Biochemistry

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Analyses of lipidic bodies from green microalgae.
(2014) Pather, Verushka.; Gupthar, Abindra Supersad.; Bux, Faizal.
This study presents the analyses of oil body components in microalgae which may be involved in oil droplet assembly including certain triacylglycerol precursors which can be processed to biodiesel, an alternative fuel source. Stress induction of microalgae, Chlorella vulgaris CCAP 211/11B and Dunaliella primolecta CCAP 11/34 was achieved by exclusion of nitrates in growth media. Contrary to other forms of nitrogen depletion, this condition did not greatly enhance lipid biosynthesis in the microalgae. Confocal microscopy and fluorescent dyes nile red and bodipy were employed for the visualization of lipidic body components. The fluorescence hues emitted by neutral lipids and phospholipids were differentiated from those due to autofluorescence and chlorophyll using ZEN software to analyse images from a Zeiss LSM 710 confocal microscope. Oil from both algae, which were subjected to transesterification and gas chromatography, revealed a predominant fatty acid, namely palmitic acid (C16:0). D. primolecta produced linolelaidic acid (C18:2n6t) under growth conditions involving both nitrate supplementation and exclusion; whilst the longest fatty acid, docosanoic acid (C22:0 chain) was produced by the alga C. vulgaris only under conditions of nitrate supplementation. Nitrate limitation had minimal effect on the oil hydrocarbon yield which increased only 0.02% and 0.01% for C. vulgaris and D. primolecta, respectively. The highest biodiesel yield of 26.11 % was recorded from D. primolecta when grown under conditions of nitrate exclusion. The protein concentrations extracted from oil of the former alga ranged from 1.87 - 1.95 Gg/ml when grown under nitrate supplemented conditions and 1.74 - 1.90 Gg/ml when nitrate was excluded from the media. The protein concentrations extracted from oil of D. primolecta ranged from 1.91 - 2.23 Gg/ml and 1.88 - 1.98 Gg/ml, respectively, when the algae were grown in the presence and exclusion of nitrates. In the adaptation of protocols for protein extraction from oil, sunflower and salmon oils were initially used. Sunflower oil extracts produced by 10% (w/v) SDS treatment, yielded protein bands of 198, 96, 70 and 58 KDa on 10% (w/v) polyacrylamide gels while 6M urea treatment yielded a band of 200 KDa. Salmon oil treated with 10% (w/v) SDS and 6 M urea yielded bands of 195 and 27 KDa, and 198 KDa, respectively, as well as common bands of 68 and 64 KDa. In comparison, the extraction of discrete proteins from algal oil proved to be difficult as the extractants SDS and urea could have denatured protein components into subunit structure.
Analysis of the Mycoplasma hominis hsp70 gene and development of a PCR ELISA assay.
(1998) Shearer, Nicollette.; Hastings, John W.
Mycoplasmas conform most closely with the theoretical concept of 'minimum cells', existing as the smallest, free-living organisms capable of self-replication. They survive as parasites of plants, insects, animals or humans, with the most common human colonising species being Mycoplasma hominis. M. hominis has been characterised as a human pathogen responsible for a variety of infections, which pose a significant threat particularly to immunocompromised patients and neonates. However little has been elucidated about the cell physiology and molecular structure of this organism. Of interest to this study were the investigation of the heat shock response of M. hominis and the diagnostic assays used for its detection. The heat shock response is a ubiquitous physiological feature of all organisms and displays unprecedented conservation. This phenomenon is particularly evident in the 70 kDa family of heat shock proteins (hsp70) which exhibits a high degree of homology between different species. The hsp70 gene from M. hominis was cloned and preliminary partial sequencing indicated the similarity with other hsp70 homologs. The regulation of hsp70 expression at the transcriptional and translational levels was investigated. The level of hsp70 mRNA was found to increase correspondingly in response to heat shock, more visibly than the level of hsp70 protein. However imrnunochemical studies of the M. hominis hsp70 translation product demonstrated further the homology with other species. To facilitate rapid diagnosis of M. hominis infections, a PCR ELISA diagnostic assay was developed and optimised. The amplification of a conserved region of the M. hominis 16S rRNA gene was linked to subsequent hybridisation to an appropriate capture probe in a microtiter plate format. The sensitivity of the assay was comparable to other molecular assays although the PCR ELISA produces more rapid results and is less labour intensive.
Anti-c-myc cholesterol-based lipoplexes: development, characterisation and evaluation as Onconanotherapeutic agents in vitro.
(2018) Habib, Saffiya.; Singh, Moganavelli.
Strategies aimed at inhibiting the expression of the c-myc oncogene could provide the basis for alternative cancer treatment. In this regard, silencing c-myc expression using small interfering RNA (siRNA) is an attractive option. However, the development of a clinically viable, siRNAbased, c-myc silencing system is largely dependent upon the design of an appropriate siRNA carrier that can be easily prepared. Nanostructures formed by the electrostatic association of siRNA and cationic lipid vesicles represent uncomplicated, well-recognised siRNA delivery systems. Therefore, this study has focused on traditional cationic liposomes as the foundation for the development of a simple, but effective anti-c-myc onconanotherapeutic agent. Novel liposome formulations contained equimolar quantities of the cytofectin, N,Ndimethylaminopropylamidosuccinylcholesterylformylhydrazide (MS09), and cholesterol (Chol); with or without 2 mol % pegylation. Liposomes which contained dioleoylphosphatidylethanolamine (DOPE) as the co-lipid were included for comparative purposes. Pegylated and non-pegylated MS09/Chol (1:1) suspensions were reproducibly prepared by lipid film hydration to give unilamellar vesicles that were stable for at least 10 months at 4 ˚C. Liposomes successfully bound siRNA to form lipoplexes of less than 200 nm in size, with zeta potentials between -16 and -44 mV. These assumed globular and bilamellar structures in which siRNA was partially protected. Although all formulations were well tolerated at ≤14 nM siRNA, pegylation severely inhibited siRNA delivery in cancer cell lines, MCF-7 and HT-29, which overexpress c-myc. The non-pegylated MS09/Chol (1:1) lipoplex, at the MS09:siRNA (w /w) ratio of 16:1, was most effectively taken up by MCF-7 and HT-29 cells, with negligible effect in non-transformed cells when applied at 12 nM siRNA. Lipoplexes directed against the c-myc transcript (anti-c-myc siRNA), mediated a dramatic reduction in c-myc mRNA and protein levels. This was accompanied by a loss of migratory potential and apoptotic cell death. Moreover, oncogene knockdown and anti-cancer effects were superior to that of a commercially available transfection reagent, Lipofectamine™ 3000. Although the DOPE-containing counterpart performed with iii comparable efficacy under standard in vitro conditions, it was incapable of siRNA delivery at physiological serum concentration. Hence, the anti-c-myc MS09/Chol (1:1) lipoplex reported exemplifies a straightforward anti-cancer agent that warrants further investigation in vivo.
Antibody-mediated inhibition of proteases of African trypanosomes.
(2006) Huson, Laura.; Coetzer, Theresa Helen Taillefer.
The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may contribute to pathogenesis of the disease, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. Oligopeptidase B, a trypanosomal serine peptidase, is also a potential virulence factor in African trypanosomes because it is released into the host circulation by dead or dying parasites, where it retains catalytic activity due to the enzyme's insensitivity to serum protease inhibitors. The vaccine potential of the catalytic domain of congopain, C2, and oligopeptidase B complexed with 0'2-macroglobulin (0'2M) was evaluated by producing antibodies in rabbits. Inhibition of congopain and oligopeptidase B activity by these antibodies was assessed. The oligopeptidase B open reading frame from T. congolense and T. vivax was cloned and expressed in Escherichia coli, from which active recombinant enzymes were purified. These recombinant enzymes exhibited trypsin-like specificity for peptide substrates, cleaving on the carboxy side of basic amino acid residues such as arginine and lysine. Enzymes were found to be optimally active between pH 8 and 10, optimally stable at pH 6, and showed activation by reducing agents and sensitivity to ionic strength. The enzymes showed typical oligopeptidase B-like inhibitor profiles, except that they were not inhibited by thiol sensitive inhibitors such as iodoacetamide and Nethylmaleimide. High yields of bovine and rabbit 0'2M were isolated by a three-step procedure of fractionation by PEG 6000, and zinc chelate and Sephacryl S-300 HR chromatography. Congopain, its catalytic domain C2, papain and cathepsin L all cleaved the bait region of bovine 0'2M and became trapped inside the 0'2M molecule, where their activity against large molecular weight substrates was inhibited. C2 could thus be complexed with 0'2M directly or used to form C2-0'2M-oligopeptidaseB complexes for immunisation purposes. iv The catalytic domain of congopain, C2, was used to immunise rabbits either without adjuvant, as a water-in-oil emulsion with Freund's adjuvant, or in a complex with either bovine or rabbit U2M. Freund's adjuvant elicited the highest anti-C2 antibody response. However, the greatest inhibition, 65%, of C2 activity against Z-Phe-Arg-AMC was obtained with antibodies produced by rabbits receiving C2-U2Mcomplexes. In a second study, C2 and oligopeptidase B were used to immunise rabbits , either in alum, or complexed to bovine U2M. Anti-C2 antibody levels were highest in rabbits immunised with the free proteins in alum, whereas anti-oligopeptidase B antibody levels were comparable for each adjuvant system. Anti-oligopeptidase antibodies produced with alum gave 100% inhibition of oligopeptidase B activity. In contrast, antibodies produced against C2-u2M-oligopeptidase B complexes had little effect on oligopeptidase B activity. However, these antibodies inhibited 55% of C2 activity. Alum was a slightly less efficient adjuvant for C2 and 50% inhibition of C2 activity was observed. It appeared that immunisation of rabbits with C2 complexed to U2M resulted in the production of antibodies that were better able to neutralise the proteolytic activity of C2 and congopain in vitro than that with conventional adjuvants . The immunisation of C2 complexed to bovine u2-macroglobulin therefore has the potential to neutralise parasite congopain in vivo, and may contribute to an anti-disease vaccine against African trypanosomosis. Complexation of oligopeptidase B to u2M offers no benefit, since antibodies produced against this complex are not able to inhibit the activity of oligopeptidase B. Immunisation with oligopeptidase B in alum is sufficient to produce efficient enzyme-inhibiting antibodies in the context of an anti-disease vaccine against African trypanosomosis.
Antidiabetic activity of Warburgia salutaris (Bertol. f.) Chiov. (Canellaceae).
(2017) Msomi, Nontokozo Zimbili.; Simelane, Mthokozisi Blessing Cedric.; Murambiwa, Pretty.
Abstract available in PDF file.
Antidiabetic and toxicological properties of some African medicinal plants used in the treatment of diabetes and its complications.
(2018) Erukainure, Ochuko Lucky.; Islam, Mohammad Shahidul.
This study investigated the antioxidant, antidiabetic and toxicity properties of antidiabetic medicinal plants comprising of Vernonia amygdalina, Cola nitida, Raffia palm (Raphia hookeri) wine, Phaseolus lunatus, Dacryodes edulis, and Clerodendrum volubile using in vitro, ex vivo, in silico and in vivo models. The leaves of V. amygdalina and D. edulis, as well as C. volubile flower were sequentially extracted with solvents of increasing polarity to yield ethyl acetate, ethanol and aqueous extracts. Cola nitida and V. amygdalina were infused in hot water to yield infusion extracts. Phaseolus lunatus was subjected to aqueous extraction to yield aqueous extract, while Raffia palm wine was concentrated to yield the concentrate. The extracts and concentrate were screened for their in vitro and ex vivo antioxidant activities, as well as their inhibitory effect on α-glucosidase, α-amylase and pancreatic lipase activities, and their ability to stimulate muscle glucose uptake and inhibit intestinal glucose absorption in vitro. The ethanol extracts of D. edulis, C. volubile and V. amygdalina were subjected to GC-MS analysis, while the aqueous extract of P. lunatus, palm wine concentrate and the infusions were analyzed with LC-MS to elucidate the active compounds that may be responsible for their bioactivities. The ethanol extracts of C. volubile and D. edulis were further subjected to liquid-liquid fractionation to yield the hexane, dichloromethane, ethyl acetate, butanol and aqueous fractions. These fractions were also assayed for their antioxidant and antidiabetic properties in vitro and ex vivo. The dichloromethane, ethyl acetate and butanol fractions were subjected to GC-MS analysis to elucidate their active compounds. The identified compounds were molecularly docked with the test enzymes in silico to further validate their bioactivities. The antidiabetic properties of palm wine concentrate, C. nitida infusion, and D. edulis butanol fraction were investigated in a type 2 diabetes rat model. The in vivo study revealed a potent hypoglycemic activity, with concomitant amelioration of oxidative stress in the serum, pancreas, testes and brain. This was further substantiated by the downregulation of Nrf2 expressions in the pancreas and brain. These results further validate the use and safety of these plants in diabetes management.
Antidiabetic properties of centella asiatica in type II diabetic rats.
(2016) Oyenihi, Ayodeji Babatunde.; Masola, Bubuya.; Mukaratirwa, Samson.
Abstract available in PDF file.
Antioxidative and antidiabetic activity and phytochemicals analysis of some selected Sudanese traditional medicinal plants.
(2021) Idris, Almahi Idris Mohamed.; Islam, Shahidul.
This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
Antioxidative and antidiabetic activity and phytochemicals, analysis of some selected Sudanese traditional medicinal plants.
(2021) Mohamed, Almahi Idris.; Islam, Shahidul.
This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
Antioxidative and antidiabetic effects of some African medicinal plants.
(2016) Mohammed, Aminu.; Islam, Mohammad Shahidul.
Three (3) medicinal plants [Aframomum melegueta K. Schum., Xylopia aethiopica (Dunal.) A. Rich. and Capsicum annuum L.] were selected based on their traditional uses in the treatment of diabetes in Africa. Various crude extracts and fractions from different parts of the plants were screened using several anti-oxidative and anti-diabetic tests in vitro. Most active fractions from each plant were used to examine in vivo anti-diabetic activity in type 2 diabetes (T2D) rat model. Additionally, possible bioactive compounds from most active extracts and fractions were analyzed by using GC-MS, TLC and NMR spectroscopy. The results showed that ethanolic extracts derived from the fruits of the plants demonstrated excellent anti-oxidative and anti-diabetic activities in vitro compared to other extracts from the same or different parts of these plants. After fractionation, ethyl acetate fraction from A. melegueta and acetone fractions from X. aethiopica and C. annuum exhibited strong radical scavenging (IC₅₀: 1-120 μg/mL) activity, inhibition of hemoglobin glycation (IC₅₀: 100-150 μg/mL), α-amylase (IC₅₀: 50-170 μg/mL) and α-glucosidase (IC₅₀: 40-87 μg/mL) activities hence were used for the in vivo study. The GC-MS analysis of the three (3) most active fractions revealed the presence of mostly phenolic compounds of 4-hydroxy-3-methoxyphenyl derivatives. Furthermore, the data of the in vivo study showed that oral intervention of the fractions (150 and 300 mg/kg bw) for 4 weeks demonstrated potent anti-diabetic actions via improving body weight gain, reducing feed and fluid intake and hyperglycemia, improving glucose tolerance ability, insulin sensitivity, amelioration of pancreatic β-cell histology and β-cell functions, improving dyslipidemia in a T2D rat model. Additionally, the pancreatic histopathological damages and other oxidative damages caused by the induction of diabetes were attenuated to near normal in the liver, kidney, heart and pancreas of the treated animals. The bioassay-guided fractionations lead to the isolation of 3 arylalkanes (6-paradol (1), 6-shagaol (2), and 6- gingerol (3)) and oleanolic acid (4) from A. melegueta fruits, when oleanolic acid (4) was the first to be isolated from A. melegueta. Moreover, 6-gingerol (3) and oleanolic acid (4) were similarly isolated for the first time from X. aethiopica fruits as well. These compounds have exhibited significant inhibitions against the α-amylase and α-glucosidase actions and thus are possible anti-diabetic agents and the anti-diabetic action of A. melegueta and X. aethiopica fruits is attributed to the presence of these compounds. This study also confirmed the use of these plants in African anti-diabetic traditional medicines by traditional healers. However, further clinical study is required to confirm these effects in human subjects.
Antiplasmodial activity of Warburgia salutaris (Bertol. F.) Chiov. (Cannelaceae).
(2017) Nyaba, Zoxolo Nokulunga.; Simelane, Mthokozisi Blessing Cedric.; Murambiwa, Pretty.
Abstract available in PDF file.
Apoptosis in peripheral blood mononuclear cells of human immunodeficiency virus (HIV) infected patients undergoing highly active antiretroviral therapy.
(2008) Karamchand, Leshern.; Chuturgoon, Anil Amichund.; Dawood, Halima.
Highly active antiretroviral therapy (HAART) is currently the only treatment that effectively reduces the morbidity and mortality of individuals infected with Human Immunodeficiency Virus-1 (HIV-1). Standard HAART regimens typically comprise 2 nucleoside reverse transcriptase inhibitors and either one non-nucleoside reverse transcriptase inhibitor or a protease inhibitor. These drugs bind to and inhibit the HIV-1 Reverse Transcriptase and Protease enzymes respectively, thereby suppressing viral replication. The nucleoside reverse transcriptase inhibitors promote mitochondrial (mt) dysfunction by strongly inhibiting mt polymerase gamma (Pol-y) and subsequently, mtDNA replication. In contrast, the non-nucleoside reverse transcriptase inhibitors, efavirenz (EFV) and nevirapine (NVP) do not inhibit Pol-y although EFV has been shown to induce mt depolarisation ( mlow) in vitro at supra-therapeutic concentrations. However, the capacity of non-nucleoside reverse transcriptase inhibitor drugs to induce mt toxicity in vivo previously remained undetermined. The objective of this study was to determine the influence of EFV and NVP on peripheral lymphocyte mt transmembrane potential (Avj/m) and apoptosis in HIV-1-infected patients treated with these non-nucleoside reverse transcriptase inhibitors. Thirty-two HIV-1-infected patients on HAART between 4 and 24 months (12 on EFV, 20 on NVP) and 16 HAART-naive HIV-1-infected patients were enrolled into this study. All participants were black South African patients. Spontaneous peripheral lymphocyte apoptosis and mlow were measured ex vivo by flow cytometry for all patients. CD4 T-helper apoptosis for the EFV and NVP cohorts was 19.38% ± 2.62% and 23.35% ± 1.51% (mean ± SEM), respectively, whereas total lymphocyte mlow was 27.25% ± 5.05% and 17.04% ± 2.98%, respectively. Both parameters for each cohort were significantly lower (P < 0.05) than that of the HAART-naive patients. The NVP cohort exhibited both a significant time dependent increase in peripheral lymphocyte ö¿mlow (P = 0.038) and correlation between Thelper apoptosis and low (P = 0.0005). These trends were not observed in the EFV cohort. This study provides evidence that both EFV and NVP induce peripheral lymphocyte ö¿ m low in HIV-1-infected patients on non-nucleoside reverse transcriptase inhibitor-based HAART, which in the case of NVP is sufficient to induce the apoptosis cascade.
Apoptosis, redox stress and cancer.
(2000) Moodley, Thunicia.; Elliott, Edith.
Apoptosis is a regulated "programme" by which cells are induced to die in a manner which does not result in pathological inflammatory reactions, and involves dismantling of the cell into membrane-bound fragments that are removed by phagocytosis. This process is induced in order to remodel tissues and maintain homeostasis in cell numbers. Apoptosis may be induced via many pathways, many of which are redox-regulated, and is dysregulated in cancer cells, mainly due to mutational inactivation of certain pathways. Cancer cells also have a non-linear response to redox imbalance, a potentially exploitable characteristic for the therapeutic selective induction of apoptosis in cancer cells in mixed cell populations. Model cell culture systems are required for the selective toxicity testing of anti-cancer drugs, many of which work by inducing redox stress. In the current study, hydrogen peroxide was selected as the redox stress-inducing agent, and the test cells were an immortal, non-invasive breast epithelial cell line (MCFlOA) and its rastransfected, pre-malignant derivative (MCF10AneoT). A reliable, sensitive, cost effective and least time-consuming system for detection of apoptosis in such a system was sort and two novel methods, cytochrome c release and caspase-3 activity assays, were finally selected and compared with results seen by conventional DNA laddering and morphological examination at the light and electron microscopic level. No single procedure was found to be reliable individually. For the model system used, a combination of electron microscopy and DNA laddering was sufficient for simply detecting apoptotic cell death and necrosis. The caspase activity assay distinguished between apoptosis and necrosis, and cytochrome c release proved the most sensitive indicator of cell response. However, since cytochrome c release may be reversible and may not necessarily proceed to the downstream events of apoptosis in the time frame used in the current assays, it is not certain that cytochrome c release ultimately leads to apoptosis. However, three forms of cytochrome c were observed on western blots, the nature and significance of which remains to be determined. A comparison of the results of different methods allowed a model for the sequence of specific apoptotic events to be proposed.
The application of layered double hydroxide nanoparticles (LDHs) as potential anticancer drug delivery systems.
(2016) Mncwabe, Zoleka.; Singh, Moganavelli.
Chemotherapy being one of the principle techniques used in cancer treatment, has been applied in the treatment of a wide spectrum of cancers. However, this mode of treatment is fraught with a myriad of challenges, reducing its effectivity and inducing the need for repeated treatments. Poor drug delivery systems or lack thereof, have led to patients suffering unpleasant side effects that not only cause collateral damage to their bodies but also reduces the quality of their lives. The current array of chemotherapeutic drugs available may be effective in certain cancers, nevertheless the need for their optimization is still necessary for better safety, stability and efficiency of treatment. Thus the current study was designed to investigate the potential of layered double hydroxide (LDH) nanoparticles in the delivery of the broad spectrum anticancer drug, 5-Fluorouracil (5-Fu). Four LDH nanoparticles, MgAl 2:1, MgAl 3:1, ZnAl 2:1and ZnAl 3:1 were successfully synthesized and intercalated with 5-Fu using the calcination reconstruction process to form nanohybrids. The LDHs and their nanohybrids, MgAl 2:1-5-Fu, MgAl 3:1-5-Fu, ZnAl 2:1-5-Fu and ZnAl 3:1-5-Fu were structurally confirmed using XRD, FTIR, UV-Vis, ICP-OES; with size, zeta potential and ultrastructural morphology investigated using nanoparticle tracking analysis (NTA) and electron microscopy (TEM and SEM). LDHs were characteristically hexagonal in shape with sizes ranging from 100 -150 nm, and high zeta potentials enforcing their colloidal stability. The successful intercalation of 5-Fu was confirmed from drug encapsulation efficiency studies to be between 40-60% in the respective LDHs. Furthermore, drug release studies revealed a steady controlled release of the drug over a 7-hour period at pH 4-7, with more than 60% of the drug being released by the end of this period. In vitro MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and SRB (Sulphorhodamine B) cytotoxicity studies on free 5-Fu and LDH bound 5-Fu in human cell lines, breast adenocarcinoma cell line (MCF-7), hepatocellular carcinoma (HepG2), colorectal adenocarcinoma (CaCo-2) and embryonic kidney (HEK293), showed a dose dependent cytotoxicity profile with the free 5-Fu being more toxic to the cells than the bound drug. This was further confirmed in fluorescent apoptotic studies (dual acridine orange and ethidium bromide staining method), where free 5-Fu had a higher apoptotic index than the LDH bound 5-Fu
Assessment of genetic diversity and DNA fingerprinting of the Cape parrot (Poicephalus robustus) using randomly amplified polymorphic DNA (RAPD)
(2004) Blue, Gillian Margaret.; Fossey, Annabel.; Perrin, Michael Richard.
The Cape parrot (Poicephalus robustus) is South Africa's only endemic parrot. It has become increasingly rare in recent years, with fewer than 500 birds left in the wild, and is now regarded as endangered. Possible factors contributing to this rapid decline in numbers include habitat loss, food shortage, disease and illegal trafficking and trading in the species. Habitat loss and food shortage have been brought about by the rapid destruction of the yellowwood trees in the afromontane forests in South Africa and have played a role in reducing the population numbers. The Psittacine beak and feather disease virus (PBFDV) has also contributed to the loss of some individuals, however it is the illegal trafficking of this rare and valuable species that has become of great concern. As the Cape parrot is becoming increasingly rare and therefore highly sought after, its commercial value has multiplied to the extent that illegal black market trapping is on the rise. The industry involved in breeding and conservation of endangered bird species, has a need for the proper establishment of studbooks, containing all available information on captive as well as tagged birds. Most of the information found in studbooks is based on morphological attributes of individual birds. Although this is useful, there is a need to add molecular information in order for complete identification of individuals, particularly in a species threatened by illegal trading and theft. A preliminary analysis of the amount of variation present in the population of interest is therefore required so that appropriate methods and techniques can be developed to identify individual birds. A RAPD analysis was conducted to assess the amount of variation in the Cape parrot and lay the foundations for the establishment of individual identification in the species. Blood samples from 30 parrots, consisting of both related and unrelated individuals, were obtained from three separate locations: Amazona in Assagay, Rehoboth Farm in Dargle, as well as from the Eastern Cape. 15 random primers were selected and used to conduct a randomly amplified polymorphic DNA (RAPD) analysis. RAPDs are extremely useful in situations where relatively inexpensive first approximations of the genetic variation are needed, such as in rare and endangered species. After successful optimisation of the technique in the species, the 15 primers were screened for all 30 individuals and the individual DNA fingerprints, analysed. Clear, distinctive and reliable DNA fingerprints were obtained for all individuals however, it was interesting to note despite the analysis of 85 loci using the 15 primers almost identical DNA fingerprints were produced between the individual birds. A population analysis into the amount of variation present between and within the three populations, as well as for the representative population as a whole, was conducted. Using various statistical programmes such as POPGENE and ARLEQUIN, heterozygosities, genetic distance measures, diversity indices, Wright's fixation index and AMOVAs were estimated. The amount of polymorphism detected in this investigation was 33 % and the heterozygosity, 0.37, which is a relatively high value for the uniformity displayed in the DNA profiles. The high GC content of the primers however, could be a possible explanation thereof. Relationship and kinship determination, sex determination as well as population assignment was possible despite not being able to identify each individual based on unique DNA fingerprints. The AMOVA analysis indicated significant variation on both the between (5.59 %) and within (94.41 %) levels of analysis. Little variation or differentiation was observed between the three subpopulations, which was confirmed with an FST value of 0.056. The variation experienced within each subpopulation was analysed using Shannon's index of phenotypic diversity. The Amazona population displayed the most variation with a value of 0.286 and the Rehoboth population, the least with 0.195. This was expected, with the individuals from the latter population comprising one extended family. Nei's measures of genetic identity revealed that the individuals from Amazona were more similar to the Eastern Cape population, which was again expected with regular exchanging of chicks between the two breeders. RAPD technology was successful in laying the foundations for individual identification in the Cape parrot. It was also successful in producing reproducible DNA fingerprints in the species that were able to determine relatedness to some extent, determine the sex of individuals and identify individuals from a particular subpopulation. Furthermore RAPD analysis gave a good indication of the variation found in the Cape parrot population, which is important for conservation purposes. In order to maximize conservation efforts and strategies in an endangered species, determining the level of genetic diversity and variation found in the remaining individuals of the population is of great importance. This information could provide powerful insight for conservation purposes and depending on the level of diversity detected, appropriate breeding programmes could be set up in order to increase the genetic variation and thereby reduce the chance of extinction of the species. The following important findings emerged from this investigation: • RAPD technology, once optimised for the species of interest, is successful in producing clear and reliable DNA fingerprints, provided the same protocol is followed carefully throughout the investigation. • An optimised protocol for fingerprinting the Cape parrot using RAPDs was established. • Possible sex identification, population assignment and a degree of kinship determination was determined using RAPDs. • Little variation was found within the representative Cape parrot population as a whole due to small population size and possible inbreeding. • As expected for an avian species, little genetic sub-division or differentiation was observed between the three populations analysed.
Assessment of hypoxoside and its derivatives as anti-cancer drugs.
(2013) Xulu, Bongiwe Ziphelele.; Elliott, Edith.; Drewes, Siegfried Ernst.; Van Heerden, Fanie Retief.
Extracts of the African potato have long been believed to have anti-cancer properties. The aim of the current research was to isolate hypoxoside (HYP) from Hypoxis hemerocallidea (African potato) and synthesize the dimethyl (DMH) and decaacetyl (DAH) derivatives and to test their selective cytotoxicity on a model consisting of a normal (MCF10A) and premalignant, invasive breast epithelial cells (MCF10A-NeoT). Hypoxoside was extracted from the H. hemerocallidea corms using ethanol, purified using a C-18 reverse phase column and the compound examined by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry and found to be of high purity. This was also the case for the synthesized compounds. To assess possible selective effects (cytotoxicity) of derivatized and underivatized hypoxoside, effects on the metabolism of premalignant and normal cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Effects on cell number (total counts) and cell death [trypan blue and propidium iodide (PI) staining for dead cells versus a lack of staining for live cells] were, thereafter, assessed. Imaging of live adherent cells was also carried out using acridine orange (AO) and PI for live and dead cells (respectively). Propidium iodide staining of detached cells was carried out for flow cytometric determination of cell death (PI indicating early apoptotic or late apoptotic/necrotic cells). After treatment of normal (MCF10A) breast epithelial cells and premalignant cHa-rastransfected (MCF10A-NeoT) derivative breast epithelial cells with HYP, DMH and the DAH derivative, the MTS assay and the Duncan‟s multiple range, analysis of variance (ANOVA) post hoc analysis of the MTS results revealed that only the 150 and 300 µM DAH derivative had a statistically significant effect on the metabolic activity of the abnormal cell line relative to the dimethyl sulfoxide (DMSO) and revealed no significant effect on the normal MCF- 10A cell line after treatment with any of the test compounds. Supravital PI staining of adherent cells seemed to indicate a far higher rate of induction of cell death in abnormal cells than evident in the MTS assay and the PI-based flow cytometry or the trypan blue exclusion assays and need re-investigating, though result trends were similar. Total cell counts, show that HYP and its derivatives appear to increase both cancer and normal cell proliferation significantly, except in the case of DAH at 150 and 300 μM in the MCF10A-NeoT, without affecting the MCF-10A cell line. The trypan blue method for detection of cell death, together with total cell counts, the Duncan‟s analysis of MTS results and a 24 hour exposure to test compounds, seems to constitute an optimal system for drug screening and indicates the statistically significant selective toxicity of the DAH compound at 150 and 300 μM in the MCF10A-NeoT, suggesting that the DAH derivative at 150 and 300 µM would have significant, selective therapeutic potential on Ras-related malignancies.
Assessment of lysine damage during food processing.
(1985) Anderson, Trevor Ryan.; Quicke, George Venn.
The fluorodinitrobenzene (FONB), succinic anhydride (SA), dansyl chloride (DAN), dye-binding lysine (OBL), total lysine (TL), ninhydrin (NIN) and Tetrahymena lysine (TET) methods were compared for their ability to assess available lysine in soyaprotein heated in the absence or presence of glucose, lactose or xylose and in formaldehyde-treated lactalbumin. The reactive lysine methods showed comparable sensitivity to lysine damage in soyaprotein heated in the absence of sugar, the results indicating the presence of acid labile isopeptides and unidentified acid stable derivatives. Results for soyaprotein heated with glucose, lactose or xylose showed that the type of sugar and the extent of heat treatment has a strong influence on the progress of the Maillard reaction. Furthermore since fructoselysine (F-L) and lactulosyl-lysine (L-L) are colourless up to 30% loss of available lysine can occur without any change in product colour. The FONB method is the most sensitive for mildly damaged glucose-soya samples followed by DAN or OBL, SA and TL whereas for mildly damaged lactose-soya samples the order is OBL, FONB, SA, TL and DAN. For severely damaged samples the DAN or SA methods were the most sensitive followed by OBL, FONB and TL. Formylation of lactalbumin occurred more readily at higher formaldehyde concentrations. Exposure time had less effect while pH (5 and 9) had no effect. Methylene derivatives reached maximum levels sooner than the methylol compounds. Lysine and tyrosine but not histidine formed methylene bridges while tyrosine was found to condense with free formaldehyde during acid hydrolysis raising questions as to the interpretation of similar studies reported in the literature. The FONB, OBL and DAN methods were all very sensitive to this type of damage with the NIN and TL methods being less sensitive and the SA method being completely unsuitable. The TET assay is unsuitable for 'early' Maillard damage since at low sample-N levels growth is stimulated by its ability to utilise unavailable F-L and L-L while at higher N-levels growth is inhibited. No single method is most suitable for all types of damage. Furthermore, all except DAN and DBL are either too long, rather complicated, require expensive equipment or involve the use of dangerous chemicals. The DAN method appears promising but the problem of converting arbitrary fluorescence units to lysine values needs to be overcome. The DBL is recommended for routine analysis since it is simple, economical and highly sensitive to all lysine damage provided care is taken to optimise dye-binding for each type of material analysed.
Bio-guided isolation of biologically active compounds from seeds of selected South African medicinal plants.
(2016) Perumal, Amanda.; Govender, Patrick.; Naidoo, Sershen.; Pillay, Karen.
Abstract available in PDF file.
A biochemical and immunological comparison of the Jaagsiekte and two related retroviruses.
(1987) York, Denis Francis.; Verwoerd, D. W.; Dennison, Clive.
Jaagsiekte is a contagious cancer affecting the lungs of sheep. Although the etiological agent is Jaagsiekte retrovirus (JSRV), two other retroviruses viz South African maedi visna virus ( SA - OMVV) and a novel Bovine retrovirus (BRV) have been associated with or implicated in the jaagsiekte disease complex. JSRV was sufficiently purified from lung rinse material using a Freon extraction, Percoll density gradient centrifugation and chranatography on a Sephacryl column, its polypeptide composition was studied by gel electrophoresis and its morphology observed electron microscopically. Monoclonal antibodies were made against purified preparations of the virus. Two hybridomas were isolated that produced MAbs which appear to be tumour cell specific. A third hybridoma, called 4A1O, produces antibodies considered to be viral specific. These MAbs have been used in the development of JS specific immunoassays. A cross reaction between JSRV and a polyclonal serum against Mason Pfizer monkey virus (MPMV) was confirmed and used in a Western blot technique to identify, monitor and differentiate JSRV from other viruses. During the study of JSRV it became apparent that another retrovirus was often present in JS infected lungs. This virus, referred to as SA - OM1V I, is a novel South African isolate of maedi visna virus (MVV). As SA - OM1V I has physicochemical characteristics similar to JSRV, it was often found in purified JSRV preparations. Being a retrovirus it is also detected by the reverse transcriptase assay which was the only method used to assay and monitor for JSRV during the early stages of our work. Using a Westen blot technique and sera against MVV and MPMV it was possible to simultaneously detect and differentiate JSRV from SA - OMVV I. A method was also developed whereby the two viruses could be separated from each other during purification. The information gained and techniques developed whilst studyiing JSRV were also used to isolate and characterize BRV. This novel virus originated from bovine cells that had been co-cultivated with white blood cells from an ox suffering from malignant catarrhal fever. Three out of four sheep inoculated with BRV developed JS. It therefore had to be· ascertained whether this virus was related to JSRV or not. The comparative study revealed that BRV was biochemically and morphologically quite different fran JSRV. Interestingly, it was shown that serum against MPMV cross reacted with a 32 kd protein of BRV indicating a serological relationship between JSRV, MPMV and BRV. The possible role of BRV in the etiology of jaagsiekte remains to be elucidated.
Biochemical and microbiological changes in sugarcane stalks during a simulated harvest-to-crush delay.
(2008) Martin, Lauren Anne.; Hunter, Charles Haig.; Watt, Derek Alexander.
Post-harvest cane deterioration in the South African sugar industry results in significant revenue loss that is estimated to be in the region of ZAR 60 million per annum. Despite these large losses, precise biological data relating to the process of cane deterioration under South African conditions is limited. Severity of deterioration is influenced by a number of factors, including the length of the harvest-to-crush delay (HTCD), ambient temperature and harvesting practices. For example, burning of cane prior to harvest may result in rind splitting, which provides entry for microbes, particularly Leuconostoc mesenteroides that may exacerbate deterioration. The effect of these factors on deterioration was examined by quantifying the biochemical and microbiological changes that occur in sugarcane stalks after harvest, with the influence of length of HTCD, degree of L. mesenteroides infection and ambient temperature receiving attention. The primary novelty of the work resides in the analysis of deterioration under tightly regulated temperatures, which were designed to reflect diurnal variations typically experienced during summer and winter in the South African sugar belt. In addition, inoculation of mature internodes with a consistent titre of L. mesenteroides was used as a means to mimic a consistent level of infection of harvested stalks by the bacterium. Metabolites selected for analysis were those both native to the stalk and produced as by-products of microbial metabolism, viz. sucrose, glucose, fructose, ethanol, lactic acid, dextran and mannitol. Simulated HTCDs under summer temperatures resulted in increasing glucose and fructose levels with time, which contrasted to the approximately constant levels of these hexose sugars under winter conditions. Commonly referred to as ‘purity’ in an industrial context, precise determination of the concentration of these hexoses in cane consignments could potentially indicate the extent of deterioration. Despite the detection of a basal concentration of lactic acid in unspoiled cane, the observed increase in concentration of this organic acid over the simulated summer HTCD suggests that this metabolite could also potentially serve as an indicator for postharvest deterioration. In contrast, the investigation indicated that ethanol was an unsuitable biochemical marker for deterioration of L. mesenteroides infected cane. An inability to detect dextran and mannitol in the samples, combined with consistent sucrose levels and variable mill room data, suggest that extreme proliferation of L. mesenteroides is facilitated primarily by in-field practices, particularly the manner in which cane is prepared prior to harvest and transport to the mill. Bacterial proliferation and infection by L. mesenteroides of inoculated stalks were monitored by standard selective culturing techniques. Despite the limited detection of L. mesenteroides-associated metabolites, culture-based analyses revealed that the bacterium was the dominant bacterial species within the samples. A number of other bacterial species were isolated and identified, however the extent to which the total number of microorganisms proliferated was limited to a maximum of 1 x 105 colony forming units per gram of fresh tissue. In conjunction with these analyses, a molecular approach known as Polymerase Chain Reaction-Mediated Denaturing Gradient Gel Electrophoresis (PCR-DGGE) was undertaken to investigate the bacterial diversity patterns associated with deteriorating sugarcane stalks throughout the delay period. In contrast to the results obtained by means of the culture-based assays, PCR-DGGE revealed that L. mesenteroides was not the dominant bacterial population, and showed that the level of bacterial diversity was relatively consistent across the differing treatments and with time. The use of complimentary culture-dependent and cultureindependent analyses thus permitted the detection of this discrepancy and indicated the utility of PCR-DGGE in the determination of bacterial community structure of postharvest sugarcane tissue. The biology of post-harvest deterioration of green sugarcane stalks is highly complex, even under rigorously controlled temperature and infection regimens. The results of this study emphasize the important effects that harvest method and environmental conditions have on post-harvest sugarcane deterioration. Towards the formulation of industry-relevant recommendations for combating post-harvest deterioration, future work will strive to mimic the effects that harsh harvesting and transport practices have on the severity of the problem.

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