A crystal structure of the human IgG1 Fc fragment-Fc gammaRIII complex.

The immune response depends on the binding of opsonized antigens to cellular Fc receptors and the subsequent initiation of various cellular effector functions of the immune system. Here we describe the crystal structures of a soluble Fcγ receptor (sFcγRIII, CD16), an Fc fragment from human IgG1 (hFc1) and their complex. In the 1:1 complex the receptor binds to the two halves of the Fc fragment in contact with residues of the Cγ2 domains and the hinge region. Upon complex formation the angle between the two sFcγRIII domains increases significantly and the Fc fragment opens asymmetrically. The high degree of amino acid conservation between sFCγRIII and other Fc receptors, and similarly between hFc1 and related immunoglobulins, suggest similar structures and modes of association. Thus the described structure is a model for immune complex recognition and helps to explain the vastly differing affinities of other FcγR–IgG complexes and the FcεRIα–IgE complex.

In vitro anti-HIV activity of five selected South African medicinal plant extracts

AIM OF THE STUDY Five South African medicinal plants, Bulbine alooides (L.) Willd. (Asphodelaceae), Crinummacowani Baker (Amaryllidaceae), Hypoxis sobolifera var. sobolifera (Jacq.) Nel (Hypoxidaceae), Leonotisleonurus (L.) R.Br. (Lamiaceae) and Tulbaghiaviolacea Harv (Liliaceae) used for the treatment of various ailments, including infectious diseases, were screened for activity against human immunodeficiency virus (HIV). MATERIALS AND METHODS Aqueous and ethanol extracts were tested for inhibitory activity in HIV-1 infected CEM.NK(R)-CCR5 cells, and against HIV-1 reverse transcriptase (RT) and HIV-1 protease (PR). RESULTS In CEM.NK(R)-CCR5 cells, ethanol extracts of Leonotisleonurus inhibited HIV-1 significantly (33% reduction in HIV-1 p24, P<0.05). HIV-1 RT inhibition (> or =50%) was shown for extracts of Bulbine alooides (aqueous and ethanol), Hypoxis sobolifera (aqueous and ethanol) and Leonotisleonurus (aqueous), but inhibitory activity was lost upon dereplication for removal of non-specific tannins/polysaccharides. HIV-1 PR inhibition was observed for extracts of Hypoxis sobolifera (aqueous), Bulbine alooides (aqueous and ethanol) and Leonotisleonurus (ethanol). Only ethanolic extracts of Bulbine alooides and Leonotisleonurus retained HIV-1 PR inhibition after dereplication with IC50 of 94 microg/ml and 120 microg/ml, respectively. CONCLUSION The dereplicated ethanolic extracts of Leonotisleonurus and Bulbine alooides showed the greatest anti-HIV potential in this study through inhibition of HIV-1 PR.

The roles of the proteasome, and cathepsins B, L, H and D, in ostrich meat tenderisation

As very little research has been conducted on ostrich meat tenderisation, this study aims at investigating the roles of the proteasome and cathepsins B, L, H, and D in the tenderisation process. The enzyme activities in meat from eight ostriches during a 12-day ageing period and the corresponding physical characteristics (e.g. pH, shear force) and myofibril patterns were determined. After 12 days, substantial high remaining activities were found, especially of the proteasome, thus implicating their possible roles in the tenderisation process. The mean shear force values, however, showed no improvement in tenderness, but the myofibril patterns showed the appearance of a M(r) 32 K component. Myofibril degradation studies of the proteasome, analysed electrophoretically, also revealed a possible role of the proteasome, but under activating conditions. This study provides further insights into the tenderisation process, particularly of ostrich meat, which may ultimately be used for the advantageous manipulation of the process.

In vitro anti-HIV-1 properties of ethnobotanically selected South African plants used in the treatment of sexually transmitted diseases

ETHNOPHARMACOLOGICAL RELEVANCE [corrected The plants selected in this study are used traditionally in the treatment of sexually transmitted diseases and traditional healers interviewed claimed these plants can also help AIDS patients. AIM To evaluating the in vitro anti-HIV properties of selected plants in various bioassays. MATERIALS AND METHODS The extracts were evaluated for their inhibition against alpha-glycohydrolase, reverse transcriptase and viral proteins (NF-kappaB and Tat) which play a significant role in the HIV life cycle. RESULTS Terminalia sericea extract (IC(50)=92mg/ml) exhibited a considerable alpha-glucosidase inhibitory activity which was better than acarbose (IC(50)=131mg/ml) under our assay conditions. In the reverse transcriptase assay, T. sericea also showed good inhibitory activity (IC(50)=43mg/ml), which was higher than that of the reference drug, Adriamycin (IC(50)=100mg/ml). The ethyl acetate extract of Elaeodendron transvaalense exhibited the most potent inhibitory activity in both the NF-kappaB and Tat assays with inhibitory activity of 76% and 75% respectively at a concentration of 15mg/ml. The acetone and chloroform extracts of E. transvaalense and Zanthoxylum davyi also showed good activity in the NF-kappaB and Tat assays.

Quantitative and qualitative analysis of sterols/sterolins and hypoxoside contents of three Hypoxis (African potato) spp.

The glycoside, hypoxoside, identified and isolated from the corms of the African potato (Hypoxis hemerocallidea) has shown promising anticancer activities. The African potato is used as an African traditional medicine for its nutritional and medicinal properties. Most research has been carried out on H. hemerocallidea (formerly known as H. rooperi), with very little or nothing on other Hypoxis spp. Thin layer chromatography (TLC) was used to confirm the presence of sterols/sterolins, whereas a GC method was developed to identify and quantify sterols (especially β-sitosterol) in chloroform extracts of H. hemerocallidea, H. stellipilis and H. sobolifera var. sobolifera. High performance liquid chromatography (HPLC) was used to identify and quantify hypoxoside content in these Hypoxis spp. TLC results showed that H. sobolifera var. sobolifera contained the most sterols and sterolins compared to the other two Hypoxis spp. Gas chromatography (GC) results show that β-sitosterol and campesterol were the two main phytosterols present in the Hypoxis extracts. H. sobolifera var. sobolifera and H. hemerocallidea contained the most β-sitosterol and hypoxoside, respectively. H. sobolifera and H. hemerocallidea contained 74.69 µg of β-sitosterol and 12.27 µg of hypoxoside per 5 mg of chloroform extracts, respectively. These results show a significant difference in the sterol/sterolin and hypoxoside contents between species of the genus Hypoxis, which may influence their degree of biological activities.

Purification and partial characterization of ostrich skeletal muscle cathepsin D and its activity during meat maturation

Cathepsin D was purified from ostrich (Struthio camelus) skeletal muscle using pepstatin-A chromatography. The enzyme was comprised of two subunits (29.1 and 14 kDa). The N-terminal amino acid sequence of both subunits were determined and showed high amino acid sequence identity to other cathepsin D homologs. Ostrich cathepsin D was optimally active at pH 4 and at a temperature of 45°C, and was strongly inhibited by pepstatin-A (K(i)=3.07×10(-9)M) and dithiothreitol. Cathepsin D activities from five ostriches were monitored over a 30-day period. Cathepsin D remained substantially active throughout the 30-day storage period with an average remaining activity of 112±8.57% at day 30 (mean value from 5 ostriches).

The amino acid sequence of pancreatic α-amylase from the ostrich, Struthio camelus.

Abstract The amino-acid sequence of α-amylase isolated from the pancreas of the ostrich, Struthio camelus was determined. The α-amylase (OPA) consisted of 497 amino acid residues with pyroglutamic acid at the N-terminus and no oligosaccharide. Amino acid identity between OPA and chicken, porcine and human pancreatic α-amylases individually, was found to be 88, 82 and 86%, respectively.

Ostrich intestinal glycohydrolases: distribution, purification and partial characterisation

Intestinal glycohydrolases are enzymes involved in assimilating carbohydrate for nutrition. The avian forms of these enzymes, in particular the maltase-glucoamylase complex (MG), are not well characterised. This study encompassed characterisation of these enzymes from ostrich intestines, and the first kinetic analysis of an avian MG. Proteolytically solubilised MG from ileal brush border membrane vesicles was purified by Sephadex G-200 gel filtration and Tris-affinity-chromatography, while jejunal sucrase-isomaltase (SI) and MG were purified by Toyopearl-Q650 and phenyl-Sepharose chromatography. Amino acid sequences and compositions of enzyme subunits, resulting from SDS-PAGE, were determined. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostrich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be detected. The jejunal MG and SI, resulting from brush-border membrane vesicles, could not be separated during purification. However, a minor form of ileal MG was purified using Sephadex G-200 chromatography. Ileal MG contained three subunits of M(r) 145,000, 125,000 and 115,000. Although the N-terminal amino acid sequences bear no homology to SI, the M(r) 115,000 subunit shows homology to porcine MG in both sequence and amino acid composition. The pH optimum of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activity 5.5. The glycohydrolases were most active at 58 degrees C, but were quickly denatured above 60 degrees C. Sucrose- and starch-hydrolysing activities were more thermostable than maltose- and isomaltose-hydrolysing activities. Kinetic parameters (K(m), kcat and kcat/K(m)) for the hydrolysis of maltooligosaccharides, starch and glycogen are reported for ileal MG. Maltotriose and maltotetraose displayed partial inhibition of ileal MG. The study revealed large similarities between ostrich SI and MG in charge, size, shape and hydrophobicity, based on their inseparability by several methods. Measurement of the specificity constants for maltooligosaccharide hydrolysis by ileal MG revealed less efficient hydrolysis of longer substrates as compared to maltose and maltotriose.

Purification and characterization of the 20S proteasome from ostrich skeletal muscle

Abstract The proteasome is a high molecular weight, multisubunit and multicatalytic enzyme. Here we report the purification and characterization of ostrich skeletal muscle 20S proteasome. It was purified to homogeneity with Mr 700 000, pI 6.67 and a ladder of 22.2 33.5 kDa bands on SDSPAGE. The amino acid composition and aminoterminal sequences showed large identities to those of other species. For the three major activities, pH and temperature optima ranged between 8.0 11.0 and 40 70C, and stabilities between 5 12 and up to 40 60C. Substrate specificity and inhibitory effects were also studied. Many similarities to other sources were shown, with a few significant differences.

Biological inferences from IgM binding characteristics of recombinant human secretory component mutants.

The polymeric immunoglobulin receptor (pIgR) or membrane secretory component (SC) selectively transports polymeric IgA and IgM across secretory epithelial cells to mucosal surfaces. The ligand binding ectodomain consists of five homologous Ig-like domains with domain I being an absolute requirement for binding. The role of DII to V in IgM binding remains unknown. Here, using in vitro refolded non-glycosylated recombinant domain deletion mutants of human SC, we show by biological and biophysical binding assays that DII to V are required for high affinity binding to IgM. Competitive binding analysis, by whole cell ELISA, showed that DII-V significantly increase the affinity of recombinant SC for IgM (K(i)=2.42 nM) as opposed to recombinant DI only (K(i)=44.8 nM). Lastly, we provide qualitative data highlighting the complexity of measuring the IgM/SC interaction using surface plasmon resonance spectroscopy.