Biochemistry and Microbiology

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Characterization of antibiotic resistance in Enterobacteriaceae isolates from the Mhlathuze River
(2003) Biyela, Precious Thabisile; Lin, J.
The wide and indiscriminate use of antibiotics often results in the establishment of a pool of antibiotic resistance in the environment. In order to establish the state of bacterial resistance to antibiotics in the Mhlathuze River, 114 enteric bacteria were isolated from water samples collected from this river over a period of two years. The isolates were identified using the culture methods and confirmed by the API 20E system. The isolates were then tested for their susceptibility or resistance to a battery of 15 antibiotics. Those that showed multiple antibiotic resistance, 43 in total were screened for the presence of classl integrons and the associated antibiotic resistance genes using the polymerase chain reaction (PCR). The resistance of the enteric bacteria isolated over a period of one year showed that resistance to the older classes of antibiotics was high (94.7 % resistance to one antibiotic and 80.8 % resistance to two antibiotics). Furthermore, antibiotic resistance data of the environmental isolates showed a strong correlation (r= 0.97) with data obtained from diarrhea patients. PCR based methods demonstrated that class 1 integrons were present in more than 50% of the environmental bacterial isolates that were resistant to multiple antibiotics. This class of integrons is capable of transferring genes responsible for resistance to beta-lactam, aminoglycoside, sulfonamide and quaternary ammonium antimicrobial agents. Conjugate plasmids were also isolated, but from a small percentage of isolates. This study showed that the Mhlathuze River (i) is a medium for the spread of bacterial antibiotic resistance genes (ii) acts as a reservoir for these genes and (iii) due to socio-economic pressures may play a role in the development and evolution of these genes along this river system.
Microbiological evaluation of the Mhlathuze River in KwaZulu-Natal
(2004) Mthembu, Nompumelelo; Lin, J.; Basson, A.K.
High levels of faecal coiifonns pose a treat to the health of the rural community that uses the river water directly for domestic use without treatment. The microbiological, physical and chemical analysis of the Mhiathuze River was investigated over a twenty-one months period. Five water samples were collected along the Mhiathuze River and analysed to monitor the indicator bacteria pollution with changing seasonal patterns. Surface water temperarure and rainfall during the period of study appeared to be some of the factors affecting the increased bacterial counts. Elevated levels of indicator microorganisms (both faecal and total coiifonns) and heterotrophic plate count bacteria were observed from March 1998 to November 1999. Bacteria isolated from the river included Escherichia coli, Pseudomonas spp., Enterobacter spp.r Serraiia spp,, Klebsiella spp. and Aeromonas hydrophila. The average monthly pH values ranged between 6.5 and 8.5. The turbidity, dissolved oxygen, hardness, ortho- and total phosphates values obtained did not show any major changes that would call for caution. A polymerase chain reaction (PCR) method was used to amplify E6S rRNA and phoP gene fragments from the isolated bacteria and directly from the water resource. Annealing temperature was adjusted to set up the optimum conditions of the PCR mixture. Performing serial dilutions of DNA carried out the sensitivity of detection for PCR products. It was deduced that amplifications with phoP and 16S rRNA primers were visualised up to 10"' and 10"° ug of DNA, respectively. Multiplex PCR with the two primers generated an amplification product of approximately 755 bp for all environmental isolated used and an additional 299 bp product for E. coll C. freundii and C. ctversus. Rsa I and Hinf I restriction enzymes were also used for double digestion ofE.coli and P. vuigaris.
Anti-diabetic properties of GUH
(2006) Gabuza, Kwazi B.; Opoku, A.R.; Louw, J.; Woodroof, C.
Objective: To investigate anti-diabetic properties of the plant extract known as GUH in comparison to commercially available drugs metformin and rosiglitazone. Methods: Male Wistar rats were fed a maintenance diet (MD) with tap water or a high fat diet (HFD) with sucrose solution for a period of twelve weeks. Thereafter a separate groups of animals were then treated with GUH at 5 different dose levels, metformin or rosiglitazone for twelve weeks. Food intake, liquid intake, weights, blood glucose, and insulin were monitored throughout the treatment period. An intravenous glucose tolerance test (IVGTT) was performed on representative animals from each treatment group prior to termination. At termination blood was taken and total cholesterol, tri-acylglycerol (TAG), low density lipoproteins LDL, and high density lipoproteins (HDL) were measured. Results: The effect of GUH in MD fed rats was not marked. However, food intake and weight gain and total cholesterol were lower than in control animals. In HFD fed animals GUH, metformin and rosiglitazone had a significant effect The extract reduced blood glucose and increased circulating insulin levels when compared to controls but results were not significantly different to metformin and rosiglitazone treated animals. As with metformin and rosiglitazone, GUH increased food intake with a concomitant weight increase. This weight increase was, nevertheless, less than with the other 2 treatments. In HFD fed animals GUH at the highest dose level increased the glucose clearance rate to a greater extent than metformin and rosiglitazone. Conclusion: The results conclusively show that the extract GUH was at least as effective, and in some instances more effective, than currently used diabetes treatments. Although further work is required to investigate the mode of action, it is evident that extracts of indigenous South African plants can be cost effective and efficacious treatments.
The study of rehabilation of hydrocarbon contaminated soil using bio-remedial microbes
(2006) Shandu, Jabulani Siyabonga; Basson, A.K.; Kelbe, B.E.
The study investigated the feasibility of bioremediation as a treatment option for chronically diesel-oH-poHuted soil at petroleum and gas depot of Oilco (a company that is a division of Shell) situated at the east side of Empangeni which is in the Northern KwaZulu-Natal province, South Africa. To examine the efficiency of bioaugmentation, the contaminated site was treated with microbes, (previously isolated from the diesel-contaminated soil) to a depth of ±1, 2 meters, ±5 meters wide and 2 meters in length, plus the woodshavim^ as their nutrient source. The effectiveness of bioremediation was observed over a period of 11 weeks and samples were taken at 15-day intervals. Over that period of 11 weeks, the changes in hydrocarbon concentrations were monitored in the soil and soil leachate and the accompanying changes in the soil microbial counts and activity. A significant reduction in the diesel-oil level could be achieved. The BTEX method was used in GCMS to check for changes in TPH. Prior to GCMS analysis the soil texture was analyzed using the Particle Size Determination method and the soil was observed to be sandy-loam (Day, 1995). For checking the soil microbial counts and activity the following groups of microbes were observed Aerobic Total Counts, Nitrofyers, Nitrosojyers and Free-living nitrogen fixing bacteria (Chan^L aL, 1993). The four groups of microbial counts were used as a biological parameter, and there was a correlation between each other as well as with the residua] hydrocarbon concentration, indicating the importance of biodegradation. The effect of biostimulation of the indigenous soil microorganisms declined with time during the study.
Effect of pumpkin seed (cucurbita pepo) protein isolate on the antioxidant enzymes in ccl4-induced liver injury in low-protein fed rats
(2007) Nkosi, Cynthia Zanele; Opoku, A.R.; Terblanche, S.E.
The effect of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of lactate dehydrogenase (LD), alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) against carbon tetrachloride (CCU)-induced acute liver injury in low-protein fed rats were investigated. A group of male Sprague-Dawley rats which were maintained on a low-protein diet for five days were divided into three subgroups. Two subgroups were injected with carbon tetrachloride and the other group with an equivalent amount of olive oil. Two hours after CCU intoxication one of the two subgroups was administered with pumpkin seed protein isolate. All three subgroups of rats were maintained on the low- protein diet for the duration of the investigation. Groups of rats from the different subgroups were sacrificed at 24, 48 and 72 hours after their respective treatments. After five days on the low-protein diet the activity levels of ail four enzymes were significantly higher than their counterparts on a norma! balanced diet. CCI4 intoxication resulted in significant increases in the activity levels of ail four enzymes investigated. The administration of pumpkin seed protein isolate after CCI4 intoxication resulted in significantly reduced activity levels of all four enzymes, it is concluded that pumpkin seed protein isolate administration was effective in alleviating the detrimental effects associated with protein malnutrition.
The usefulness of multiple antibiotic resistance (MAR) indexing technique in differentiating fecal coliform bacteria from different sources
(2008) Mthembu, Mathews Simon; Djarova, T.; Biyela, P.T.; Basson, A.K.
Pollution of water sources with human fecal matter and associated intestinal pathogens poses a great risk to public health. Fecal contamination of water is not the only problem to communities that consume untreated water. The extent of the microbial contamination of water sources also needs to be considered when designing treatment regimes for the production of potable water. The more polluted the source of drinking water is, the more extensive and expensive treatment regimes have to be used to produce microbial risk-free water. For decades fecal coliform counts have been used as indicators of fecal contamination and the potential presence of intestinal pathogens in surface waters. However, fecal coliforms fail to provide information about the source of fecal contamination. Knowing the source of fecal contamination is vital in managing this problem in surface waters. This study explored the use of two techniques, multiple antibiotic resistance (MAR) indexing and caffeine detection as means of differentiating E. coli isolates from various sources. A total of 322 E. coli were isolated from domestic and wild animals as well as human sewage by using conventional culture methods. Standard chemical and biochemical tests were used to identify these isolates. All isolates were assayed against a battery of 10 antibiotics using the micro-dilution method. The results obtained were used to generate antibiotic resistance profiles which in turn were used to statistically group the isolates into different subsets. Caffeine detection by Thin Layer Chromatography (TLC) was used to differentiate between human and non-human derived E. coli isolates. The correct classification rate was 78% when MAR indexing was used and 50% when using caffeine detection. Sixty percent of E. coli from humans were correctly classified and 95.5% of E. coli from animals were correctly classified as non-humans sources respectively. The results of this study underscore the validity of MAR indexing as a method of bacterial source tracking. MAR indexing has great discriminatory power without the complexities and the high costs often associated with established genotype-based methods. Caffeine detection indicated an average classification rate (50%). With further research, caffeine detection may give another option for source tracking when genotyping methods are limited by either costs or lack of expertise. The use of combined techniques may provide a much more reliable and cost-effective option for bacterial source tracking when each technique used provide similar results.
The antimicrobial activity of five food spices when tested against various gram-positive –and gram-negative microorganisms
(2008) Seepersad, Kashimee; Basson, A.K.; Djarova, T.; Shandu, J.S.
The discovery of antibiotics by Alexander Fleming in the early nineteen hundreds not only created an enormous breakthrough in medical treatment but along with it introduced the emergence of new and now what is considered an ever increasing number of multi-drug resistant pathogens. Like antibiotics, herbs and spices have been used traditionally by many, for the treatment of various aliments ranging from stomach indigestion, lesions of the skin to beauty therapy. At present it is estimated that about 80% of the world population rely on botanical preparations as medicines to meet their health needs as opposed to treatment by conventional medicine with spices creating a shelf of its own in the global medical cabinet. In this study, the antimicrobial potential of five spices (commonly known as ginger, cinnamon, turmeric, nutmeg and chilli) was analysed against various Gram positive- and Gram negative microorganisms namely, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Salmonella spp, Shigella spp and Staphylococcus aureus. Analysis of the results of sensitivity tests (disc and agar well diffusion assays) indicated each of the microorganisms to be completely inhibited, intermediately inhibited or completely resistant towards a particular spice extract. The formation of zones of inhibition present where inhibition had occurred indicated that the spice tested was effective as an antimicrobial agent when screened. Zones of absolute inhibition (greater than fifteen millimetres in diameter) were obtained during positive agar well and disc diffusion assaying with neomycin used as the antimicrobial agent of choice. Inhibition zones observed to be in the upper limit range (pertaining to the study) of 20 mm – 27 mm in diameter. Comparative studies using the test spices indicated that chilli, turmeric, nutmeg, cinnamon and ginger each demonstrated zones of inhibition within this limit at one or more laboratory testing. Chilli was the most active antimicrobial agent when tested and in some instances demonstrated antimicrobial effectiveness greater than that exhibited by the positive control neomycin. Turmeric, nutmeg, cinnamon and ginger however each demonstrated inhibition within the same range as that of neomycin. The observations of such inhibition amongst the spices were comparatively significant and demonstrated the potential use of these spices as antimicrobial agents with an efficacy that can be compared to that of the already recognized and widely used antibiotic, neomycin. The minimum inhibitory concentration (MIC) was successfully determined for each of the spice extracts. The reactions observed during MIC determination were confirmatory of the antimicrobial activity present in the extracts of each spice. Analyses of the results conclude that the active compounds present in the selected spices were effective against certain microbial species. This observation demonstrated that spice can and may be used in the treatment of bacterial infections. This could in the future be an alternative treatment to antibiotics for one or all of the microbial species investigated and in so doing allow the healing powers of spices to be acknowledged.
Anti-nutritional constituent of Colocasia Esculenta (Amadumbe) : a traditional crop food in Kwazulu-Natal
(2008) McEwan, Ronalda; Opoku, A.R.; Djarova, T.; Oyedeji, O.A.
Colocasia esculenta L. Schott belongs to the family Aracea and is grown for its edible corms as a staple food throughout subtropical and tropical regions of the world. Amadumbe (the Zulu name for Colocasia esculenta) is consumed by and holds an important place in the diet of local rural people in Kwazulu-Natal, South Africa. Three Amadumbe phenotypes were evaluated for their nutritional qualities. Like all known tubers, the locally grown Amadume contained high carbohydrate levels, adequate protein and low lipid content. Essential fatty acids (linoleic and linolenic) were identified as components of the Amadumbe lipids. Amadumbe was generally low in mineral content, apart from potassium and magnesium levels that were relatively high. Some anti-nutrients (protease inhibitors, lectin, phenolic compounds, alkaloids, oxalates, phytates, cyanogens and saponin) present in Amadumbe were also identified and quantified. The anti-nutrient levels were generally low and thus may not pose an immediate effect on the health of consumers. Reduction of the anti-nutrients through processing (cooking, frying, roasting) was observed to enhance the nutritional value of these tubers. However, their presence suggests that a steady consumption may lead to toxic levels. Two proteins (Al and B2) with a-amylase inhibitor activity, and a steroidal saponin (gamma-sitosterol) were extracted and partially characterised. The a-amylase inhibitors were extracted and partially purified through ammonium sulphate precipitation and chromatographic fractionation on diethylaminoethyl (DEAE)-Sephacel and Sephadex G-100. The molecular weights of the two inhibitors were estimated to be 17 000 and 19 000 dalton, respectively. The inhibitors were fairly heat-stable, with optimum activity at 40° C? pH 6.0. Both inhibitors showed activity against mammalian a-amylases, but were devoid of activity against fungal amylases. Inhibitor A also showed activity against plant amylases. The steroidal saponin extracted from Amadumbe was characterized through TLC, HPLC, GC-MS, IR and NMR spectroscopic analysis and identified to be gamma- sitosterol, an isomer of beta-sitosterol which is known to have a variety of high biological activity. Studies of the effect of beta-sitosterol on absorptive and digestive enzymes in Sprague-Dawley rats revealed that oral administration of beta-sitosterol had no apparent gross or microscopic lesions in the liver, kidney or small intestine. The administered p-sitosterol significantly decreased serum aspartate aminotransferase (ALT) and alanine aminotransferase (AST) levels. Na+/K -ATPase and intestinal disaccharidases activities were also significantly reduced in beta-sitosterol fed rats. These results do suggest that even though Amadumbe is a neglected crop in South Africa, it is a highly nutritional crop; the consumption of it could be beneficial to diabetic and hypertensive patients.
Antimicrobial activity testing of traditionally used plants for treating wounds and sores at Ongoye area KwaZulu-Natal, South Africa
(2009) Mthethwa, Ntombeziningi Shirley; de Wet, H.; Basson, A.K.
This study focused on the investigation of plants used for the treatment of wounds and sores by local people living around the Ongoye forest, KwaZulu-Natal. An ethnobotanical survey was conducted in eighty homesteads in this area. The ethnobotanical data revealed that 33 plant species were used in treating sores and wounds, but only 15 plant species were collected from the wild and homesteads and three plant species were bought from a muthi market. According to the ethnobotanical information Hypericum aethiopicum (unsukumbili) was the most used plant for treating sores and wounds in this area. The survey showed that women (62.5%) possessed more knowledge than the men (37.5%) who were interviewed at the homesteads regarding the medicinal uses of plants. Acetone, methanol, cold and hot water extracts from the different plant parts (bark, leaves, stems and the whole plant) were done on 18 species. These plants species are: Acanthospermum australe, Acorus calamus, Albizia adianthifolia, Baccharoides adoensis, Clerodendrum hirsutum, Combretum erythrophyllum, Faurea saligna, Gerbera ambigua, Gunnera perpensa, Hypericum aethiopicum, Hypoxis hemerocallidea, Lippia javanica, Pentanisia prunelloides, Sclerocarya birrea, Solanum aculeastrum, Trichilia dregeana, Warburgia salutaris, Ziziphus mucronata. The above-mentioned plants were screened for antibacterial activity against the following bacteria strains: Bacillus subtilis (6051), Escherichia coli (7751, U1405s, U16406, U16403), Klebsiella pneumoniae (13883), Staphylococcus aureus (12600, P5020, P4790, T1266), ‘Salmonella spp., Shigella flexneri and Shigella sonnei’. The antibacterial activities were determined by disk-diffusion, agar-well diffusion, minimum inhibitory concentrations (MIC) and bio-autographic methods. The plant extracts were also screened for the following phytochemicals: alkaloids, flavonoids, saponins, anthraquinones, cardiac glycosides and tannins. The following plants were the most effective against the micro-organisms tested: Gunnera perpensa, Hypericum aethiopicum, Hypoxis hemerocallidea, Lippia javanica, Pentanisia prunelloides, Trichilia dregeana and Warburgia salutaris. The bio-autographic results showed several compounds separated on the TLC with activity against the test organism, Staphylococcus aureus (ATCC2600). This study thus lends some support to traditional knowledge and may serve as a basis for selecting the most active medicinal plants to use in traditional medicine practices in the future.
Lactogenic activity of Gunnera perpensa L. (Gunneraceae) from South Africa
(2010) Simelane, Mthokozisi B.C; Opoku, A.R.; Djarova, T.
Gunnera perpensa L. (Gunneraceae) is a medicinal plant used by Zulu traditional healers to induce labor, expel the placenta after birth, to relieve menstrual pains, and to stimulate milk production. Phytochemical screening of the rhizomes revealed the presence of alkaloids, flavonoids, steroids, saponins, tannins and glycosides. Methanol extracts of G. perpensa exhibited strong scavenging of 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) and 3-ethylbenzothiazoline-6-sulfonate (ABTS), but showed poor radical scavenging of nitric oxide, superoxide and hydroxyl radicals. At a concentration of 5 mg/100 ml, the extract was able to inhibit lipid peroxidation of the whole rat brain homogenate (71%) and lipoxygenase (30%) activity. The plant extract also contained reduced form of nicotinamide adenine dinucleotide (NADH, 3.8 ρm/g), and total phenol (248.45 mg/g). The total antioxidant capacity was 36% relative to ascorbic acid (AA) and 64% relative to butylated hydroxyl toluene (BHT). The effect of an aqueous extract of the rhizome of the plant on milk production in rats was also investigated. Female lactating rats that received oral doses of aqueous extract of G perpensa produced more milk than controls (P<0·05). Pup weight gain was also significantly higher than that in the control group (P<0.05). In addition, the mammary glands of rats treated with the extract showed lobuloalveolar development. The extract of G perpensa was found to stimulate the contraction of the uterus; the highest amplitude was 5.06±08mm. G perpensa extract inhibited (23%) fish-brain acetylcholinesterase activity. The plant extract did not significantly influence prolactin, growth hormone, progesterone, cortisol, ALT, AST, and albumin levels. It is inferred that the plant extract exerts its activity on milk production and secretion by stimulating lobuloalveolar cell development and the contraction of myoepithelial cells in the alveoli. The cytotoxicity of the extract (LC50) to brine shrimp larvae was 137.62 mg/ml and to two human cell lines (HEK293 and HEPG2) it was 279.43μg/ml and 222.33μg/ml respectively. It is apparent that the antioxidant and lactogenic activity of G. perpensa contributes to its effectiveness in folk medicine.
In vitro anti-platelet aggregation activity of the extracts of protorhus longifolia
(2011) Mosa, Rebamang Anthony; Opoku, A.R.; Oyedeji, A.O.
Platelet aggregation beyond purpose of haemostasis is the underlying cause of blood-clotting related diseases. Concoctions of P. longifolia are used by Zulu traditional healers to manage such diseases. This work aimed at investigating the anti-platelet aggregation activity of the extracts of this plant and to identify and characterise the active components present and responsible for the anti-platelet aggregation activity. Phytochemical screening of the plant material revealed the presence of various secondary metabolites (tannins, flavonoids, alkaloids and terpenoids). Crude extracts (obtained by sequential extraction of plant material with hexane, chloroform, ethyl acetate, methanol and water) and two triterpenes (3-oxo- 5á-lanosta-8,24-dien-21-oic acid, and 3â-hydroxylanosta-9,24-dien-24-oic acid) isolated and characterized (using various chromatographic and spectrometric techniques-IR, MS, 1H-NMR, and 13C-NMR) from the crude chloroform extract were screened for antioxidant, anti-platelet aggregation, anti-inflammatory activities, and cytotoxicity. The antioxidant activity of the plant components was determined on DPPH and ABTS+ radicals. Their reduction potential and chelating activity on Fe2+ were also determined. Except the methanol extract (IC50 of 0.07 and 0.16 mg/ml), the crude extracts and the isolated compounds showed poor (< 50%) antioxidant activities as they weakly scavenged DPPH and ABTS+ radicals, exhibited low reduction potentials and poor Fe2+ chelating activities. The anti-platelet aggregation activity of both the crude extracts and isolated compounds was separately investigated on thrombin, ADP and epinephrine induced rat platelet aggregation. The extracts and the isolated triterpenes exhibited a concentration dependent anti-platelet aggregation activity induced by the three agonists. The highest activity by the hexane extract (IC50 of 0.59 mg/ml) was observed on the thrombin-induced platelet aggregation. In addition, the isolated compound also exhibited in vitro anticoagulant activity on the whole rats blood. The acute anti-inflammatory activity of the isolated triterpene was determined using the carrageenan- induced rat paw oedema model. The compound (500 mg/kg body weight) significantly (p<0.05) inhibited the acute inflammation of rat paw. The hexane and chloroform extracts showed weak cytotoxic effects on brine shrimps with LC50 39.6 and 54.7mg/ml respectively. Also the pure compound- 3â-hydroxylanosta-9,24-dien-24-oic acid exhibited weak cytotoxic effects on HEK293 and HEPG2 cell lines (IC50 8520 and 7960 ìg/ml respectively). These results support the use of P. longifolia in folk medicine in the management of blood-clotting related diseases.
The development of a molecular chaperone-based system to improve the heterologous production of Plasmodium falciparum AdoMetDC protein in E. coli.
(2011) Makhoba, Xolani Henry; Shonhai, A.
S-Adenosylmethionine decarboxylase/ornithine decarboxylase from Plasmodium falciparum (PfAdoMetDC/ODC) has been described as an ideal antimalarial drug target. However, the production of this large bifunctional protein to facilitate its biochemical characterization is hampered by the poor yields of recombinant protein. It has previously been proposed that co-expression of target recombinant malaria proteins with molecular chaperones of malarial origin in Escherichia coli could improve the yield of the target recombinant proteins. A cytosolic heat shock protein 70 (Hsp70) from Plasmodium falciparum (PfHsp70-1), has been previously shown to exhibit chaperone function when heterologously expressed in E. coli cells whose endogenous Hsp70 function was compromised. PfHsp70-1 presumably protected the E. coli cells against thermal stress by preventing protein aggregation. In the current study, PfAdoMetDC and PfAdoMetDC/ODC were expressed along with PfHsp70-1 in E. coli BL21 (DE3) star cells to improve the yield and quality of the PfAdoMetDC/ODC proteins. E. coli BL21 (DE3) star cells were transformed with plasmid constructs pMRBAD/PfHsp70-1 (encoding PfHsp70-1) and either pASK-IBA/PfAdoMetDC (encoding PfAdoMetDC, either as wild type or codon harmonized version) or pASK-IBA/PfAdoMetDC/ODC (encoding PfAdoMetDC/ODC, either as wild type or codon harmonized version), followed by induction to facilitate the production of the chaperone and the target proteins, respectively . Protein expression and solubility studies were conducted followed by purification of the PfAdoMetDC and PfAdoMetDC/ODC proteins using strep-tectin column. The co-expression of PfAdoMetDC/ODC with PfHsp70-1 did not necessarily improve the production of the former. Although there is no evidence that PfHsp70-1 improved the solubility of PfAdoMetDC protein, it greatly improved the purity of PfAdoMetDC protein obtained following elution through the strep-tectin column. Whilst PfAdoMetDC were expressed from a codon harmonized coding sequence alone (expressed in the absence of PfHsp70-1) was purified associated with contaminants, the same protein purified after co-expression with PfHsp70-1 was obtained at a much higher purity level. This could have been due to PfHsp70-1 shielding nascent PfAdoMetDC, thus preventing its association with the contaminating species. On the other hand, PfHsp70-1 improved the expression and yield of PfAdoMetDC/ODC (wild type), but it had no effect on the yield of the protein expressed of the plasmid haboring the codon harmonized version. Thus PfHsp70-1 may have facilitated the folding and production of the PfAdoMetDC/ODC (wild type) protein whose folding could have been impeded due to potential delayed translation due to codon mismatch.
An in vitro study of the effects of free fatty acids on insulin mediated glucose uptake and metabolism by myocytes and fibroblast derived adipocytes
(2011) Mazibuko, Sithandiwe Eunice; Muller, C.; Opoku, A.R.; Pheiffer, C.
The incidence of type 2 diabetes (T2D) is increasing at an alarming rate, especially in developing countries, including South Africa. In developing countries, changes in lifestyle, especially the increase in the consumption of a westernized diet, rich in fats and sugar, is associated with hyperlipidemia, obesity and development of T2D. Skeletal muscle and adipose tissues are the major tissues involved with post-prandial peripheral glucose disposal in response to insulin. Insulin resistance reduces the ability to clear glucose from the circulation resulting in hyperinsulinemia and hyperglycemia with the development of T2D. High levels of plasma free fatty acids (FFAs), particulary saturated FFAs such as palmitate, are associated with insulin resistance in muscle and adipose tissue. Monounsaturated FFAs, such as oleate and essential FFAs such as omega 3 and omega 6 are claimed to be beneficial and to improve insulin sensitivity. Aim This study aims to determine the in vitro effect of the free fatty acids (FFAs) palmitate, oleate, omega 3 and omega 6 on insulin-stimulated glucose metabolism in myocytes and adipocytes. Materials and methods Mouse C2C12 and rat L8 cells were differentiated into myocytes and myotubules by serum deprivation, while mouse 3T3-L1 fibroblasts were differentiated into adipocytes by culturing in 3-isobutyl-1-methylxanthine (IBMX), dexamenthasone and insulin. Myocytes and adpipocytes were cultured in DMEM containing 5.5 mM or 20 mM glucose supplemented with 0.75 mM of each of the respective FFAs (i.e. palmitate, oleate, omega 3 and omega 6) for 24 hours. Thereafter media was replaced with fresh media containing 0.75 mM of each of the FFAs with or without insulin stimulation in the last hour. Glucose uptake was measured using a 2-deoxy-[3H]-D-glucose method. Glycogen and glucose-6-phosphate (G6P) concentrations were determined using commercial kits. Glucose oxidation was measured by 14CO2 release from cells incubated with glucose D-[14C(U)]. Cell viability and mitochondrial dehydrogenase activity was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide method. Quantitative real-time PCR was used to measure messenger RNA levels of selected genes involved in the insulin signaling pathway. Results The different FFAs tested had varying effects in C2C12 and L8 myocytes, and 3T3-L1 adipocytes cultured in media containing 5.5 mM or 20 mM glucose. Palmitate decreased both basal and insulin-stimulated glucose uptake in C2C12 myocytes cultured in 5.5 mM and 20 mM glucose. Oleate decreased insulin stimulated glucose uptake in cells cultured in 5.5 mM glucose but had no effect at 20 mM glucose. The essential FFAs had no effect on glucose uptake. In L8 myocytes, palmitate decreased both basal and insulin-stimulated glucose uptake at 5.5 mM glucose, while only insulin-stimulated glucose uptake was reduced at 20 mM glucose. Neither oleate, omega 3 nor omega 6 affected basal glucose uptake in cells cultured in 5.5 mM or 20 mM glucose. However, at 20 mM glucose these three FFAs reduced insulin-stimulated glucose uptake. In contrast to myocytes, none of the FFAs affected basal or insulin-stimulated glucose uptake in 3T3-L1 adipocytes cultured at 5.5 mM glucose. However at 20 mM glucose only palmitate reduced both basal and insulin-stimulated. In terms of glucose metabolism, palmitate decreased basal and insulin-stimulated G6P concentrations in C2C12 myocytes cultured in 5.5 mM and 20 mM glucose. Oleate increased basal G6P concentrations at 5.5 mM glucose, but had no effect at 20 mM glucose. Omega 3 increased and omega 6 decreased basal G6P concentrations at both 5.5 mM and 20 mM glucose. At 5.5 mM glucose, oleate had no effect on insulin-stimulated G6P concentrations, while omega 3 and omega 6 decreased G6P concentrations. At 20 mM glucose these three FFAs increased insulin-stimulated G6P concentrations. In L8 myocytes palmitate, oleate and omega 3 reduced basal G6P concentrations, while omega 6 increased basal G6P concentrations when cultured in 5.5 mM glucose. At 20 mM glucose palmitate and oleate decresed both basal and insulin-stimulated G6P concentrations, while only insulin-stimulated G6P concentrations were decreased by omega 3 and omega 6. In 3T3-L1 adipocytes basal G6P was not affected by all the FFAs at 5.5 mM glucose. However, insulin-stimulated G6P was reduced by palmitate and omega 3. All of the FFAs increased basal G6P concentrations at 20 mM glucose in 3T3-L1 adipocytes. Only oleate decreased insulin-stimulated G6P at 20 mM glucose. In C2C12 myocytes none of the FFAs had an affect on basal or insulin-stimulated glycogen concentrations at 5.5 mM or 20 mM glucose. In L8 myocytes cultured in 5.5 mM glucose palmitate, oleate and omega 3 increased basal glycogen concentrations. Palmitate, oleate and omega 6 decreased insulin-stimulated glycogen concentrations. At 20 mM glucose, oleate, omega 3 and omega 6 increased basal glycogen concentrations while none of the FFAs had an effect on insulin stimuated glycogen concentration. In 3T3-L1 adipocytes cultured in 5.5 mM glucose only omega 3 and omega 6 reduced basal glycogen concentrations. None of the FFAs effected glycogen concentrations at 20 mM glucose. In C2C12 myocytes cultured in 5.5 mM glucose, none of the FFAs affected basal glucose oxidation. Palmitate decreased and oleate inceased insulin-stimulated glucose oxidation. In L8 myocytes only palmitate decreased basal glucose oxidation. Palmitate, oleate and omega 6 reduced insulin-stimulated glucose oxidation at 5.5 mM glucose. In 3T3-L1 adipocytes none of the FFAs affected basal glucose oxidation at 5.5 mM glucose. At 20 mM glucose all of the FFAs reduced both basal and insulin-stimulated glucose oxidation in all three cell lines used. In C2C12 myocytes, palmitate downregulated insulin-stimulated mRNA expression of Irs1, Pi3k and Glut4 although this was not statistically significant.
Antiplasmodial/Antipyretic activity of some Zulu medicinal plants
(University of Zululand, 2011) Nethengwe, Mulalo Flexy; Opoku, A.R.; Shonhai, A.; Oyedeji, O.A.
Malaria is one of the major diseases that have partially paralysed the world‘s health presently. There is, therefore, an urgent need to identify new antimalarial drugs as the plasmodium species continues to gain resistance to presently used drugs. In developing communities where malaria is prevalent, people depend strongly on traditional medicine as a source of inexpensive treatment for this disease. Gardenia thunbergia T.A Sprague, Siphonochilus aethiopicus (Schweif.) B.L Burtt, Schotia brachypetala Sond., Acorus calamus L., Withania somnifera (L) Dunal in DC., Elaeodendron transvalense (Burtt Davy) R.H. Archer, Hypoxis hemerocallidea Fisch., C.A. Mey.&Ave-Lall., Vernonia adoensis Sch. Bip. Ex Walp. and Acanthospermum australe (Loefl.) Kuntze) are some of the medicinal plants commonly used by Zulu traditional healers in South Africa to treat malaria. Aim The study aims to determine the phytochemicals present in the plants, larvicidal, antioxidant, in vivo antipyretic and in vitro antiplasmodial activities as well as the cytotoxicity of the nine plants. The study also aims to isolate and purify the active compound in the most active plant extract. Material and methods Plants obtained from the muti market were botanically identified and were screened for phytochemicals; the appropriate portions of each plant were separately extracted v into dichloromethane, methanol and water solvents. Larvicidal activity against Culex quinquefascitus larvae was determined by incubating the larvae with the plant extracts for 24 hours, where after percentage mortality was calculated. The antioxidant activity of the methanol extracts of the plants was determined by measuring the decrease in the colour of an oxidative system in the presence of the plants extracts. The various antioxidant activities investigated included the free radical (DPPH, ABTS, super oxide, nitric oxide and hydroxyl) scavenging activity, Fe2+ chelating, reducing power, and total antioxidant capacity. Antipyretic activity was determined by treating different groups of pyretic rats with different concentrations of the plants extracts (100 mg/kg, 500 mg/kg and 1000 mg/kg). The pyretic condition was induced by subcutaneous injection of 12% brewer‘s yeast. Temperatures before and after treatment were also compared. The antimalarial activity of the plants extracts were also screened against the chloroquine sensitive plasmodium falciparum D10 strain. Tests were done in triplicate for three concentrations (20 μg/ml, 10 μg/ml and 5 μg/ml). The active extracts were screened for cytotoxicity using the MTT assay. The most active in vitro antiplasmodial extract was subjected to isolation, purification and characterization using chromatographic and spectrometric techniques-IR, GC-MS, 1H-NMR, and 13C-NMR. Results Phytochemical screening revealed the presence of saponins, terpernoids, flavonoids, anthroquenones, cardiac glycosides; alkaloids (the major active constituents of most antimalarial drugs) were also observed in A. australe which, amongst others, exhibited the most in vitro antiplasmodial activity. The plant extracts either killed or reduced spontaneous movement in Culex quinquefascitus larvae after 24 hours following treatment. Methanol extracts exhibited antioxidant (DPPH, ABTS scavenging, Fe2+ chelating) activity, albeit to varying degree of efficiency. The dichloromethane and methanol extracts significantly (p≤ 0.05) reduced pyrexia with activity increasing in a concentration dependent manner. The antiplasmodial activity against chloroquine sensitive strain of Plasmodium falciparum (D10) showed that the methanol extracts of G. thunbergia, V. adoensis and the dichloromethane extracts of E. transvalense, and W. somnifera were active (IC50 of 1.04-5.07μg/ml). Although A. australe exhibited high in vitro antiplasmodial activity, the major compounds (Sitosterol and Stigmasterol) present in the extract did not exhibit any observable antiplasmodial activity. Conclusion The results support the use of some of these plants in folk medicine and suggest that these plants contained constituents that could be developed as potent antimalarial drugs (mosquito larvicide, anti-fever and anti-plasmodial).
In vitro anti-platelet aggregation activity of the extracts of bulbine natalensis
(2011) Lazarus, Geraldine Genevive; Opoku, A.R.; Oyedeji, A.O.
Bulbine natalensis Baker is a medicinal plant with succulent, aloe-like leaves from the Asphodelaceae family, commonly used by Zulu traditional healers to treat blood clotting-related diseases. The aim of this research was to test the anti-platelet aggregation effect of B.natalensis’ extracts on rat platelet aggregation separately induced by thrombin, ADP, collagen, epinephrine, papain, bromelain and trypsin. Fresh plant material was extracted sequentially using hexane, chloroform, ethyl acetate, methanol, and water. The chloroform extract had the highest yield of 0.20%. Phytochemical screening revealed the presence of anthraquinones, cardiac glycosides, saponins, tannins, flavonoids and alkaloids. The hexane extract revealed the highest contents of total phenols (5.028 mg/g); flavonoid (3.293 mg/g) and proanthocyanidin (2.565 mg/g). Hexane extract also displayed the highest ABTS scavenging activity (IC50 4.72 mg/ml) followed by the ethyl acetate extract (IC50 5.39 mg/ml). The water extract exhibited the highest reducing power; the activity was even greater than the standard antioxidant BHA. Studies reveal that B.natalensis is a good antioxidant. Brine shrimp lethality test indicated that all the extracts were highly toxic to the larvae. The ethyl acetate extract was the most toxic (LC50 2.21 mg/ml). Anti-platelet aggregation activities on rat platelets were observed. The chloroform extract inhibited ADP-induced clotting by 100% at doses 1 and 3 mg/ml with IC50 values of 5.32 mg/ml before tannin removal and >10 mg/ml after tannin removal. Two compounds (F6/1 and F6/5) were isolated and purified from the chloroform extract. It was apparent that the compounds were unstable and could not be
The detection of Plasmodium falciparum in human saliva samples
(2011) Pooe, Ofentse Jacob; Shonhai, A.; Mharakurwa, S.
Malaria still persists as a major public health scourge, claiming a global toll of up to 500 million clinical cases, mostly on African children. Current effective malaria diagnostic methods necessitate blood withdrawal from patients for accurate P. falciparum identification. Risks introduced by blood withdrawal can causes communities to be less cooperative to donate blood during malaria surveillance studies. Previous studies showed that saliva could be used to detect malaria by molecular techniques. This study, therefore, sought to establish the constituents of human saliva that harbours parasite DNA in malaria infected subjects. Furthermore the study optimised the use of Saliva as an alternative malaria DNA source in malaria infected patients. A total of 88 subjects were enrolled in this study, 35 (40, 7 %) were males and 51 (59, 3 %) were females. The age range was from 3 months to 99 years old (mean = 29.6 years; median = 18 years). Blood was drawn from each subject for subsequent use in microscopic examination and PCR tests. Saliva samples were also collected for PCR on Saliva derived parasite DNA. DNA was extracted using commercial kit on different saliva fractions from 46 malaria positive (thick film positive & blood PCR positives) individuals and 45 malaria negative individuals. Nested PCR was used to amplify malaria the Plasmodium falciparum dihydrofolate reductase (pfdhfr) gene. Generally PCR conducted on DNA purified from both blood and saliva was more sensitive than conventional microscopy, as previously reported. The pellet fraction of saliva was a more reliable and sensitive source of amplifiable parasite DNA compared to the soluble saliva fraction. After PCR optimisation amplification was enhanced 94.1% (sensitivity) and 97% (specificity) against DNA derived from blood as the gold standard. This study further confirms that saliva samples are a reliable non-invasive alternative to blood for the PCR detection of malaria, as previously reported. The source of DNA was primarily in the pellet fraction though; however, refinement is still needed to identify the exact source of DNA in that fraction.
The anti-platelet aggregation activity of Rapanea melanophloeos -A Zulu medicinal plant
(2011) Gwala, Phiwamandla Emmanuel; Opoku, A.R.; Oyedeji, A.O.
Rapanea melanophloeos (L.) Mez is a medicinal plant that is used by Zulu traditional healers to manage blood-clot related diseases.Various extracts (methanol, nhexane, chloroform, ethyl acetate and water) prepared from the bark of R. melanophloeos were screened for phytochemicals, antioxidant, and anti-platelet aggregation activity. Phytochemical screening of the plant showed the presence of tannins, terpenoids, alkaloids, saponins, cardiac glycosides, flavonoids and phlobatannins. Steroids and anthraquinone were however not detected. The extracts strongly (>70%) scavenged 1, 1 -diphenyl-2-picryhydrazyl and 2, 2-azinobis 3-ethyl-benzothiazoline-6-sulfonic acid free radicals. The extracts had 91% chelating effect on Fe2+ ions and exhibited concentration dependent reducing power. The extracts showed varying degrees of inhibition (21% to 97%) of rat platelet aggregation induced separately by thrombin, adenosine diphosphate (ADP) and epinephrine. The extracts further exhibited antiplatelets aggregation activity on enzymes (trypsin, papain and bromelain) treated platelets. The lethality of the extracts was tested on brine shrimps larvae. Hexane and chloroform extracts had LC50 values of 1068.731 mg/ml and 3648, 349mg/ml respectively. A triterpene (3β-Hydroxylanosta-9, 24-dien-21-oic acid) wasisolated and characterized (through various chromatographic techniques, extensive 1D and 2D NMR spectroscopy) from the ethyl acetate extract. The triterpene exhibited antiplatelet aggregation, and inhibition of acetylcholinesterase activity. The cytotoxicity of the triterpene on two cell lines (HEK293 and HEPG2) gave LC50 values of 851.5 μg/ ml and 796.0 μg/ ml respectively. The results suggested that the extracts of Rapanea melanophloeos could be considered as herbal treatment for disease associated with blood clotting.
The chemical composition, antimicrobial and antioxidant properties of the essential oils of Tulbaghia violacea Harv and Eucalyptus grandis W.Hill ex Maiden.
(2012) Sewanu, Soyingbe Oluwagbemiga; Opoku, A.R.; Oyedeji, A.O.
Tulbaghia violacea Harv. and Eucalyptus grandis W. Hill ex Maidan are medicinal plants used by Zulu traditional healers for managing respiratory track diseases such as asthma and bronchitis. This study was designed to evaluate the chemical composition, antioxidant and antibacterial activities of the essential oils hydrodistilled separately from the rhizomes of Tulbaghia violacea and the leaves of Eucalyptus grandis. Chemical profile of the oils were carried out using GC and GC-MS. The main constituents of the essential oils of Tulbaghia violacea, were found to be 2,4- Dithiapentane (51.04%), p- Xylene (4.43%),Chloromethylmethyl sulfide (8.62%), O- Xylene (6.08%), Thiodiglycol (6.17%), and p- xylol (5.88%); these together constituted 82.22% of the extracted oil. The main constituents (81.44%) of the essential oils of Eucalyptus grandis were m- Xylene (33.04%), Ethylbenzene (11.59%), Eucalyptol (15.50%), p- Xylene (9.61%), Limonene (3.48%), Operea 1(3.30%), p-cymene (2.75%) and Toluene (2.17%). While the oils of Tulbaghia violacea showed very weak activity (≤ 50%) in the scavenging of DPPH and ABTS radicals, they strongly (63% and 61%) scavenged nitric oxide and chelated Fe2+ ions respectively. The essential oils of Eucalyptus grandis, on the other hand, had a better scavenging activity for DPPH and ABTS, and the other free radicals tested (≥ 50%), but poorly chelated Fe2+ ions. The antimicrobial activity of the essential oils carried out on both Gram positive and Gram negative bacteria showed that the oils of Tulbaghia violacea were affective against 8 of the 16 microorganisms tested with minimum inhibitory concentration (MIC) values ranging from 2.5 mg/ml - 5.0 mg/ml; the oils of Eucalyptus grandis were active against 13 of the 16 organisms tested with the MIC‘s ranging from 0.625 mg/ml – 5.0 mg/ml, and the minimum bactericidal concentration (MBC) value determined for 4 of the bacteria used, ranging from 2.5 mg/ml – 10 mg/ml. The essential oils of Eucalyptus grandis were also tested against 8 bacteria that were resistant to antibiotics (CIPRO: Levo, Clindamycin, Gentimicin, Penicillin, OxaClox, Oxameth, Cotrimoxazole and Ampicillin) and were seen to show high activity against 7 of the 8 with MIC ranging from 5 mg/ml – 10 mg/ml. The studies on the effect of the essential oils on the DNA of the susceptible microorganisms revealed that the oils could not damage the microbial DNA. The cytotoxicity levels of the T. Violacea essential oils against HEK293 and HepG2 cell lines were low (IC50values of 1218 μM and 1641 μM respectively). It is apparent that the bioactivity of the essential oils of T. violacea and E. grandis contribute to the use of these plants in folk medicine.
Effects of Citrullus lanatus seed (Egusi) protein isolate on lipid peroxidation in malnourished rat fed on high fat diets
(University of Zululand, 2012) Ogunyinka, Bolajoko Idiat; Opoku, A.R.
Proteins play vital role in the normal physiology of an individual and the deficiency of it in the diet leads to many conditions often referred to as PEM. Due to high mortality rate among PEM children around the world, programs are aimed at improving the health and wellbeing of such vulnerable children. Animal proteins still remain the best source of good quality protein; however, those that are highly susceptible to PEM cannot afford protein of animal origin. There is need, therefore, to look at readily available proteins of plant origin. The potential of Citrullus lanatus (Egusi) seeds protein isolate, as an alternate protein source, on malnourished rats was investigated. De-hulled, deffated egusi seeds were extracted with water (pH 10). The extracted proteins were precipitated at pH 5, centrifuged and freeze-dried to obtain the protein isolate. Proximate composition shows that egusi seed isolate is rich in protein (63.4 %), and minerals. Functional properties indicate that the protein isolate has good oil absorbability and water binding capacity. The amino acid composition revealed the presence of a wide spectrum of essential amino acids. The administration of the protein isolate (20% incorporated into low diet) to malnourished rats resulted in the alleviation of the detrimental effects associated with protein malnutrition. The variables investigated were the serum enzymes (ALP, AST, ALT, GGT, LDH), serum lipid (cholesterol, TAG), haematological parameter (WBC, RBC, Hb, PCV, MCV, MCH, MCHC, RDW, Platelets, Neutrophils, Monocytes, Lymphocytes, LUC, Eosinophils, Basophils), and the histology of the liver and kidney. It is concluded that egusi protein isolate could be a candidate in the search for solution to PEM.
Analysis of the effects of Gold nanoparticles on the functional integrity of select serum proteins and heat shock proteins of mammalian origin
(2012) Luthuli, Duncan Sifiso; Shonhai, A.; Revaprasadu, N.
Gold nanoparticles (AuNPs) are a natural starting point for understanding nanoparticle-protein interaction due to their possible applications in biomedical functions, such as disease diagnosis and drug delivery. This has driven interest to understand the effects of AuNPs on the functional and structural integrity of heat shock proteins (Hsp) and serum proteins. When AuNPs are used for medical purposes through the intravenous route, they may be modified by serum proteins and these modifications may give rise to pathologies, or alter the intended purpose of the nanoparticle. Furthermore, Hsp are ubiquitous proteins that occur in cells and are upregulated under stress. It is envisaged that Hsp may also interact with AuNPs delivered to cells and/or the blood circulatory system. In this study, I sought to analyse the interaction between AuNPs and bovine serum albumin (BSA), citrate synthase (CS), malate dehydrogenase (MDH) as well as human heat shock protein 70 (Hhsp70). AuNPs were synthesised by a citrate reduction method in the presence of cysteine as the capping agent, and analysed using UV/visible spectroscopy and transmission electron microscopy (TEM). The effects of AuNPs on the stability of BSA, MDH, Hhsp70 and CS to heat stress were assessed spectroscopically, both in the presence and absence of AuNPs. I further investigated the effects of AuNPs on the function of Hhsp70 in suppressing the aggregation of MDH. Data observed in this study suggested that, the interaction between AuNPs and proteins (BSA and Hhsp70) may be facilitated by sulfhydryl (SH) groups present in them. It was also observed that AuNPs have capabilities of suppressing heat induced aggregation of MDH and CS. Thus AuNPs have chaperone activity as they are capable of maintaining proteins in their soluble, functional forms during heat stress.

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