PROF. MULAA FRANCIS JAKIM

The oleochemical industry is currently still dominatedby conventional chemistry, with biotechnology onlystarting to play a more prominent role, primarily withrespect to the biosurfactants or lipases, e.g. as detergents,or for biofuel production. A major bottleneckfor all further biotechnological applications is theproblem of the initial mobilization of cheap and vastlyavailable lipid and oil substrates, which are thento be transformed into high-value biotechnological,nutritional or pharmacological products. Under theEU-sponsored LipoYeasts project we are developingthe oleaginous yeast Yarrowia lipolytica into a versatileand high-throughput microbial factory that, byuse of specific enzymatic pathways from hydrocarbonoclasticbacteria, efficiently mobilizes lipidsby directing its versatile lipid metabolism towardsthe production of industrially valuable lipid-derivedcompounds like wax esters (WE), isoprenoid-derivedcompounds (carotenoids, polyenic carotenoid ester),polyhydroxyalkanoates (PHAs) and free hydroxylatedfatty acids (HFAs). Different lipid stocks (petroleum,alkane, vegetable oil, fatty acid) and combinationsthereof are being assessed as substrates in combinationwith different mutant and recombinant strains ofY. lipolytica, in order to modulate the composition andyields of the produced added-value products.

PUFA from oil extracted from Nile perch viscera were enriched by selective enzymatic esterification of the free fatty acids (FFA) or by hydrolysis of ethyl esters of the fatty acids from the oil (FA-EE). Quantitative analysis was performed using RP-HPLC coupled to an evaporative light scattering detector (RP-HPLC-ELSD). The lipase from Thermomyces lanuginosus discriminated against docosahexaenoic acid (DHA) most, resulting in the highest DHA/DHA-EE enrichment while lipase from Pseudomonas cepacia discriminated against eicosapentaenoic acid (EPA) most, resulting in the highest EPA/EPA-EE enrichment. The lipases discriminated between DHA and EPA with a higher selectivity when present as ethyl esters (EE) than when in FFA form. Thus when DHA/EPA were enriched to the same level during esterification and hydrolysis reactions, the DHA-EE/EPA-EE recoveries were higher than those of DHA/EPA-FFA. In reactions catalysed by lipase from T. lanuginosus, at 26 mol% DHA/DHA-EE, DHA recovery was 76% while that of DHA-EE was 84%. In reactions catalysed by lipase from P. cepacia, at 11 mol% EPA/EPA-EE, EPA recovery was 79% while that of EPA-EE was 92%. Both esterification of FFA and hydrolysis of FA-EE were more effective for enriching PUFA compared to hydrolysis of the natural oil and are thus attractive process alternatives for the production of products highly enriched in DHA and/or EPA. When there is only one fatty acid residue in each substrate molecule, the full fatty acid selectivity of the lipase can be expressed, which is not the case with triglycerides as substrates.

p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: "Times New Roman"; }div.Section1 { page: Section1; } 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) by-products. 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 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 PUFAs to natural phenolics, the PUFAs are protected against oxidation while PUFA improves the hydrophobicity of the phenolic which could enhance its function in lipid systems.

Oil was extracted from fatty material obtained from Nile perch viscera using the protease Protex 30L. Enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the glyceride fraction was carried out by hydrolysis of extracted oils with lipases from Candida rugosa, Thermomyces lanuginosus and Pseudomonas cepacia. The unusual fatty acid distribution of the oil influenced the apparent lipase specificity to a large extent. In the unhydrolysed oil, only 16% of EPA was in sn-2 position while 51% of palmitic acid was located in this position of the triacylglycerol (TAG) molecules. Non-regioselective lipase from C. rugosa was the most effective in combined enrichment of both EPA and DHA. This was partly because it was able to hydrolyse off palmitic acid from the sn-2 position, which 1-, 3-specific lipases were unable to do. Hydrolysis with C. rugosa lipase enriched EPA from 3 to 6 mol% and DHA from 9 to 23 mol%, with recoveries of 42 and 55%, respectively. The 1-, 3-specific lipase from T. lanuginosus was ineffective in enriching EPA, but gave best DHA enrichment, 38 mol% with a recovery of 39%. DHA was rather equally distributed in sn-1, -2 and -3 positions of TAG. The results show that both the fatty acid specificity and regiospecificity of the lipase as well as the fatty acid distribution of the oil should be considered when choosing the strategy for fatty acid enrichment.

PUFA from oil extracted from Nile perch viscera were enriched by selective enzymatic esterification ofthe free fatty acids (FFA) or by hydrolysis of ethyl esters of the fatty acids from the oil (FA-EE).Quantitative analysis was performed using RP-HPLC coupled to an evaporative light scattering detector(RP-HPLC-ELSD). The lipase from Thermomyces lanuginosus discriminated against docosahexaenoicacid (DHA) most, resulting in the highest DHA/DHA-EE enrichment while lipase from Pseudomonascepacia discriminated against eicosapentaenoic acid (EPA) most, resulting in the highest EPA/EPA-EEenrichment. The lipases discriminated between DHA and EPA with a higher selectivity when present asethyl esters (EE) than when in FFA form. Thus when DHA/EPA were enriched to the same levelduring esterification and hydrolysis reactions, the DHA-EE/EPA-EE recoveries were higher than thoseof DHA/EPA-FFA. In reactions catalysed by lipase from T. lanuginosus, at 26 mol% DHA/DHA-EE,DHA recovery was 76% while that of DHA-EE was 84%. In reactions catalysed by lipase fromP. cepacia, at 11 mol% EPA/EPA-EE, EPA recovery was 79% while that of EPA-EE was 92%. Bothesterification of FFA and hydrolysis of FA-EE were more effective for enriching PUFA compared tohydrolysis of the natural oil and are thus attractive process alternatives for the production of productshighly enriched in DHA and/or EPA. When there is only one fatty acid residue in each substratemolecule, the full fatty acid selectivity of the lipase can be expressed, which is not the case withtriglycerides as substrates.

A Gram-negative obligate alkaliphilic bacterium (BOG6T) that secretes carotenoids was isolated fromthe outflow of Lake Bogoria hot spring located in the Kenyan Rift Valley. The bacterium is motile bymeans of a polar flagellum, and forms red colonies due to the production of xanthophyll carotenoidpigments. 16S rRNA gene sequence analysis showed this strain to cluster phylogenetically within thegenus Paracoccus. Strain BOG6T is aerobic, positive for both catalase and oxidase, and nonmethylotrophic.The major fatty acid of the isolate is C18: 1w7c. It accumulated polyhydroxybutyrategranules. Strain BOG6T gave astaxanthin yield of 0.4 mg/g of wet cells indicating a potential forapplication in commercial production of carotenoids. On the basis of its genotypic characteristics, fattyacid composition and physiological reaction profiles, it is proposed that the isolate may be assigned tothe genus Paracoccus as Paracoccus bogoriensis sp. nov. The type strain is BOG6T (=DSM16578=LMG22798). The GenBank 16S rDNA nucleotide sequence accession number is AJ580352.

An optical immunosensor (AQUA-OPTOSENSOR) and ELISA (enzyme-linked immunosorbent assay) for the analysis of pyrethroids and DDT in river water and/or sediment, are described. The optical immunosensor consists of a bench-top optical read-out-device and disposable single-use sensor chips. ELISA was carried out in the coating antigen format. As examples, phenothrin (pyrethroid) and p,p'-DDT were chosen. Herein we describe the overall strategy, the set-up and principle of the immunosensor platform, and show representative results for immunosensor and ELISA analysis. The immunosensor employs fluorophore (Oyster®-645)-labeled monoclonal antibodies (mouse mAb Py-1 and rat mAb DDT 7C12), and makes use of the evanescent field, thus operating without washing steps. ELISA in the coating antigen format uses a second antibody labeled with peroxidase. Both, phenothrin and p,p'-DDT can be analyzed with these immunochemical techniques in the low ppb levels. Advantages and drawbacks of both immunochemical platforms are discussed.

The thermal unfolding of Amy 34, a recombinant alpha-amylase from Bacillus halodurans, has been investigated using differential scanning calorimetry (DSC). The denaturation of Amy 34 involves irreversible processes with an apparent denaturation temperature (T(m)) of 70.8 degrees C at pH 9.0, with four transitions, as determined using multiple Gaussian curves. The T(m) increased by 5 degrees C in the presence of 100-fold molar excess of CaCl2 while the aggregation of Amy 34 was observed in the presence of 1000-fold molar excess of CaCl2. Increase in the calcium ion concentration from 1- to 5-fold molar excess resulted in an increase in calorimetric enthalpy (DeltaH(cal)), however, at higher concentrations of CaCl2 (up to 100-fold), DeltaH(cal) was found to decrease, accompanied by a decrease in entropy change (DeltaS), while the T(m) steadily increased. The presence of 100-fold excess of metal chelator, EDTA, resulted in a decrease in T(m) by 10.4 degrees C. T(m) was also decreased to 61.1 degrees C and 65.9 degrees C at pH 6.0 and pH 11.0, respectively.

The thermal unfolding of Amy 34, a recombinant alpha-amylase from Bacillus halodurans, has been investigated using differential scanning calorimetry (DSC). The denaturation of Amy 34 involves irreversible processes with an apparent denaturation temperature (T(m)) of 70.8 degrees C at pH 9.0, with four transitions, as determined using multiple Gaussian curves. The T(m) increased by 5 degrees C in the presence of 100-fold molar excess of CaCl2 while the aggregation of Amy 34 was observed in the presence of 1000-fold molar excess of CaCl2. Increase in the calcium ion concentration from 1- to 5-fold molar excess resulted in an increase in calorimetric enthalpy (DeltaH(cal)), however, at higher concentrations of CaCl2 (up to 100-fold), DeltaH(cal) was found to decrease, accompanied by a decrease in entropy change (DeltaS), while the T(m) steadily increased. The presence of 100-fold excess of metal chelator, EDTA, resulted in a decrease in T(m) by 10.4 degrees C. T(m) was also decreased to 61.1 degrees C and 65.9 degrees C at pH 6.0 and pH 11.0, respectively.

Proteins isolated from the midgut of Glossina pallidipes were used to immunize rabbits and their efficacy as vaccine candidate(s) against the fly, and their potential to block transmission of Trypanosoma brucei rhodesiense assessed. Two fractions, detergent (DET) and aqueous (AQ) fractions were separated using a non-ionic detergent (Triton X-114) and a series of bioassay experiments carried out using serum obtained from rabbits immunized with either of the two fractions. The mortality rates of tsetse flies fed on serum from rabbits immunized with DET and AQ was 56 and 35%, respectively, as compared to 20% mortality in controls. The DET antigen(s) caused considerably higher mortality (chi(2)=1.194, P<0.05) than that on controls. These findings suggest that midgut proteins contain antigens that are lethal to tsetse flies, and are potential candidates for the development of anti-tsetse vaccine. When flies fed on serum derived from DET immunized rabbits were fed on T. b. rhodesiense infected blood, only 20% of them picked the infection. Very few flies (20%) fed on serum derived from DET immunized rabbits had infection of T. b. rhodesiense. In the control flies 45% of them had infection in the midgut with a higher and actively motile parasite load. Assessment of fecundity indicated significantly higher (chi(2)=2.117, P<0.05) larviposition for the control flies when compared to the AQ group of flies (chi(2)=1.054, P<0.05). Significant differences in abortions and pupal weights were also observed. These results suggest that midgut proteins contain antigens with potential for use in development of vaccine to block transmission of trypanosomes through tsetse.

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.

Fourteen obligate alkaliphilic and halotolerant bacterial isolates, exhibiting extracellular amylase activity at 55 degrees C and pH 10, were isolated from hot springs around Lake Bogoria, Kenya. From 16S rDNA sequence analysis, nine isolates shared 100% identity with Bacillus halodurans strain DSM 497T, while the rest shared 99% identity with alkaliphilic Bacillus species A-59. PCR of the intergenic spacer region between 16S and 23S rRNA genes (ISR-PCR) divided the isolates into two groups, while tDNA-PCR divided them into three groups. Bacillus halodurans DSM 497T had a different ISR pattern from the isolates, while it had a tDNA-PCR profile similar to the group that shared 99% identity with alkaliphilic Bacillus species A-59. All isolates hydrolysed soluble starch as well as amylose, amylopectin and pullulan. The amylase activity (1.2-1.8 U ml(-1)) in the culture broths had an optimum temperature of 55-65 degrees C, was stimulated by 1 mm Ca2+, and was either partially (16-30%) or completely inhibited by 1 mM EDTA. Activity staining of the cell-free culture supernatant from the isolates revealed five alkaline active amylase bands.

Proteins isolated from the midgut of Glossina pallidipes were used to immunize rabbits and their efficacy as vaccine candidate(s) against the fly, and their potential to block transmission of Trypanosoma brucei rhodesiense assessed. Two fractions, detergent (DET) and aqueous (AQ) fractions were separated using a non-ionic detergent (Triton X-114) and a series of bioassay experiments carried out using serum obtained from rabbits immunized with either of the two fractions. The mortality rates of tsetse flies fed on serum from rabbits immunized with DET and AQ was 56 and 35%, respectively, as compared to 20% mortality in controls. The DET antigen(s) caused considerably higher mortality (chi(2)=1.194, P<0.05) than that on controls. These findings suggest that midgut proteins contain antigens that are lethal to tsetse flies, and are potential candidates for the development of anti-tsetse vaccine. When flies fed on serum derived from DET immunized rabbits were fed on T. b. rhodesiense infected blood, only 20% of them picked the infection. Very few flies (20%) fed on serum derived from DET immunized rabbits had infection of T. b. rhodesiense. In the control flies 45% of them had infection in the midgut with a higher and actively motile parasite load. Assessment of fecundity indicated significantly higher (chi(2)=2.117, P<0.05) larviposition for the control flies when compared to the AQ group of flies (chi(2)=1.054, P<0.05). Significant differences in abortions and pupal weights were also observed. These results suggest that midgut proteins contain antigens with potential for use in development of vaccine to block transmission of trypanosomes through tsetse.

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.

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.

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.

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.

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.

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.

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.

Cultivated Plasmodium falciparum gametocytes reach maturity in vitro in approximately 14-16 days, during which they pass through five morphologically distinct developmental stages. Purification of the earlier developmental stages has not been previously reported. We have modified the standard discontinuous Percoll gradient method for the separation of stage IV and V gametocytes to obtain enriched preparations of those and the earlier P. falciparum gametocyte stages. In contrast to the stages II, III, and IV, the mature stage V gametocytes from our gradient readily transformed into gametes. Such preparations may be useful in research studies on the mechanisms that underlie gametocytogenesis.

Cultivated Plasmodium falciparum gametocytes reach maturity in vitro in approximately 14-16 days, during which they pass through five morphologically distinct developmental stages. Purification of the earlier developmental stages has not been previously reported. We have modified the standard discontinuous Percoll gradient method for the separation of stage IV and V gametocytes to obtain enriched preparations of those and the earlier P. falciparum gametocyte stages. In contrast to the stages II, III, and IV, the mature stage V gametocytes from our gradient readily transformed into gametes. Such preparations may be useful in research studies on the mechanisms that underlie gametocytogenesis.

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.

In a bid to determine the HIV-1 subtype variants in transmission in Nairobi and its possible association with clinical status, we screened 207 confirmed HIV-1 positive patients visiting HIV/AIDS laboratory at the Virus Research Centre in Nairobi between January and March 1994. We used a selfmade ELISA obtained from an established panel of HIV-1 V3 loop peptides (ANRS, France) and derived from seven isolates: MN, HXB2, SC, Z6, Z2, ELI and CDC4. Test samples were obtained from 95 blood donors and medical examination attendees, 57 patients with chronic diarrhoea, 31 confirmed pulmonary tuberculosis, 16 with pneumonia and 12 herpes zoster. Out of the total, 21.5% had antibodies against the MN strain, 19.1% had against the Z2 strain while reaction against the HXB2 strain was observed in 17.2%. SC, CDC4, Z6 and ELI had prevalences of 11.5%, 6.2%, 5.3% and 3.8% respectively. Fifteen per cent of the tested sera showed no reaction to any of the used peptides. Strong and significant associations were observed between the total number of strains a sample react to and the clinical state. We infer that both the North American consensus strains (MN and HXB2) and the African isolates (Z2 and Z6) are predominant in Nairobi. The correlation between antibody reactivity and clinical state is an interesting observation that necessitates an expanded study and, the use of strain specific peptides maybe a sensitive and easier method for use for molecular epidemiological purposes. PIP: During January-March 1994, in Nairobi, Kenya, the sera of pre-university students, suspected AIDS/advanced HIV-infection cases, and blood donors were screened for HIV-1 antibodies at the Virus Research Centre. All confirmed HIV-1 positive samples were categorized according to the patient's clinical status. A self-made ELISA was obtained from an established panel of HIV-1 V3 loop peptides and derived from seven isolates (MN and HXB2 [North American strains], SC, CDC4, Z2 and Z6 [African strains], and ELI). The sera of the 22 confirmed HIV-1 negative students were used as negative controls. There were 207 confirmed HIV-1 cases (95 blood donors and 112 suspected AIDS/advanced HIV-infection cases). 64 (31%) and 112 (54%) samples reacted to at least 3 strains and no more than 2 strains, respectively. The remaining 31 (15%) samples did not react to any of the 7 peptide strains. Samples with CD4 cell counts greater than 500 x 1 million reacted significantly to more peptide strains than those with CD4 counts below 200 x 1 million (88% vs. 7%). Reactivity to specific strains were 21.5% for MN, 19.1% for Z2, 17.2% for HXB2, 11.5% for SC, 6.2% for CDC 4, 5.5% for Z6, and 3.8% for ELI. Anti-HXB2 antibodies were more common in blood donors than suspected AIDS/advanced HIV-infection cases (22% vs. 13%). AIDS/advanced HIV-infection cases were more likely to have no antibodies than blood donors (21% vs. 7%). A significant association existed between the number of peptide strains a patient could react to and the clinical state (p 0.01). Specifically, 77% of samples with no V3 antibodies to the seven strains had AIDS or advanced HIV infection while 55% of those which had cross reactivity with three or more strains were asymptomatic. Further research is needed to better understand this correlation. These findings suggest that use of strain specific peptides may be a sensitive and easier method for use for molecular epidemiological purposes.

In a bid to determine the HIV-1 subtype variants in transmission in Nairobi and its possible association with clinical status, we screened 207 confirmed HIV-1 positive patients visiting HIV/AIDS laboratory at the Virus Research Centre in Nairobi between January and March 1994. We used a selfmade ELISA obtained from an established panel of HIV-1 V3 loop peptides (ANRS, France) and derived from seven isolates: MN, HXB2, SC, Z6, Z2, ELI and CDC4. Test samples were obtained from 95 blood donors and medical examination attendees, 57 patients with chronic diarrhoea, 31 confirmed pulmonary tuberculosis, 16 with pneumonia and 12 herpes zoster. Out of the total, 21.5% had antibodies against the MN strain, 19.1% had against the Z2 strain while reaction against the HXB2 strain was observed in 17.2%. SC, CDC4, Z6 and ELI had prevalences of 11.5%, 6.2%, 5.3% and 3.8% respectively. Fifteen per cent of the tested sera showed no reaction to any of the used peptides. Strong and significant associations were observed between the total number of strains a sample react to and the clinical state. We infer that both the North American consensus strains (MN and HXB2) and the African isolates (Z2 and Z6) are predominant in Nairobi. The correlation between antibody reactivity and clinical state is an interesting observation that necessitates an expanded study and, the use of strain specific peptides maybe a sensitive and easier method for use for molecular epidemiological purposes. PIP: During January-March 1994, in Nairobi, Kenya, the sera of pre-university students, suspected AIDS/advanced HIV-infection cases, and blood donors were screened for HIV-1 antibodies at the Virus Research Centre. All confirmed HIV-1 positive samples were categorized according to the patient's clinical status. A self-made ELISA was obtained from an established panel of HIV-1 V3 loop peptides and derived from seven isolates (MN and HXB2 [North American strains], SC, CDC4, Z2 and Z6 [African strains], and ELI). The sera of the 22 confirmed HIV-1 negative students were used as negative controls. There were 207 confirmed HIV-1 cases (95 blood donors and 112 suspected AIDS/advanced HIV-infection cases). 64 (31%) and 112 (54%) samples reacted to at least 3 strains and no more than 2 strains, respectively. The remaining 31 (15%) samples did not react to any of the 7 peptide strains. Samples with CD4 cell counts greater than 500 x 1 million reacted significantly to more peptide strains than those with CD4 counts below 200 x 1 million (88% vs. 7%). Reactivity to specific strains were 21.5% for MN, 19.1% for Z2, 17.2% for HXB2, 11.5% for SC, 6.2% for CDC 4, 5.5% for Z6, and 3.8% for ELI. Anti-HXB2 antibodies were more common in blood donors than suspected AIDS/advanced HIV-infection cases (22% vs. 13%). AIDS/advanced HIV-infection cases were more likely to have no antibodies than blood donors (21% vs. 7%). A significant association existed between the number of peptide strains a patient could react to and the clinical state (p 0.01). Specifically, 77% of samples with no V3 antibodies to the seven strains had AIDS or advanced HIV infection while 55% of those which had cross reactivity with three or more strains were asymptomatic. Further research is needed to better understand this correlation. These findings suggest that use of strain specific peptides may be a sensitive and easier method for use for molecular epidemiological purposes.

In a bid to determine the HIV-1 subtype variants in transmission in Nairobi and its possible association with clinical status, we screened 207 confirmed HIV-1 positive patients visiting HIV/AIDS laboratory at the Virus Research Centre in Nairobi between January and March 1994. We used a selfmade ELISA obtained from an established panel of HIV-1 V3 loop peptides (ANRS, France) and derived from seven isolates: MN, HXB2, SC, Z6, Z2, ELI and CDC4. Test samples were obtained from 95 blood donors and medical examination attendees, 57 patients with chronic diarrhoea, 31 confirmed pulmonary tuberculosis, 16 with pneumonia and 12 herpes zoster. Out of the total, 21.5% had antibodies against the MN strain, 19.1% had against the Z2 strain while reaction against the HXB2 strain was observed in 17.2%. SC, CDC4, Z6 and ELI had prevalences of 11.5%, 6.2%, 5.3% and 3.8% respectively. Fifteen per cent of the tested sera showed no reaction to any of the used peptides. Strong and significant associations were observed between the total number of strains a sample react to and the clinical state. We infer that both the North American consensus strains (MN and HXB2) and the African isolates (Z2 and Z6) are predominant in Nairobi. The correlation between antibody reactivity and clinical state is an interesting observation that necessitates an expanded study and, the use of strain specific peptides maybe a sensitive and easier method for use for molecular epidemiological purposes. PIP: During January-March 1994, in Nairobi, Kenya, the sera of pre-university students, suspected AIDS/advanced HIV-infection cases, and blood donors were screened for HIV-1 antibodies at the Virus Research Centre. All confirmed HIV-1 positive samples were categorized according to the patient's clinical status. A self-made ELISA was obtained from an established panel of HIV-1 V3 loop peptides and derived from seven isolates (MN and HXB2 [North American strains], SC, CDC4, Z2 and Z6 [African strains], and ELI). The sera of the 22 confirmed HIV-1 negative students were used as negative controls. There were 207 confirmed HIV-1 cases (95 blood donors and 112 suspected AIDS/advanced HIV-infection cases). 64 (31%) and 112 (54%) samples reacted to at least 3 strains and no more than 2 strains, respectively. The remaining 31 (15%) samples did not react to any of the 7 peptide strains. Samples with CD4 cell counts greater than 500 x 1 million reacted significantly to more peptide strains than those with CD4 counts below 200 x 1 million (88% vs. 7%). Reactivity to specific strains were 21.5% for MN, 19.1% for Z2, 17.2% for HXB2, 11.5% for SC, 6.2% for CDC 4, 5.5% for Z6, and 3.8% for ELI. Anti-HXB2 antibodies were more common in blood donors than suspected AIDS/advanced HIV-infection cases (22% vs. 13%). AIDS/advanced HIV-infection cases were more likely to have no antibodies than blood donors (21% vs. 7%). A significant association existed between the number of peptide strains a patient could react to and the clinical state (p 0.01). Specifically, 77% of samples with no V3 antibodies to the seven strains had AIDS or advanced HIV infection while 55% of those which had cross reactivity with three or more strains were asymptomatic. Further research is needed to better understand this correlation. These findings suggest that use of strain specific peptides may be a sensitive and easier method for use for molecular epidemiological purposes.

In a bid to determine the HIV-1 subtype variants in transmission in Nairobi and its possible association with clinical status, we screened 207 confirmed HIV-1 positive patients visiting HIV/AIDS laboratory at the Virus Research Centre in Nairobi between January and March 1994. We used a selfmade ELISA obtained from an established panel of HIV-1 V3 loop peptides (ANRS, France) and derived from seven isolates: MN, HXB2, SC, Z6, Z2, ELI and CDC4. Test samples were obtained from 95 blood donors and medical examination attendees, 57 patients with chronic diarrhoea, 31 confirmed pulmonary tuberculosis, 16 with pneumonia and 12 herpes zoster. Out of the total, 21.5% had antibodies against the MN strain, 19.1% had against the Z2 strain while reaction against the HXB2 strain was observed in 17.2%. SC, CDC4, Z6 and ELI had prevalences of 11.5%, 6.2%, 5.3% and 3.8% respectively. Fifteen per cent of the tested sera showed no reaction to any of the used peptides. Strong and significant associations were observed between the total number of strains a sample react to and the clinical state. We infer that both the North American consensus strains (MN and HXB2) and the African isolates (Z2 and Z6) are predominant in Nairobi. The correlation between antibody reactivity and clinical state is an interesting observation that necessitates an expanded study and, the use of strain specific peptides maybe a sensitive and easier method for use for molecular epidemiological purposes. PIP: During January-March 1994, in Nairobi, Kenya, the sera of pre-university students, suspected AIDS/advanced HIV-infection cases, and blood donors were screened for HIV-1 antibodies at the Virus Research Centre. All confirmed HIV-1 positive samples were categorized according to the patient's clinical status. A self-made ELISA was obtained from an established panel of HIV-1 V3 loop peptides and derived from seven isolates (MN and HXB2 [North American strains], SC, CDC4, Z2 and Z6 [African strains], and ELI). The sera of the 22 confirmed HIV-1 negative students were used as negative controls. There were 207 confirmed HIV-1 cases (95 blood donors and 112 suspected AIDS/advanced HIV-infection cases). 64 (31%) and 112 (54%) samples reacted to at least 3 strains and no more than 2 strains, respectively. The remaining 31 (15%) samples did not react to any of the 7 peptide strains. Samples with CD4 cell counts greater than 500 x 1 million reacted significantly to more peptide strains than those with CD4 counts below 200 x 1 million (88% vs. 7%). Reactivity to specific strains were 21.5% for MN, 19.1% for Z2, 17.2% for HXB2, 11.5% for SC, 6.2% for CDC 4, 5.5% for Z6, and 3.8% for ELI. Anti-HXB2 antibodies were more common in blood donors than suspected AIDS/advanced HIV-infection cases (22% vs. 13%). AIDS/advanced HIV-infection cases were more likely to have no antibodies than blood donors (21% vs. 7%). A significant association existed between the number of peptide strains a patient could react to and the clinical state (p 0.01). Specifically, 77% of samples with no V3 antibodies to the seven strains had AIDS or advanced HIV infection while 55% of those which had cross reactivity with three or more strains were asymptomatic. Further research is needed to better understand this correlation. These findings suggest that use of strain specific peptides may be a sensitive and easier method for use for molecular epidemiological purposes.

In a bid to determine the HIV-1 subtype variants in transmission in Nairobi and its possible association with clinical status, we screened 207 confirmed HIV-1 positive patients visiting HIV/AIDS laboratory at the Virus Research Centre in Nairobi between January and March 1994. We used a selfmade ELISA obtained from an established panel of HIV-1 V3 loop peptides (ANRS, France) and derived from seven isolates: MN, HXB2, SC, Z6, Z2, ELI and CDC4. Test samples were obtained from 95 blood donors and medical examination attendees, 57 patients with chronic diarrhoea, 31 confirmed pulmonary tuberculosis, 16 with pneumonia and 12 herpes zoster. Out of the total, 21.5% had antibodies against the MN strain, 19.1% had against the Z2 strain while reaction against the HXB2 strain was observed in 17.2%. SC, CDC4, Z6 and ELI had prevalences of 11.5%, 6.2%, 5.3% and 3.8% respectively. Fifteen per cent of the tested sera showed no reaction to any of the used peptides. Strong and significant associations were observed between the total number of strains a sample react to and the clinical state. We infer that both the North American consensus strains (MN and HXB2) and the African isolates (Z2 and Z6) are predominant in Nairobi. The correlation between antibody reactivity and clinical state is an interesting observation that necessitates an expanded study and, the use of strain specific peptides maybe a sensitive and easier method for use for molecular epidemiological purposes. PIP: During January-March 1994, in Nairobi, Kenya, the sera of pre-university students, suspected AIDS/advanced HIV-infection cases, and blood donors were screened for HIV-1 antibodies at the Virus Research Centre. All confirmed HIV-1 positive samples were categorized according to the patient's clinical status. A self-made ELISA was obtained from an established panel of HIV-1 V3 loop peptides and derived from seven isolates (MN and HXB2 [North American strains], SC, CDC4, Z2 and Z6 [African strains], and ELI). The sera of the 22 confirmed HIV-1 negative students were used as negative controls. There were 207 confirmed HIV-1 cases (95 blood donors and 112 suspected AIDS/advanced HIV-infection cases). 64 (31%) and 112 (54%) samples reacted to at least 3 strains and no more than 2 strains, respectively. The remaining 31 (15%) samples did not react to any of the 7 peptide strains. Samples with CD4 cell counts greater than 500 x 1 million reacted significantly to more peptide strains than those with CD4 counts below 200 x 1 million (88% vs. 7%). Reactivity to specific strains were 21.5% for MN, 19.1% for Z2, 17.2% for HXB2, 11.5% for SC, 6.2% for CDC 4, 5.5% for Z6, and 3.8% for ELI. Anti-HXB2 antibodies were more common in blood donors than suspected AIDS/advanced HIV-infection cases (22% vs. 13%). AIDS/advanced HIV-infection cases were more likely to have no antibodies than blood donors (21% vs. 7%). A significant association existed between the number of peptide strains a patient could react to and the clinical state (p 0.01). Specifically, 77% of samples with no V3 antibodies to the seven strains had AIDS or advanced HIV infection while 55% of those which had cross reactivity with three or more strains were asymptomatic. Further research is needed to better understand this correlation. These findings suggest that use of strain specific peptides may be a sensitive and easier method for use for molecular epidemiological purposes.