Department of Biochemistry, Cell and Molecular Biology

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Start date: 1990End date: 1999

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    Sensitivity of Asexual Blood Stages Of . Plasmodium Berghet (Nk 65) to Chloroquine
    (University of Ghana., 1994-03) Aryee, N.A.
    The erythrocytic developmental cycle of Plasmodium berghei can be conveniently divided into the ring, tropho-zoite and schizont stages based on morphology. The sensitivity and effect of chloroquine on density-gradient isolated fractions of each of these stages was investigated using the plasmodial strain P.berghei (NK65), a rodent model as an experimental tool. This plasmodial strain was found to be routinely lethal in infected mice in the absence of administered therapeutic levels of chloroquine. P.berghei infected blood was separated into the various developmental stages using discontinuous Percoll gradient centrifugation. The various stage-isolated fractions were found to be infective and sensitive to these same levels of chloroquine. However chloroquine was found to show insignificant differential sensitivity with regards to the developmental stage of berghei (NK 65) strain.
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    Assessment of Sensitivity of P. Falciparum to Antifolate Antimalarials In Southern Ghana By Polymerase Chain Reaction (PCR) Assay Systems
    (University of Ghana, 1996-07) Mak-Mensah, E. E.
    Drug treatment of malaria is being hampered by the emergence of drug resistant strains of malaria parasites. Rapid methods for monitoring patients' response to treatment, applicable to field conditions, would aid management and control of drug resistant P. falciparum infections. In this study, mutation specific polymerase chain reaction assays using 3'-mismatched oligonucleotide primers which annealed to the wild or mutated parasites gene encoding dihydrofolate reductase-thymidylate synthase (DHFR-TS) were used to survey P.falciparum strains in two Regions of Southern Ghana (Volta and Greater Accra Regions). Mutations were identified directly from blood samples obtained from patients attending Out Patients Departments in Hospitals. Amplified P.falciparum DHFR-TS nucleotide sequences of 337bps were obtained when reaction products were analyzed by electrophoresis. Of 151 smear positive samples analysed, 13 (8.6%) contained the Asn-10S codon AAC that confers pyrimethamine resistance, 133 (88%) samples contained only the wild type Ser-10S codon AGC and none contained the Val-16 codon GTA found in cycloguanil-resistant pyrimethamine sensitive parasites. Out of the 13 pyrimethamine resistant cases, 10 were found in samples obtained from the Volta Region while the rest (3) were from Korle-Bu Teaching Hospital in the Greater Accra Region. No PCR product was found in 5 (3.3%) of the 151 samples. After second (nested) PCR, only one sample showed amplified product: contamination was negligible. PCR amplification of the DHFR-TS presents a rapid alternative to in vitro drug testing for monitoring the resistance of P.falciparum to antifolate antimalarials.
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    G-Protein Transduction M Iend Siaactcehda Srigonmaylc Es Cerevisiae
    (University of Ghana, 1995-09) Dzudzor, B.; Gyang, N.F.; University of Ghana, College of Basic and Applied Sciences, School of Biological Sciences, Department of Biochemistry, Cell and Molecular Biology
    Yeast mating type locus gene alpha2 (MATa2), Yeast G protein complementing gene ( YGC1) and minichromosome maintenance gene (M C M 1) have been identified by isolation of plasmids that are able to complement or suppress a gpa1::HIS3 mutation. MATa2 and YGC1 rescue both MATa and MATa-gpa1::HIS3 haploid cell types whereas MCM1 complements only MATa gpal::HIS3 cell type. MATa2 is known to be a general repressor and a determinant of both haploid and diploid cell types. MCM1 is known to be a general transcriptional activator. YGC1 has not been characterised, hence its function or mode of action is not previously known. G protein alpha subunit (GPA1) is a yeast G protein a subunit that negatively controls the budding yeast pheromone signal transduction pathway. Disruption of GPA1 results in constitutive arrest of the signal pathway that leads to cell cycle arrest at the early G1 phase of the cell cycle. Both Southern analysis and sequencing showed that MATa2, YGC1, MCM 1 have no homology to GPA1. Disruption of MATa2 (that is mata2::URA3) leads to constitutive arrest of the cell cycle at the G 1 phase. MATa2 also has no sequence homology to G P A 2 , the other G protein a subunit in yeast, known to be involved in cAMP pathway in yeast. It has been shown here that MATa2 rescues gpal::HIS3 cells even in single copy, centromere plasmids. Mating efficiency is largely reduced in cells kept alive with MATa2. MATa2 does not have the pheromone response elements (PREs) common to the STE genes (whose disruption leads to insensitivity to mating factors). The plasmid TGC was also constructed and used in creation of the yeast haploid strains LG1 and LG2. This was an attempt to screen a mammalian cDNA library for possible analogs of GPA1. These strains were used to isolate two mammalian analogs that complement the gpa1::HIS3 mutation. The results indicate that MATa2, YGC1 and MCM1 are components or modulate component(s) of the signaling pathway. It also showed that MATa2 is even a more potent negative regulator of the signaling pathway than GPA1, since overexpression is not a prerequisite for negatively regulating the pathway. MATa2 does not belong to the G protein family since it has no GTP/GDP binding and/or exchange domains.
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    Fi.Avoh01ds of Rpipei.Ta Frrrii01 Hi'a • Ahti-Diabetic Pr0pert1 1(3 and Tilv. Reduction 01? Li Vj5i>. Hicr030hal ['I’otjij Fl.'.
    (University of Ghana, 1993-09) Mills-Robertsoh, F.C.; Addy, M.E.; University of Ghana, College of Basic and Applied Sciences School of Biological Sciences Department of Biochemistry, Cell and Molecular Biology
    The an t. i - d i a b&t i c pr oper t ies of f .1 9 von oide: -x 11 a : te :i f roid B.r.idj!_l.i.a f.&rj:_a￡Li. a p 1 sn t uged 3 1 Ihe 0 en f: r e For Sc 1 ,-n t i f ie Re s e a r c h I n t o F1 an t Medicine (C S RP M ) t o t r e a. t cl i ab e t e s , were vvaluated using streptozotocin -induced d iabet io :ni0e 10 represent insulin dependent diabetes mellitus (I DDK; arid genetically diabetic mice to represent non- ins;.! I in dependent i i ab e t es ;ri e 11 i t u s (NTDDM ) A 1 s o an a I y z ed wa s the ef f e r_-1 c f r extra c 1: 0 n t h e re d u c t ion of 1 i v e r m i c r 0 s 0 in a 1 p r 0 t ? :l r 1 f. . . The possible side■-effeets of the f 1 avon0 id ex1 1 ac-1 01i 1 i ve r and . Jn ey fun c t i on 5 were also ex am in ed . T he t e- s t an i n; n Is were .riven intraperitonea 1 injection of the flavonoid extract dissolved in 0.35% sa 1 in e ,=• o 1 u t i on w l'i i 1 s t t he on t r u 1 s were • i veri 0.85% saline solution w i t h ou t 11"; e 0x t r ac t . 'j' h e r rju Its 3 h 0 w e d t h a t t h e f 13 v 0 n 0 i d e 11 a c t w a s n 01 i; y p o ,e. 3 y c 3 e m i o a s i t 3.;i no ef f eo t on t he p 1 asraa glucose levels nf n jn • d j abe tic ■nice . A11 h 0 u g h the ext r a c t d i d n o t p r e v en t t h e d e v e ! o p in en t o f y erglyc s em i a in t he TDDM r.iod e 1 af t er t. he ST1 in j e t i on . i t ' *J r educe the non • f ast ing p 1 asma gIu00se levels si.eni - i an 11 y . w h erea s s u >:h e f f e c t o n f a s 1: i r j g 10 v ■; 1 w a s a I s n t 01 • ''101 signif ioant. In the cnee of the HIDDH m■: *.• 1 tii^ x '• v3.01 ■j e c r e as e d signifies r-1L y t h e n c ■ > s - f a s l.. j r 1 c. y \ .a, :i-; ! 1 <. ^ - f >.•1 . about but thf;i e u n j.c < i,|ie . ting p J a s rii a g 1 u c o s e . T h e 7- e s u .11 s t I <•> z:; o iv e ■! < b , 1 • i j ' h e s s t a ri i 111 a 1 s :i 1 j b c I: h ]. DDM u i: d f 11D D f 1 m fit L::: , 1,;--j. , w ≪., ^ /\ [) p f; y ■'.o0 hr Gine V 3 !>Ci r ed n L a e ar• K i v i I, y an !..!,■;■:?■ i.: •. ! s , ■. 11 hong h b <:• I: h t e s t ? n - J 0 - j n t r 0 I r- 11 - •' j ■■■ s 11 - -• r 0 !- \ * i \ v e r ; o s o in a 1 p r o I:, e i n o T he re s u 11 s i n d i c a t e t !'i at, the I avono ids , wh ioh are not hypog1 y c ae m ic , redu c-e pos t pr a n d i a 1 P asEta glucose levels in d iabet ic pa ti en ts . There is also an indication that t h e y red u c e the ac t i v i t y o f d r ug met ab o J. i s in g •res, probably the ones involving the cytochrome F450DM . j.jiryme. t?u toxic side-effects on the liver and kidney were !' rved.
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    Fi.Avoh01ds of Rpipei.Ta Frrrii01 Hi'a • Ahti-Diabetic Pr0pert1 1(3 and Tilv. Reduction 01? Li Vj5i>. Hicr030hal ['I’otjij Fl.'.
    (University of Ghana, 1993-09) Mills-Robertsoh, F.C.; Addy, M.E.; University of Ghana, College of Basic and Applied Sciences School of Biological Sciences Department of Biochemistry, Cell and Molecular Biology
    The an t. i - d i a b&t i c pr oper t ies of f .1 9 von oide: -x 11 a : te :i f roid B.r.idj!_l.i.a f.&rj:_a￡Li. a p 1 sn t uged 3 1 Ihe 0 en f: r e For Sc 1 ,-n t i f ie Re s e a r c h I n t o F1 an t Medicine (C S RP M ) t o t r e a. t cl i ab e t e s , were vvaluated using streptozotocin -induced d iabet io :ni0e 10 represent insulin dependent diabetes mellitus (I DDK; arid genetically diabetic mice to represent non- ins;.! I in dependent i i ab e t es ;ri e 11 i t u s (NTDDM ) A 1 s o an a I y z ed wa s the ef f e r_-1 c f r extra c 1: 0 n t h e re d u c t ion of 1 i v e r m i c r 0 s 0 in a 1 p r 0 t ? :l r 1 f. . . The possible side■-effeets of the f 1 avon0 id ex1 1 ac-1 01i 1 i ve r and . Jn ey fun c t i on 5 were also ex am in ed . T he t e- s t an i n; n Is were .riven intraperitonea 1 injection of the flavonoid extract dissolved in 0.35% sa 1 in e ,=• o 1 u t i on w l'i i 1 s t t he on t r u 1 s were • i veri 0.85% saline solution w i t h ou t 11"; e 0x t r ac t . 'j' h e r rju Its 3 h 0 w e d t h a t t h e f 13 v 0 n 0 i d e 11 a c t w a s n 01 i; y p o ,e. 3 y c 3 e m i o a s i t 3.;i no ef f eo t on t he p 1 asraa glucose levels nf n jn • d j abe tic ■nice . A11 h 0 u g h the ext r a c t d i d n o t p r e v en t t h e d e v e ! o p in en t o f y erglyc s em i a in t he TDDM r.iod e 1 af t er t. he ST1 in j e t i on . i t ' *J r educe the non • f ast ing p 1 asma gIu00se levels si.eni - i an 11 y . w h erea s s u >:h e f f e c t o n f a s 1: i r j g 10 v ■; 1 w a s a I s n t 01 • ''101 signif ioant. In the cnee of the HIDDH m■: *.• 1 tii^ x '• v3.01 ■j e c r e as e d signifies r-1L y t h e n c ■ > s - f a s l.. j r 1 c. y \ .a, :i-; ! 1 <. ^ - f >.•1 . about but thf;i e u n j.c < i,|ie . ting p J a s rii a g 1 u c o s e . T h e 7- e s u .11 s t I <•> z:; o iv e ■! < b , 1 • i j ' h e s s t a ri i 111 a 1 s :i 1 j b c I: h ]. DDM u i: d f 11D D f 1 m fit L::: , 1,;--j. , w ≪., ^ /\ [) p f; y ■'.o0 hr Gine V 3 !>Ci r ed n L a e ar• K i v i I, y an !..!,■;■:?■ i.: •. ! s , ■. 11 hong h b <:• I: h t e s t ? n - J 0 - j n t r 0 I r- 11 - •' j ■■■ s 11 - -• r 0 !- \ * i \ v e r I avono ids , wh ioh are not hypog1 y c ae m ic , redu c-e pos t pr a n d i a 1 P asEta glucose levels in d iabet ic pa ti en ts . There is also an indication that t h e y red u c e the ac t i v i t y o f d r ug met ab o J. i s in g •res, probably the ones involving the cytochrome F450DM . j.jiryme. t?u toxic side-effects on the liver and kidney were !' rved.
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    Cytochrome P-450 Monooxygenase Complex and Glutathione-Stransferase in Sarotherodon Melano Theron as Biomarkers of Pollution.
    (University of Ghana, 1998-09) Renner, C.; M.E.; University of Ghana, College of Basic and Applied Sciences, School of Biological Sciences Department of Biochemistry, Cell and Molecular Biology
    Samples of Sarotherodon melanotheron obtained from water bodies of different pollution histories, that is, a site on the Volta lake near Ada, the Fosu lagoon at Cape Coast and aquaria containing the herbicide, 'Roundup', were used to investigate induction of hepatic cytochrome P-450 monooxygenase enzyme system (CYP) and glutathione-stransferase (GST) in response to pollutants. Total microsomal protein concentration was determined by the Folin- Lowry method. The activity of NADPH cytochrome P-450 reductase, a component of monooxygenase enzyme system, was measured, using reduction of exogenous cytochrome C. Another component, cytochrome P-450, was measured using CO-difference spectrum. The overall monooxygenase activity was determined using 7-ethoxyresorufin-0- deethylase (EROD) and 7-pentoxyresorufin-O-depentylase (PROD) assays, which indicated specifically, the induction of cytochrome P-4501A and P-4502B isoenzymes respectively. Glutathione-s-transferase activity was measured using l-chloro-2,4-dinitrobenzene as the substrate. The results for all the biochemical parameters measured, indicated that the levels of those of the fish from Fosu lagoon were excessively higher than the values recorded for fish from Volta lake and the aquaria, indicating a relatively higher pollution level in the Fosu lagoon. For the CYP the levels measured for both fish from the Fosu lagoon and the aquaria were almost similar, indicating higher induction of CYP by 'Roundup' . For the EROD and PROD determinations for the monooxygenase system, activities were detected in livers of fish from Fosu lagoon and the Volta lake. No such activities were detected in fish exposed to 'Roundup', even though CYP induction was high and these monooxygenase enzyme activities were found prior to herbicide exposure. The activity of EROD was 3.5 fold higher than that of PROD, indicating less PB-types of inducers than that of 3-MC type in the Fosu lagoon. This study has indicated that the Fosu lagoon is polluted with 3-MC types and PB-types of inducers and that the 'Roundup'-induced CYP is not only different from the CYP isozymes responsible for the EROD and PROD assays, it actually inactivates them. GST induction indicated in this study would support the effects of the induced CYP in biotransformation of the xenobiotics which enter the fish in polluted waters.
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    Molecular Analysis of the Genomic Dna of Six Varieties of Cowpea, Vigna Unguiculata L. Walp
    (University of Ghana, 1996-09) Crabbe, E.; Wilson, M.D.; Acquaah, R.A.; University of Ghana, College of Basic and Applied Sciences, School of Biological Sciences, Department of Biochemistry, Cell and Molecular Biology
    Three non-chemical conditions of preservation, storage at room temperature (that is benchtop), in a freezer and at simulated herbarium were examined to determine which is most suitable for the preservation of cowpea leaves for molecular studies. Under each storage condition, the effect of duration of storage and the difference in leaf age (that is flush or mature) on DNA yield and purity were assessed. Periodically, DNA was extracted from both flush and mature leaf samples from 6 cowpea (Vigna unguiculata L. Walp.) varieties, Amantin, Asontem, Ayiyi, Bengpla, Ejura Red and Soronko beginning from the day of harvest (day 1), through day 7, 21, 35 and 49 after storage. At room temperature and simulated herbarium conditions, the yield and purity of the DNA extracted from both flush and mature leaves decreased during the period of the study. Time was found to correlate inversely with the purity and yield of DNA in all cases. Under these conditions, the yield of DNA extracted from day 1 samples (fresh samples) of both leaf types was significantly different from the DNA yield from day 7 to 49 samples. Also, no significant difference was found in DNA yield from day 7 to 49 samples. However, in most cases, more DNA was obtained from flush leaves than from their corresponding mature leaves. Of the three conditions, leaves stored at room temperature yielded the least amount of DNA. Samples kept under simulated herbarium conditions yielded more DNA than kept in the freezer but the difference was not significant. Also, a fairly constant yield and purity was obtained for DNA extracted from frozen samples. Frozen samples yielded relatively purer DNA than their corresponding samples stored either at room temperature or at simulated herbarium though the observed differences were not significant. Therefore, storage in a freezer provided the best non-chemical preservation condition among the three assessed. Only 2 (GA and AR) out of 15 random primers assayed using the technique of Random amplified polymorphic DNA-polymerase Chain Reaction, (RAPD-PCR) amplified segments of the genomic DNA extracted from the 6 varieties. Further analysis (Restriction Fragment Length Polymorphism studies) was done on the GAprimed 1200 bp PCR product using eight restriction endonucleases, Alul, Dral, ￡coRI, EcoRV, Haelll, Hinfl, Kpnl and Pvul. Four enzymes, Alul, Haelll. Hinfl and Kpnl digested the product. However, for each enzyme, identical banding patterns were observed on both agarose and polyacrylamide gels in all the six varieties Single stranded conformation polymorphism analysis (SSCP) was conducted on the two ARprimed products, 369 bp and 545 bp. Only the 545 bp was denatured but the profiles of the bands in all the six varieties were also identical. Therefore, these molecular techniques could not be used to identify the individual varieties
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    Phage - Mediated Transfer of Genetic Material
    (University of Ghana, 1993) Owusu-Biney, A.; Rodrigues K. F.; University of Ghana, College of Basic and Applied Sciences, School of Biological Sciences, Department of Biochemistry, Cell and Molecular Biology
    Ten phages were isolated randomly from sewage sources and disposal points on the University of Ghana,Legon campus, purified and characterised as delivery systems for the resistance marker genes, Strr and Benr. The phages were found to have proteins of relative molecular weights ranging from 9,000 - 100,000. Eight phages were found to be morphologically related to the tailed phages. The other two were tailless phages. All the coliphages isolated were morphologically related to the T-even phages, whilst Sf RBCL 15 and Sd RBCL 5 were related to the P-phages. The tailless phages were found to be morphologically related to the p phages (06 and 0X174 phages). The coliphages were found to be closely related to each other. They did exhibit some detectable cross reactivity to Sd RBCL 5, Sd RBCL 2 3 and Sf RBCL 15. The coliphages were not related to the S. tvphi phages. Selected pathogenic bacteria from the Noguchi Memorial Institute for Medical Research and the Medical School of the university of Ghana were screened for the presence of the antibiotic resistance gene marker. All the bacteria, except three, were found to be highly resistant to the marker antibiotics used : Salmonella Group D was relatively more sensitive to all three antibiotics ; Staphylococcus aureus was sensitive to Benzylpenicillin and Salmonella typhi was sensitive to Tetracycline. The isolated phages with the exception of St RBCL 2 0 exhibited the ability to transduce the resistance marker genes from one bacterium to sensitive bacteria at a high frequency.
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    Nadph Dependent Cytochrome P-450 Reactions- Mode Of Inhibition By The N—Butanol Fraction Of Desmodium Adscendens
    (University of Ghana, 1996-03) Kamassah, A.A
    The n-butanol fraction (nBF) of Desrrodlvm a d scen d en s , a plant used for the management of asthma, is' an inhibitor of NADFH-dependent cytochrorre P-450 (CYP) reactions. Its mechanism of action as an inhibitor is hcwever not kncwn. In this study, flavoprotein reductase activity, spectral changes associated with binding and spectral properties of reduced cytochrcme c and CYP were used to investigate the mode of inhibition. nBF reduced cytochrane c but not CYP directly. In the presence of NADFH, the rate of formation of CYP2* (the reduced form of CYP) v\as enhanced ty the addition of nBF. These effects were observed in reactions without substrate, indicating that in the absence of a substrate, nBF does noc prevent the NADPH reduction of CYP, but rather enhances it. In the presence of a substrate, as exemplified ty a spectrophotometric assay of the ERCD reaction, nBF v*as found to change the spectrum of oxidized CYP. The results indicate that in the presence of nBF, the binding site of CYP is altered to prevent substrate binding.