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- ItemOpen AccessPurification and Physicochemical Characterization of Lectin from the Seeds of Treculia Africana Decne(2015-08-21) Adeniran, Olukemi AdetutuThe purification and characterization of the lectin from the seeds of Treculia africana Decne were carried out in order to determine some of its physicochemical properties as well as evaluate its toxicity in mice so as to understand the phenomenon of food intolerance caused by lectins. The protein in Treculia africana seeds was extracted by stirring the powdered seeds in phosphate buffered saline for 3 hours, kept overnight, and followed by centrifugation at 3000 rpm. The purification of the hemagglutinating protein was by ion-exchange chromatography on a DEAE-cellulose column followed by gel filtration on a Sephadex G-100 column. The apparent and subunit molecular weight were determined by gel filtration and SDS-polyacrylamide gel electrophoresis respectively. Detection of covalently bound carbohydrate was done by staining the gels after electrophoresis with periodic acid Schiff s reagent. The specificities of the crude extract and the purified protein were defined with human blood groups and sugars. The effects of temperature and pH on the hemagglutinating activity were determined by incubating aliquots of the protein at different temperature and pH values. The metal ions (Cu2+, Fe2+, Cr2+, Ni2+. Zn2+, Co2+, mg2+, Mn2+ and Ca2+) concentrations were determined by Atomic Absorption Spectroscopy. The effect of chelating agent (ethylenediaminetetra acetic acid EDTA) on the metal ions was investigated by dialyzing the protein against 10 mM EDTA and assaying for hemagglutination. Amino acid composition was analyzed with the Technicon sequential multi-sample amino acid analyzer. A 15-day acute toxicity of the lectin on mice was evaluated using standard procedures. Histopathological study was performed on some organs (brain, kidney, liver, lung, spleen and testis) of mice given intraperitoneal doses of the lectin. The results revealed that phosphate buffered saline extract of the seeds of Treculia africana agglutinated human red blood cells non-specifically. The hemagglutinating activity was inhibited by mannose with minimum inhibitory concentration of 0.8 mM and slightly enhanced by sorbose, dulcitol, glucose, and sorbitol. The purified lectin showed a single band in both denaturing and non-denaturing polyacrylamide gel electrophoresis. The subunit molecular weight as determined by SDS-PAGE and the native molecular weight as determined by gel filtration were 22,000 and 41,000 Daltons respectively. Amino acid composition analysis revealed that the protein contained 155 residues per subunit and was characterized by a high content of arginine, glutamic acid, aspartic acid, proline, cysteine, tyrosine and phenylalanine. Treatment of the protein with chelating agent (EDTA) had no inhibitory effect on the hemaglutinating activity. Analyses of metal ion contents revealed that the protein contained 42.13 mg/ml Cu2+, 3.30 mg/ml Fe3+, 21.01 mg/ml Mg2+, 9.24. mg/ml Mn2+ and 3.85 mg/ml Ca2+. The protein showed maximum activity over the pH range 3 – 7 and is relatively thermostable up to 50°C. Periodic acid Schiff's reagent staining showed that the protein was non-glycosylated. Although, the lectin was toxic with LD50 of 47.211 μg/g body weight of mice causing death instantaneously, there was no visible damaging effect on the organs of the mice following histopathological studies. In conclusion, the study revealed that seeds of Treculia Africana contained a lectin that was non-specific in its hemagglutinating property. The lectin exhibited properties and acute toxicity characteristic of ribosome-inactivating proteins (RIPS) with no damaging effect on the organs of the tested animals.
- ItemOpen AccessThe Catalytic Properties of the Isoenzymes of Glutathione Transferase from Fruit Bat, Eidolon Helvum (Kerr) Liver(2015-06-26) Ejim-Eze, Emmanuel EmekaThis study investigated the physiochemical and catalytic properties of the isoenzymes of glutathione transferase (GST) in the fruit bat (Eidolon helvum, Kerr) liver, with a view to having an understanding of the survival strategy of this animal, which feeds on various plant materials many of which contain toxic secondary metabolites. A 15% homogenate (15 g in 85 ml buffer) of a fresh liver sample, obtained from live bats, was prepared in 10 mM Tris-HCI buffer pH 8.0, containing 1 mM EDTA, 1 mM 2-mercaptoethanol and 10% glycerol, using a warring blender. The crude homogenate was centrifuged at 20,000 rpm for 30 minutes at 4°C using a Beckman ultracentrifuge to obtain a cloudy supernatant. The supernatant was further centrifuged at 45,000 rpm for 1hr to obtain a clear crude enzyme extract. The extract was purified on DEAE-Sephacel anion exchange chromatography and CM-TrisacryI cation exchange chromatography, followed by gel filtration on Sephadex G-100. Determination of native and subunit molecular weights of the purified enzyme was carried out by gel filtration on Sephadex G-100 and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) respectively. In order to classify the various purified GST isoenzymes obtained, the specific activities towards various substrates [1,2-dichloro-4-nitrobenzene (DCNB), 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), ethacrynic acid, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-CI) and p-nitrophenyl acetate (p-NPA)] were determined by monitoring the enzyme-catalyzed conjugation of the various substrates with reduced glutathione (GSH} in a spectrophotometer under standard conditions. The apparent Michealis-Menten constant (Km) and maximal velocity (Vmax) for GSH, and some of the utilizable electrophilic substrates, were also determined by varying the concentrations of the substrates while keeping that of GSH constant and vice-versa. The data also obtained were analyzed by double reciprocal plot. Similarly, the effect of the inhibitors: cibacron blue, hematin, and S-hexylglutathione, which could be used to discriminate between the different GST classes were studied by assaying the enzyme at different concentrations of the inhibitors. The results showed that the DEAE-Sephacel anion- exchange chromatography resolved the crude enzyme extract into two major peaks of activity designated as A and B which were separately pooled. Further purification of pool A on CM-Trisacryl cation-exchange chromatography resolved it into four peaks of activity which were designated, following the established convention, as AA, AB, AC and AD; AA being the isoenzyme that eluted at the highest ionic strength and AD, the lowest. However, further purification of pool B on this column gave only one peak of GST activity, which was designated as isoenzyme B. The native molecular weight of the isoenzymes estimated from a calibration curve ranged from 40 to 44 kDa. The subunit molecular weights of each of the isoenzymes estimated by SDS-PAGE were between 21 to 23 kDa. On the basis of this, the native isoenzymes could therefore be said to be dimeric proteins. The fruit bat GST isoenzymes conjugated CDNB, NBD-CI and p-NPA to varying degrees with GSH, but could not utilize ethacrynic acid, DCNB and EPNP. Hematin inhibited all the isoenzymes to the same extent, but isoenzymes AB, AC and AD were more sensitive to cibacron blue than isoenzymes AA and B. In conclusion, the occurrence of GST in multiple forms with different catalytic properties could assist fruit bats in detoxication of various allelochemicals encountered during feeding.
- ItemOpen AccessInteractive Roles of Terpenoid Extract from the Leaves of Neem Plant (Azadirachta Indica A. Juss) On Lead-Induced Toxicity in Pregnant Rabbits(2015-06-19) Areola, Jacob OladayoThis study extracted terpenoid from the dried leaves of Neem plant (Azadirachta indica,A.JUSS). It also assessed the possibility of ameliorating the toxic effects and accumulation of lead in both the mother and the litters. This was with a view to investigating its interactive roles in lead-induced toxicity in pregnant rabbits and resulting litters. Fresh leaves were collected from Azadirachta indica trees planted on Obafemi Awolowo University campus, Ile-Ife, Nigeria. The leaves were air dried for 21 days and milled to fine powder. Terpenoid was extracted from dried leaves of Azadirachta indica by a procedure that consisted of solvent extraction with methanol (7.2 L), liquid-liquid partitioning with petroleum ether and ethyl acetate and evaporated under reduced pressure. The residue was phytochemically screened for terpenoids. Pregnant rabbits (does) weighing between 3.0 and 3.4 kg were randomly divided into 4 groups. Group I served as control and were treated orally with olive oil (2 ml/kg body weight per day). Group II served as Lead control and were treated with Lead acetate solution (50 mg / kg body weight per day). Group III served as positive control and were treated with ascorbic acid (400 mg / kg body weight per day) and Group IV served as terpenoid treated group and were treated with terpenoid extract (300 mg / kg body weight per day). One hour later, Lead acetate solution (50 mg / kg body weight per day) was administered to animals in groups III and IV. The animals were treated for 11 days starting from day 14 of pregnancy. The does and the litters (young rabbits) were sacrificed 4 weeks after parturition. Blood was collected into tubes containing anti-coagulants for plasma preparation while the blood for Lead analysis was collected into tubes without anticoagulants. Then, liver, kidney, heart and lungs were removed aseptically. The tissues were digested with (Conc.HNO3 : HC1 1 : 4 v/v) and portion of the liver was homogenized in phosphate buffer (100 mM, pH 7.4). Lead concentration in the tissues were determined by Atomic Absorption Spectroscopy (AAS). The total protein concentration in the plasma and liver homogenates were determined using biuret reaction method. The plasma albumin concentration was estimated as described in Sigma Chemical Limited catalogue. Hepatic marker enzymes (Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) activities were also determined spectrophotometrically at 505nm. The results revealed that, 15.8 g of terpenoid was obtained from 2.4 kg of dried leaf of neem plant representing 0.66% of the starting material. A total number of 32 litters were produced by pregnant rabbits in all the groups. There was no abortion and deformity of litters through out the duration of the experiment. The terpenoid extract was able to reduce Lead concentration in the blood, liver and kidney of pregnant rabbits by 45.45%, 30% and 20.9% respectively. The level of Lead reduction was found to be statistically significant (t = 3.365, p<0.05) but the extract had no significant effect on its concentration in the tissues of the litters. Lead concentration in terpenoid treated group was significantly lower than lead treated group (t = 3.365, p<0.05). All other parameters (Liver protein, plasma protein and albumin concentration, ALT and AST activities) were not significantly different in all the groups (t = 3.365, p>0.05). In conclusion, the terpenoid extract from the leaf of Azadirachta indica was not teratogenic and toxic to rabbits at the dosage used in these investigations. The extract was also able to reduce the lead burden in pregnant rabbits but could not produce the same effects on the litters.
- ItemOpen AccessIncidence of Urinary Tract Infection (UTI) Among Pregnant Women in Ile-Ife, Nigeria(2015-03-18) Adebayo, Oluwabanwo JamesThis study investigated the incidence of urinary tract infection among pregnant women. It determined the current microbial isolates that are responsible for infection as well as their susceptibility to antibiotics. The period or trimester when pregnant women are prone to urinary tract infection and the age range when infection is prevalent were also determined, this was with a view to providing information that will prevent complications in pregnancy arising from urinary tract infection. Urine samples were obtained from 300 randomly selected pregnant women attending the Antenatal Clinic of Obafemi Awolowo University Teaching Hospital Complex Ile Ife. Each patient was given a sterile, wide-necked, leak proof container and requested to give 10-20ml sample of mid-stream urine with little or no contamination and provide information on the month or term of pregnancy. The samples were then labelled with the date, the name of the patient and time of collection. Thereafter the specimen was transferred to the laboratory. The freshly collected clean-catch midstream urine was mixed by gently rotating the container . Using a sterile calibrated wire loop that holds 1/1000 ml (0.001 m1), a loopful of urine was inoculated into cystine lactose electrolyte-deficient (CLED) agar, chocolate agar and MacConkey agar. The plates were then incubated at 37°C for 24 hours. The organisms that were isolated from colonies on these media were subsequently identified and characterized using various biochemical tests, while their antibiotic sensitivity patterns were determined using the agar-disc diffusion method. This result showed that 34.3 % of pregnant women examined had significant bacteriuria suggestive of UTI. The most frequent isolate was Escherichia coli (41.7 %), followed by Staphylococcus aureus (26.2 %), Klebsiella spp. (8.7 %), Proteus mirabilis (6.8 %), Candida albicans (9.7 %), Pseudomonas aeruginosa (2.9 %) and Streptococcus faecalis (3.9 %). Urinary tract infection was common in the last trimester of pregnancy among the younger age group of 17 to 26 years. Antibiotic sensitivity tests showed that the Gram negative isolates were sensitive to nitrofurantoin, gentamicin, nalidixic acid and ofloxacin, while the Gram-positive isolates were sensitive to gentamicin, erythromycin and cloxacilin. High antibiotic resistance was observed by the urinary bacterial isolates to most of the antibiotics used, these however cannot be referred to as multiple antibiotic resistance. Klebsiella spp, P. mirabilis, P. aeruginosa and S. aureus were resistant to the following antibiotics, amoxycillin, cotrimoxazole, augmentin and tetracycline. E. coli was resistant to amoxicillin and cotrimoxazole while S. faecalis showed strong resistance to tetracycline. The study concluded that the leading microbial strains responsible for urinary tract infection among pregnant women at Ile Ife were Escherichia toll, Klebsiella and Staphylococcus aureus and most of the isolates were sensitive to nitrofurantoin, gentamicin and nalidixic acid.
- ItemOpen AccessSynthesis and Characterization of Poly-8-Methylamino-Adenylic.(Obafemi Awolowo University, 1987) Elekwa, Iheanyichukwu; Falayan, J.O.A polynucleotide, poly-8-methylamino adenylic acid, was synthesized and hybridized with poly U. Adenosine-5-monophosphate purchased from BDN Chemical Corporation was brominated with bromine in Na-acetate buffer pH 3.9, stirred for 24 hours. The excess bromine was removed with some drops of concentrated sodium sulphite solution. It was then evaporated to dryness, on a rotatory evaporator at 37 oC and characterised. The product 8-Bromo-adenosine-5-Monphosphate, was methylaminated with methylamine in water, and refluxed for 48 hours at 65oC on an oil bath. Excess methylamine was removed with dilute sulphuric acid, while the product was evaporated to dryness and characterized. The synthesized methylamino adenosine-5-monophosphate was converted to its diphosphate analogue by the phophoro-morpholidate process. Orthophosphoric acid was used to introduce the phosphate group. The converted methylamino-adenosine-5-diphosphate was polymerized with the enzyme polynucleotide phophorylase PNPase, (EC 2.7.7.8) from Micrococcus luteus that was purchased from Boehringner Chemical Corporation. This enzyme PNPase is only specific for the diphosphate form. The polymerization was carried out in a reaction medium of MgCl2.6H2O EDTA (Ethylene diaminetetra-acetic acid) and tris buffer pH 9.0. It was incubated for 21 hours at 36oC, and characterized. The Poly-8-methyamino adenylic acid so synthesized was hybridized with poly U purchased from BDH- Chemical Corporation. The Job’s continuous variation method in KH2PO4 + K2HPO4 buffer pH 7.00 at 37oC was adopted. The polymer formed at 1:1 hybrid with Poly U. This satisfies some of the requirements for synthetic polynucleotide inducers. Some physico-chemical methods used in the characterization, included infra-red, ultra-violet, proton nuclear magnestic resonance and chromatographic methods. Complete UV spectra were recorded in the range of 200-400nm on Sp 8-400 (uv/vis) spectrophotometer. The infra-red spectra recorded using a sodium chloride cell in the range of 200-4000 cm-1 on Sp 3 -300 infra-red spectrophotometer. And, the Proton Nuclear Magnetic Resonance was recorded on Perkin Elmer Spectrophometer. The spots on the paper chromatography were detected and developed under UV light (at 254 nm wavelength).