Analysis of AVR4 promoter by sequential response-element deletion

Several reports have associated the variability in physico-chemical properties of avidin protein to dynamism inherent in the consensus regulatory networks within the promoter region of avidin genes. An Avr4 promoter region ligated to chloramphenicol acetyltransferase plasmid vector (pBLCAT2) to produce recombinant plasmid Avr4pBLCAT2 was sequentially deleted to produce five distinct mutants: Avr4pBLCAT2907-176, Avr4pBLCAT2809-176, Avr4pBLCAT2789-176, Avr4pBLCAT2429-176 and Avr4pBLCAT2 302-176. The transformants elicited different chloramphenicol acetyl transferase (CAT) activities. (African Journal of Biotechnology: 2003 2(7): 185-188

Genetic diversity of ochratoxigenic Aspergillus section Nigri, using RAPD and VCG techniques

This study evaluates the genetic diversity of ochratoxigenic Aspergillus section Nigri using RAPD and VCG techniques. Results obtained revealed OPX 07 as the most informative of the tested RAPD markers generating 12 polymorphic bands while the least bands were generated by OPR 19. Of the 40 Aspergillus section Nigri (20 each of Aspergillus niger and Aspergillus carbonarius), 22 VCGs and 27 RAPD haplotypes were delineated. The two techniques demonstrated similar resolution except in few cases where the RAPD technique further sub divided some VCGs into simpler haplotypes. The average percentage of variable VCG and RAPD reactions were 25 and 50% in that order of sequence while 75 and 50% of the isolates were resolved as same isolates by these techniques respectively. It was also found that the Simpson index of genetic diversity approached one for the isolates from the four geopolitical zones of Ogun State, Nigeria with the mean genetic diversity within isolates (GL) contributing significantly approximately 89% of the total diversity observed within the isolates (F=22.23, p<0.05). The remaining 11% of variation could only be allotted to diversity among isolates (GS). On the whole, the total genetic diversity (HT) was found to be approximately 48%. In conclusion, RAPD markers provided better resolution than the classical VCG typing technique.Keywords; Genetic Diversity, Ochratoxigenic Aspergillus, RAPD and VC

Transcriptional factor influence on OTA production and the quelling attribute of Sirna on the OTA producing strains of Aspergillus section Nigri

This study determined the influence of some transcriptional factors on ochratoxin A production as well as investigates the quelling attributes of some designed siRNA on the OTA producing Aspergillus section Nigri using standard recommended techniques. Results obtained following comparison of the pks gene promoter sequences from 15 isolates depicts differences in length and homology with the pks gene ranging from 218bp in a strain of the Aspergillus niger to 700bp in Aspergillus carbonarius. The alignment of the pks gene promoter region revealed that six and two of the aligned genes have Aba A binding site corresponding to CATTCT and CATTCC respectively while Brl A binding site was absent in all the isolates. Pac C binding site corresponding to CCTGGC and GCCAAG was also found in two and three of the pks gene promoter region respectively. The three designed siRNA shows significant impact on OTA inhibitions with no significant statistical differences (80.9, 74.4 and 75.3% for pks_Ia, pks_Ib and pks_Ic respectively) (F= 3.830, p>0.05). It can be concluded that Are A and Aba A are potential enhancers for ochratoxin A biosynthesis and none of the investigated transcriptional factors is enough for the activation of ochratoxin A production. However, pks gene was seen as a good target gene for inactivation in order to develop efficient means for ochratoxin A control using RNA silencing technology.Key words: Transcriptional factors, Ochratoxin A, siRNA, Quelling , Aspergillus section Nigr

Enhanced antimalarial effects of chloroquine by aqueous Vernonia amygdalina leaf extract in mice infected with chloroquine resistant and sensitive Plasmodium berghei strains

Background: The emergence and spread of Plasmodium falciparum with resistance to chloroquine (CQ), the safest and cheapest antimalarial drug coupled with the increasing cost of alternative drugs especially in developing countries have necessitated the need to optimize antimalarial actions of plant extracts and restore chloroquine efficacy. Objective: The present study determines the ability of Vernonia amygdalina leaf extract to enhance the prophylactic and therapeutic efficacy of chloroquine against Plasmodium berghei malaria in mice. Methods: Chloroquine sensitive (P. bergheiS) and resistant (P.bergheiR) ANKA clones of Plasmodium berghei maintained by serial passage in mice were used to develop respective experimental rodent malaria models based on intraperitoneal injection of 106 parasitize erythrocyte suspension in PBS (pH 7.2) and subsequent development of parasitaemia. These models were then used to investigate the prophylactic enhancement of chloroquine (CQ) at 5 mg/kg via combination with selected doses (31.25, 62.5, 125mg/kbw) of Vernonia amygdalina leaf extracts using a 4-day suppression test. Effect of these combinations on the therapeutic efficacy of CQ at 30mg/kg over 3 days were evaluated. Treatment outcomes including parasite clearance (PCT) and rescrudescent time (RT) were compared with CQ-chlorpheniramine combination. The acute toxicity of the extract-CQ combinations was also determined enzymatically. Results: Prophylatically, chloroquine (5mg/kg) in combination with vernonia extracts achieved a dose-dependent (57.2 - 72.7%) suppression of parasitaemia due to CQ sensitive and resistant P berghei strains in the experimental animals. Therapeutically, chloroquine (30mg/kg for 3 days) combined with vernonia to dose-dependently shorten the parasite clearance times (2.6 -4.4 vs. 4.8 days; P < 0.05 for CQ-V62.5/125 combination), prolong the recrudescent times (8.9 -18.9 vs. 7.2 days; P < 0.05) and improve day 14 cure rate (66.7 -100 vs. 58.3%) in the treated P. bergheiS infected mice compared to CQ monotherapy. Whereas CQ monotherapy failed, resolution of parasitaemia due to the CQ resistant parasite with day 14 cure rates of 25 -100% were also observed with these combinations. In therapeutic terms, the potencies of CQ-V125 combination were comparable to those of CQ-chlorpheniramine (0.25mg/kg, 12hourly, 7 days) in the infected animals. Toxicity testing indicates that these combinations elicited mild to - moderate increases in the liver enzymes measured when administered orally to mice for 7 days. Conclusion: This study indicates that Vernonia amygdalina leaf extract dose -dependently restore the efficacy of CQ against CQ resistance P. berghei malaria in mice

INHIBITION OF SWARMING BY UREA AND ITS DIAGNOSTIC IMPLICATIONS AMONG UROPATHOGENIC PROTEUS SPECIES FROM LAGOS, NIGERIA

The anti-swarming property of urea and effects on antibiotic susceptibility among 52 uropathogenic Proteus strains from Lagos, Nigeria were investigated. Urea caused a reduction in swarming and number of swarmed cells at 0.5% (n = 42, DOCZ = 15.5mm), 0.75% (n= 24, DOCZ = 10.7mm), 1% (n = 17, DOCZ = 3.4mm) and 1.25% (n = 8, DOCZ = 1.7mm).  Compared to DOCZ obtained at 0.5% urea, the further reduction in DOCZ at other urea concentrations was found to be significant (p < 0.05).  Urea at less than 0.75% allowed identification of E. coli, K. pneumoniae and S. saprophyticus in mixed cultures containing Proteus spp, while colonies of Pseudomonas aeruginosa were distinctly identified at 1% urea with swarming restrained at 1.25% urea.  At 1.25% urea, antibiotic susceptibility testing by agar diffusion method revealed significant increase and decrease in the number of Proteus strains that showed resistance to amoxicillin and nitrofurantoin. Compared with the control, significant increases in the MICs of gentamicin or nitrofurantoin and streptomycin were found at $0.5$ 0.75% urea respectively (