Graeme Bradley

Nuclear translocation of the phosphoprotein Hop (Hsp70/Hsp90 organizing protein) occurs under heat shock, and its proposed nuclear localization signal is involved in Hsp90 binding

The Hsp70-Hsp90 complex is implicated in the folding and regulation of numerous signaling proteins, and Hop, the Hsp70-Hsp90 Organizing Protein, facilitates the association of this multichaperone machinery. Phosphatase treatment of mouse cell extracts reduced the number of Hop isoforms compared to untreated extracts, providing the first direct evidence that Hop was phosphorylated in vivo. Furthermore, surface plasmon resonance (SPR) spectroscopy showed that a cdc2 kinase phosphorylation mimic of Hop had reduced affinity for Hsp90 binding. Hop was predominantly cytoplasmic, but translocated to the nucleus in response to heat shock. A putative bipartite nuclear localization signal (NLS) has been identified within the Hsp90-binding domain of Hop. Although substitution of residues within the major arm of this proposed NLS abolished Hop-Hsp90 interaction as determined by SPR, this was not sufficient to prevent the nuclear accumulation of Hop under leptomycin-B treatment and heat shock conditions. These results showed for the first time that the subcellular localization of Hop was stress regulated and that the major arm of the putative NLS was not directly important for nuclear translocation but was critical for Hop-Hsp90 association in vitro. We propose a model in which the association of Hop with Hsp90 and the phosphorylated status of Hop both play a role in the mechanism of nucleo-cytoplasmic shuttling of Hop.

Antioxidant and phytochemical properties of Carpobrotus edulis (L.) bolus leaf used for the management of common infections in HIV/AIDS patients in Eastern Cape Province

BackgroundCarpobrotus edulis (Mesembryanthemaceae), also known as igcukuma in Xhosa language is a medicinal plant used by the traditional healers to treat common infections in HIV/AIDS patients. Based on this information, we researched on the plant phytoconstituents, as well as its inhibitory effect using aqueous and three different organic solvent extracts in order to justify its therapeutic usage.MethodsAntioxidant activity of the extracts were investigated spectrophotometrically against 1,1- diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) diammonium salt, hydrogen peroxide (H2O2), nitric oxide (NO), and ferric reducing power, Total phenols, flavonoids, flavonols, proanthocyanidins, tannins, alkaloids and saponins were also determined using the standard methods.ResultsQuantitative phytochemical analysis of the four solvent extracts revealed a high percentage of phenolics (55.7 ± 0.404%) in the acetone extract, with appreciable amount of proanthocyanidins (86.9 ± 0.005%) and alkaloids (4.5 ± 0.057%) in the aqueous extract, while tannin (48.9 ± 0.28%) and saponin (4.5 ± 0.262%) were major constituents of the ethanol extract. Flavonoids (0.12 ± 0.05%) and flavonols (0.12 ± 0.05%) were found at higher level in the hexane extract in comparison with the other extracts. The leaf extracts demonstrated strong hydrogen peroxide scavenging activity, with the exception of water and ethanol extracts. IC50 values of the aqueous and ethanolic extract against DPPH, ABTS, and NO were 0.018 and 0.016; 0.020 and 0.022; 0.05 and 0.023 mg/ml, respectively. The reducing power of the extract was found to be concentration dependent.ConclusionThe inhibitory effect of the extracts on free radicals may justify the traditional use of this plant in the management of common diseases in HIV/AIDs patients in Eastern Cape Province. Overall, both aqueous and ethanol were found to be the best solvents for antioxidant activity in C. edulis leaves.

Natriuretic peptides and immunoreactants modify osmoticum-dependent volume changes in Solanum tuberosum L. mesophyll cell protoplasts

In plants, as in vertebrates, natriuretic peptide (NP) hormones can influence water and solute homeostasis. Here we demonstrate that a synthetic peptide identical to the C-terminus (amino acids 99–126) of the rat atrial natriuretic peptide (rANP) modulates osmotically induced swelling of mesophyll cell protoplasts (MCPs) in a concentration and time-dependent manner. Osmotically-induced volume changes in MCPs are enhanced by plant extracts with NP immunoreactivity and this effect is concentration-dependent. In contrast, pre-treatment of the plant extracts with rabbit anti-human ANP (99–126) antiserum suppresses enhanced osmoticum-induced swelling. Isolated plant peptides (irPNP) that have been immunoaffinity purified with rabbit anti-human ANP (99–126) antiserum also enhance osmotically-induced swelling. While rANP and irPNP cause increases in cGMP levels in MCPs, elevated cGMP levels do not cause increases in osmoticum-dependent swelling but exert an inhibitory effect. These findings are consistent with a NP-dependent, cGMP-independent effect on plant cell volume regulation and a role in homeostasis for peptides that are recognized by antibodies directed against the C-terminus of vertebrate ANPs.

Stronger induction of callose deposition in barley by Russian wheat aphid than bird cherry-oat aphid is not associated with differences in callose synthase or beta-1,3-glucanase transcript abundance

The effects of infestation by the bird cherry-oat aphid (BCA), (Rhopalosiphum padi L) and the Russian wheat aphid (RWA) (Diuraphis noxia Mordvilko) on callose deposition and transcription of genes related to callose accumulation were investigated in barley (Hordeum vulgare L. cv. Clipper). The BCA, which gives no visible symptoms, induced very limited callose deposition, even after 14 days of infestation. In contrast, RWA, which causes chlorosis, white and yellow streaking and leaf rolling, induced callose accumulation already after 24 h in longitudinal leaf veins. The deposition was pronounced after 72 h, progressing during 7 and 14 days of infestation. In RWA-infested source leaves, callose was also induced in longitudinal veins basipetal to the aphid-infested tissue, whereas in sink leaves, more callose deposition was found above the feeding sites. Eight putative callose synthase genes were identified in a database search, of which seven were expressed in the leaves, but with similar transcript accumulation in control and aphid-infested tissue. Five out of 12 examined beta-1,3-glucanases were expressed in the leaves. All five were upregulated in RWA-infested tissue, but only two in BCA-infested tissue, and to a lesser extent than by RWA. The results suggest that callose accumulation may be partly responsible for the symptoms resulting from RWA infestation and that a callose-inducing signal may be transported in the phloem. Furthermore, it is concluded that the absence of callose deposition in BCA-infested leaves is not because of a stronger upregulation of callose-degrading beta-1,3-glucanases in this tissue, as compared to RWA-infested leaves.

Protective effect of T. violacea rhizome extract against hypercholesterolemia-induced oxidative stress in Wistar rats.

The present study examines the effect of methanolic extract of T. violacea rhizomes on high cholesterol (2%) diet fed rats (HCD). At the end of 4 weeks, serum total protein, albumin, reduced glutathione (GSH), and markers of oxidative stress viz., catalase (CAT), superoxide dismutase (SOD), thiobarbituric acid reactive substances (TBARS—a marker of lipid peroxidation), glutathione-S-transferase (GST) and glutathione peroxidase (GPx) in the serum, aorta, liver and heart of HCD and normal rats were assessed and compared. A significant (p < 0.05) elevation in TBARS, and a reduction (p < 0.05) in serum total protein, albumin, GSH and antioxidant enzyme activities was observed in tissues of HCD fed rats compared with the normal group. Co-administration of crude extracts of T. violacea rhizomes protected the liver, heart, serum and aorta against HCD-induced lipid peroxidation in a dose dependant manner. The activities of the extract (500 mg/kg) compared favorably with gemfibrozil. The extracts also protected against HCD-induced reduction in serum total protein, GSH and restored the activities of antioxidant tissues (liver, heart and aorta) enzymes to near normal values. This result suggested that consumption of T. violacea rhizome may help to protect against hypercholesterolemia- induced oxidative stress diseases in the heart and liver.

Ostrich (Struthio camelus) carboxypeptidase B : purification, kinetic properties and characterization of the pancreatic enzyme

Carboxypeptidase B has been isolated from numerous mammalian and invertebrate species. In contrast, very little is known about carboxypeptidases of avian origin. To provide information for a comparative study, we have undertaken an investigation of the kinetic and physical properties of ostrich carboxypeptidase B. Carboxypeptidase B from the pancreas of the ostrich was purified by water extraction of acetone powder and aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. The effects of pH and temperature on CPB activity were examined. K(i)-values for numerous inhibitors (PCI, ABSA, hipp-D-lys, epsilon-aminocaproic acid, D-arg and 3-phenylproprionic acid) and kinetic parameters (K(m), k(cat) and k(cat)/K(m)) for several substrates (hipp-arg, hipp-lys, FAAA, FAAL and hipp-AA) were determined. N-terminal sequencing and amino acid analysis were also performed. Purified ostrich carboxypeptidase B was assessed to be homogeneous by SDS-PAGE with a M(r) value of approx. 35,000. For ostrich carboxypeptidase B the K(m) values for the different substrates were of the same order as those reported for other species, whereas the k(cat) values were 8- to 21-fold lower than the reported values. FAAA and hipp-AA were the preferred substrates. PCI was the most effective inhibitor, with a K(i) in the nM region, and no inhibition was shown with 3-phenylpropionic acid. The N-terminal sequence showed a high degree of homology when aligned with CPB from other species. Amino acid analysis showed significantly lower levels of Asx and Cyh and higher levels of Trp and Leu when compared with other species. Ostrich carboxypeptidase B would appear to show many physical, chemical and kinetic properties similar to those of other known carboxypeptidases.

Ostrich (Struthio camelus) carboxypeptidase A: Purification, kinetic properties and characterization of the pancreatic enzyme

1. Carboxypeptidase A beta and carboxypeptidase A tau-type from the pancreas of the ostrich were purified by water extraction of acetone powder, aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. 2. The final preparations were homogeneous when subjected to SDS-PAGE and PAGE. The M(r) values obtained from SDS-PAGE for CPA beta and CPA tau-type were 34,600 and 34,400, respectively. 3. The effects of inhibitors (1,10 phenanthroline and indole-3-acetic acid), pH and temperature on CPA activity were examined. Ki-values for CPI, PPA, D-phe, D-trp and aminobenzylsuccinic acid were determined. 4. Km, kcat and kcat/Km values were determined for hipp-phe, cbz-gly-phe, cbz-(gly)2-phe, cbz-gly-leu, cbz-(gly)2-leu and cbz-(gly)2-val. 5. N-terminal sequencing and amino acid analysis were performed for CPA beta and CPA tau-type.

Antioxidant properties and cytotoxicity evaluation of methanolic extract of dried and fresh rhizomes of Tulbaghia violacea

The rhizomes of Tulbaghia violacea Harv. used in Eastern Cape of South Africa for the treatment of heart diseases and several human ailments was investigated for antioxidant activity. The phytochemical content and toxicity of the fresh and dried rhizomes was also evaluated. Antioxidant activity was determined by spectrophotometric methods such as 2, 2-diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide and lipid peroxidation scavenging activities and ferric reducing power assay. Toxicity evaluation was carried out using brine shrimps cytotoxicity test. Results obtained indicated that methanolic extract of the fresh rhizomes contained higher concentration of flavonoid, flavonol, phenolics, tannin and proanthocyanidin than dried sample. Antioxidant studies revealed that the two extracts exhibited potent antioxidant activities in concentration dependent manner. The fresh extract had higher radicals scavenging activity than the dried extract with 50% inhibition of DPPH, hydrogen peroxide and lipid peroxidation at a concentration of 35.0 ± 0.12, 19.3 ± 0.11 and 17.9 ± 0.15 µg/ml, respectively. The dried extract demonstrates lower ferric reducing ability with an absorbance of 0.61 when compared with fresh extract (0.79), butylated hydroxytoluene (BHT) (1.80) and rutin (1.20) standard at 50 µg/ml. The fresh and dried methanolic extracts of the plant exhibited high degree of cytotoxic activity with IC 50 values of 18.18 and 19.24 µg/ml, respectively. The results obtained in this present study indicated that the rhizome of T. violacea may serve as potential source of natural antioxidant, antimicrobial and anticancer agents.

Arginine deiminases: Therapeutic tools in the etiology and pathogenesis of Alzheimer's disease

There is, at present, no definitive pre-mortem diagnostic tool for Alzheimers disease, (AD) which relates to a poor understanding of its etiology. Brains of AD patients contain large amounts of the toxic plaque-forming β-amyloid1–42 fragment in addition to elevated concentrations of the amino acid l-arginine. This work proposes that lowering levels of arginine in the astrocytes surrounding amyloid plaques may serve as a therapeutic tool in this neurodegenerative disorder. Arginine deiminase (ADI), from Pseudomonas aeruginosa, and peptidylarginine deiminase [PAD II], from bovine brain, are inhibited by amyloid peptides that contain arginine (amyloid1–42) and those that have no arginine (amyloid12–28/22–35). Enhanced activity of PAD II is noted with free l-arginine.

Ethnomedicinal survey of medicinal plants used for the management of HIV/AIDS infection among local communities of Nkonkobe Municipality, Eastern Cape, South Africa

An ethnomedicinal survey was conducted on medicinal plants used for the management of HIV/AIDS infection in the local communities of Nkonkobe Municipality, Eastern Cape Province. Information was obtained through a well-structured questionnaire and interviews administered to traditional healers and herbalists in various regions. The survey revealed 18 species belonging to 12 families. The members of Asphodelaceae (22.2%), Apocynaceae (16.7%), Asteraceae (11.1%), Mesembryanthemaceae (5.6%) and Hypoxidaceae (5.6%) were frequently mentioned by the traditional healers and rural dwellers of the regions. The decoction of Aloe ferox, Bulbine asphodeloides and Carpobrotus edulis roots and leaves are commonly administered for the management of HIV/AIDS infection. Other opportunistic diseases treated using the roots of these plants include tuberculosis, diabetes mellitus, sores, high blood pressure, intestinal worms and constipation. Most of the extracts of these plants are administered orally for a very long period of time, depending on the age and physical well-being of the patients. Generally, children are given half the dosage for the adults depending on the severity of the illness.