Francis Mulaa

Population genetics of the potentially invasive African fruit fly species, Ceratitis rosa and Ceratitis fasciventris (Diptera: Tephritidae)

A set of 10 microsatellite markers was used to survey the levels of genetic variability and to analyse the genetic aspects of the population dynamics of two potentially invasive pest fruit fly species, Ceratitis rosa and C. fasciventris, in Africa. The loci were derived from the closely related species, C. capitata. The degree of microsatellite polymorphism in C. rosa and C. fasciventris was extensive and comparable to that of C. capitata. In C. rosa, the evolution of microsatellite polymorphism in its distribution area reflects the colonization history of this species. The mainland populations are more polymorphic than the island populations. Low levels of differentiation were found within the Africa mainland area, while greater levels of differentiation affect the islands. Ceratitis fasciventris is a central–east African species. The microsatellite data over the Uganda/Kenya spatial scale suggest a recent expansion and possibly continuing gene flow within this area. The microsatellite variability data from C. rosa and C. fasciventris, together with those of C. capitata, support the hypothesis of an east African origin of the Ceratitis spp.

Comparative analysis of microsatellite loci in four fruit fly species of the genus Ceratitis (Diptera: Tephritidae)

The possibility to cross-species amplify microsatellites in fruit flies of the genus Ceratitis was tested with the polymerase chain reaction (PCR) by analysing 23 Ceratitis capitata (Wiedemann) microsatellite markers on the genomic DNA of three other economically important, congeneric species: C. rosa (Karsch), C. fasciventris (Bezzi) and C. cosyra (Walker). Twenty-two primer pairs produced amplification products in at least one of the three species tested. The majority of the products were similar, if not identical in size to those expected in C. capitata. The structures of the repeat motifs and their flanking sequences were examined for a total of 79 alleles from the three species. Sequence analysis revealed the same repeat type as the homologous C. capitata microsatellites in the majority of the loci, suggesting their utility for population analysis across the species range. A total of seven loci were differentially present/absent in C. capitata, C. rosa, C. fasciventris and C. cosyra, suggesting that it may be possible to differentiate these four species using a simple sequence repeat-based PCR assay. It is proposed that medfly-based microsatellite markers could be utilized in the identification and tracing of the geographical origins of colonist pest populations of the four tested species and in the assessment of their risk and invasive potentials; thereby assisting regulatory authorities in implementing quarantine restrictions and other pest control measures.

Antifolate drug resistance and point mutations in Plasmodium falciparum in Kenya

Due to increased chloroquine resistance, the antifolate/sulpha drug combinations are becoming increasingly important in the chemotherapy of falciparum malaria. However, point mutations in the dihydrofolate reductase gene lead to resistance to the antifolate drugs. We therefore investigated the prevalence of the 6 reported point mutations in this gene among field isolates of Plasmodium falciparum from Kenya, to determine if the mutations correlated with resistance to pyrimethamine and the biguanides cycloguanil and chlorcycloguanil. We used a mutation-specific polymerase chain reaction technique to test for these reported mutations in 21 Kenyan isolates and 4 reference lines. We also amplified and directly sequenced the dihydrofolate reductase coding sequence from these parasites to confirm the results and test for other possible mutations. Of the reported mutations, we found S108N, which is the central mutation of pyrimethamine resistance, and mutations N51I and C59R, which modulate the levels of resistance and may confer decreases in response to cycloguanil that are folate and p-aminobenzoic acid dependent. No isolate possessed the paired point mutations S108T and A16V, or I164L and S108N, which have been associated with cycloguanil resistance in previous studies. These results provided supportive evidence for the combined use of a cycloguanil-class drug (e.g., chlorproguanil) and a sulpha drug (e.g., dapsone) against P.falciparum malaria in Kenya.

Enzymatic Synthesis of Lipophilic Rutin and Vanillyl Esters from Fish Byproducts.

Lipase-catalyzed synthesis of lipophilic phenolic antioxidants was carried out with a concentrate of n-3 polyunsaturated fatty acids (PUFAs), recovered from oil extracted from salmon ( Salmon salar ) byproduct. Vanillyl alcohol and rutin were selected for the esterification reaction, and obtained esters yields were 60 and 30%, respectively. The antioxidant activities of the esters were compared with those of commercial butylated hydroxytoluene (BHT) and α-tocopherol using DPPH radical scavenging and thiobarbituric acid assays. In the DPPH assay, rutin esters showed better activity than vanillyl esters, and on the contrary in lipophilic medium, vanillyl esters were found to be superior to rutin esters. In bulk oil system, the antioxidant activities of rutin and vanillyl derivatives were lower than that of BHT and α-tocopherol, but in emulsion, they showed better activity than α-tocopherol. By attaching to natural phenolics, the PUFAs are protected against oxidation, and PUFA improves the hydrophobicity of the phenolic, which could enhance its function in lipid systems.

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g−1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8–9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a Km of 6.6 mM for H2O2 and Vmax of 707 mM H2O2 min−1 g−1 wet wt. cells, and showed saturation kinetics at 50 mM H2O2. The cells were entrapped in calcium alginate and used for H2O2 degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.

Repetitive sequences upstream of the pfg27/25 gene determine polymorphism in laboratory and natural lines of Plasmodium falciparum ☆

The structure of the genomic region located upstream of the gametocyte-specific gene pfg27/25 of Plasmodium falciparum was analysed in laboratory lines and field isolates of the parasite. The gene is located in a subtelomeric region of chromosome 13 in parasite clones 3D7 and HB3. Analysis of laboratory lines and field isolates of P. falciparum indicated that polymorphism upstream of pfg27/25 is mainly due to the structure of a repetitive DNA region located at about half a kilobase from the pfg27/25 coding sequence. Different types of repetitive sequences are present in this region, whose copy number is variable in different parasite lines. In addition a GC-rich sequence element contained in this region, which is proposed to be the startpoint of pfg27/25 mRNA, presents either a direct or a reverse orientation in different parasite lines. Genomic deletions upstream of the pfg27/25 gene are also described in two laboratory lines of the parasite, which eliminate two newly identified malaria genes. orf P and orf Gap, from the genome of these parasites. One of them, orf Gap, deleted from the reference parasite clone 3D7, is abundantly expressed as mature mRNA in asexual parasites. PCR analysis on 64 field isolates of P. falciparum indicated that orf P and orf Gap sequences are present in all tested samples of naturally propagating parasites.

Evidence for the involvement of a tsetse midgut lectin-trypsin complex in differentiation of bloodstream-form trypanosomes

We have previously described a bloodmeal-induced molecule (lectin-trypsin complex) from the midgut of the tsetse fly, Glossina longipennis, with both lectin and trypsin activities (Osir et al., 1995). In this paper, we report on the isolation of a similar molecule from the midguts of Glossina fuscipes fnscipes and provide direct evidence for its involvement in the development of African trypanosomes. The molecule (native Mr ∼65,700) has two non-covalently linked subunits, Mr ∼28,800 and Mr ∼35,700. The native molecule was found to be capable of inducing differentiation of bloodstream-form trypanosomes into procyclic (midgut forms) in vitro. In the assays, specific antibodies against procyclin were used to monitor the transformation of the bloodstream-form trypanosomes into procyclic forms. This induction was specifically inhibited by D-glucosamine. Cis-aconitate was also capable of inducing the transformation process with the same efficiency as that of the lectin-trypsin complex. While increasing the concentrations of the lectin-trypsin complex (≥100 μg protein/ml) in the incubation assays resulted into higher transformation rates, it also led to high parasite mortality. These results provide evidence for the involvement of the midgut lectin-trypsin complex in the differentiation of bloodstream-form trypanosomes within tsetse midgut.RésuméNous avons précédemment décrit une molécule induite par le repas de sang (un complexe lectine-trypsine) présente dans l’intestin moyen de la mouche tsé-tsé ’Glossina longipennis, ayant une activité lectine et trypsine (Osir et al., 1995). Dans ce papier, nous décrivons l’isolement d’une molécule similaire présente dans l’intestin moyen de Glossina fuscipes fuscines et démontrons son implication dans le développement des trypanosomes africains. La molécule (pure Mr ∼65,700) présente deux sous unités liées non covalentes, Mr ∼28,800 et Mr ∼35,700. La molécule pure est capable d’induire la différenciation des formes sanguines du trypanosome en formes pro-cycliques (intestin moyen) in vitro. Lors des essais, des anticorps spécifiques de la procycline ont été utilisés pour contrôler la transformation des formes sanguines du trypanosome en forme procycliques. Cette induction a été inhibée spécifiquement avec du D-glucosamine. Le cis-aconitate est également capable d’induire le processus de transformation avec une efficacité comparable à celle du complexe lectine-trypsine. Alors que l’augmentation des concentrations du complexe lectine-trypsine (≥100 μg protéine/ml) dans les essais d’incubation permet d’augmenter les taux de transformation, il induit également une importante mortalité du parasite. Ces résultats démontrent la participation du complexe lectine-trypsine de l’intestin moyen dans la différenciation des formes sanguines des trypanosomes dans l’intestin moyen de la mouche tsé-tsé.

Protease-, Pectinase- and Amylase- Producing Bacteria from a Kenyan Soda Lake

Background: Alkaline enzymes are stable biocatalysts with potential applications in industrial technologies that offer high quality products. Objective: The growing demand for alkaline enzymes in industry has enhanced the search for microorganisms that produce these enzymes. Methods: Eighteen bacterial isolates from Lake Bogoria, Kenya, were screened for alkaline proteases, pectinases and amylases; characterized and subjected to quantitative analysis of the enzymes they produced. Results: The screening analysis ranked 14, 16 and 18 of the bacterial isolates as potent producers of alkaline proteases, pectinases and amylases, respectively. The isolates were classified into two groups: Group 1 (16 isolates) were facultatively alkaliphilic B. halodurans while group 2 (2 isolates) were obligately alkaliphilic B. pseudofirmus. Further analysis revealed that group 1 isolates were divided into two sub-groups, with sub-group I (4 isolates) being a phenotypic variant sub-population of sub-group II (12 isolates). Variation between the two populations was also observed in their enzymatic production profiles e.g. sub-group I isolates did not produce alkaline proteolytic enzymes while those in sub-group II did so (0.01-0.36 U/ml). Furthermore, they produced higher levels of the alkaline pectinolytic enzyme polygalacturonase (0.12-0.46 U/ml) compared to sub-group II isolates (0.05-0.10 U/ml), which also produced another pectinolytic enzyme-pectate lyase (0.01 U/ml). No clear distinction was however, observed in the production profiles of alkaline amylolytic enzymes by the isolates in the two sub-populations [0.20-0.40 U/ml (amylases), 0.24-0.68 U/ml (pullulanases) and 0.01-0.03 U/ml (cyclodextrin glycosyl transferases)]. On the other hand, group 2 isolates were phenotypically identical to one another and also produced similar amounts of proteolytic (0.38, 0.40 U/ml) and amylolytic [amylases (0.06, 0.1 U/ml), pullulanases (0.06, 0.09 U/ml) and cyclodextrin glycosyl transferases (0.01, 0.02 U/ml)] enzymes. Conclusion: The facultatively alkaliphilic B. halodurans and obligately alkaliphilic B. pseudofirmus isolates are attractive biotechnological sources of industrially important alkaline enzymes. (Less)

Structure of the 40S ribosomal subunit of Plasmodium falciparum by homology and de novo modeling

Generation of three dimensional structures of macromolecules using in silico structural modeling technologies such as homology and de novo modeling has improved dramatically and increased the speed by which tertiary structures of organisms can be generated. This is especially the case if a homologous crystal structure is already available. High-resolution structures can be rapidly created using only their sequence information as input, a process that has the potential to increase the speed of scientific discovery. In this study, homology modeling and structure prediction tools such as RNA123 and SWISS–MODEL were used to generate the 40S ribosomal subunit from Plasmodium falciparum. This structure was modeled using the published crystal structure from Tetrahymena thermophila, a homologous eukaryote. In the absence of the Plasmodium falciparum 40S ribosomal crystal structure, the model accurately depicts a global topology, secondary and tertiary connections, and gives an overall root mean square deviation (RMSD) value of 3.9 Å relative to the template׳s crystal structure. Deviations are somewhat larger in areas with no homology between the templates. These results demonstrate that this approach has the power to identify motifs of interest in RNA and identify potential drug targets for macromolecules whose crystal structures are unknown. The results also show the utility of RNA homology modeling software for structure determination and lay the groundwork for applying this approach to larger and more complex eukaryotic ribosomes and other RNA-protein complexes. Structures generated from this study can be used in in silico screening experiments and lead to the determination of structures for targets/hit complexes.

Protein polymorphism in two populations of the brown ear tick, Rhipicephalus appendiculatus neumann (acari: ixodidae)

Two-dimensional gel electrophoresis was used to compare the protein profiles of two geographically isolated populations of the tick Rhipicephalus appendiculatus Neumann (Acari: Ixodidae) in Kenya. Most of the protein spots were common to both populations, but a number were specific to each population. Since proteins are encoded by genes, the presence of population-specific proteins suggests that there may be genetic differences between the two populations. It is proposed that some of these population-specific proteins might be related to the differences in susceptibility of the ticks to Theileria parva infection.RésuméL’électrophorèse à gel bi-dimensionnel a été utilisée pour comparer les profiles protéjques de deux populations géographiquement isolées de tiques Rhipicephalus appendiculatus Neumann (Acari: Ixodidae) au Kenya. Bien que la plupart des taches protéiques étaient communes aux deux populations, certaines d’entre elles étaient cependant spécifiques à l’une ou l’autre population. Etant donné que les protéines sont codées par géne, la présence de protéines spécifiques à une population suggère l’existence possible de différences spécifiques entre ces deux populations. Ces protéines spécifiques pourraient être liées, en quelque sort, aux différences de prédisposition des tiques aux infections de Tlicileria parva.