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Purpose and methods In this study, the analgesic activity of the crude alcohol (acetone-methanol) and aqueous (in PBS, pH 7.2) extracts of the marine molluscs, Pachymelania aurita and Tympanotonus fuscatus, has been evaluated using the formalin test (for chronic antinociceptive) and the tail-flick (acute antinociceptive) pain models in male swiss albino mice. Results The results show that the extracts of P. aurita and T. fuscatus demonstrated high safety margins as single doses of up to 2000 mg/kg bwt proved to be well tolerated and non-lethal, although the alcohol extract of P. aurita caused necrosis in the liver and kidney when administered at a dose level of 2000 mg/kg bwt. In the formalin test, treatment with the aqueous extracts of P. aurita and T. fuscatus as well as the alcohol extract of T. fuscatus 30 min before the subcutaneous injection of 5% formalin to the paw of the mice resulted in a significant time- and dose-dependent reduction in total and phase 2a pain-related behavior and thus nociception. The extracts had no analgesic effect in tail-flick test up to the highest dose tested. Conclusion Hence, the results from both models indicate that the site of their analgesic action is probably peripheral.

Introduction Globally anti-tuberculosis drug resistance is one of the major challenges affecting control and prevention of tuberculosis. Kenya is ranked among 30 high burden TB countries globally. However, there is scanty information on second line antituberculosis drug resistance among tuberculosis patients. Therefore, this study aimed at determining Mycobacterium tuberculosis drug resistant strain distribution pattern in 10 counties of Western Kenya among HIV positive and negative patients. Method A cross-sectional study was conducted in Western Kenya, which comprises 10 counties. A multistage sampling method was used where a single sub-county was randomly selected followed by sampling one high volume health facility from each sub-county. Consenting study subjects with at least two smear positive sputum at the time of enrolment were randomly selected. The collected sputum was decontaminated with N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH) and then stained with Ziehl Neelsen Stain before visualizing the presence of bacilli under microscope at ×100 magnification with oil immersion. Further, the identified bacilli were cultured and susceptibility test carried out using known first and second line antimycobacterial tuberculosis. HIV testing was carried out using Determine® HIV-1/2 rapid test (Abbot Diagnostics, Maidenhead, United Kingdom). Those who had smear converted were dropped from the study. Finally, drug susceptibility pattern across the 10 counties of Western Kenya was evaluated. Results Our study showed that Mycobacterium tuberculosis drug resistance among HIV negative and positive cases in Western Kenya was prevalent in all the 10 counties surveyed. Based on the drug susceptibility tests, 53.2% and 42.7% of the study samples were resistant to at least one antituberculosis drug among HIV negative and HIV positive patients respectively. The data analysis revealed that among the HIV-positive and HIV-negative patients, resistance to INH was predominant (28.5%, and 23.6%, respectively), followed by RIF (16.4% and 14.6% respectively). Second-line drug resistant strains identified among HIV negative patients included Ethionamide (0.3%), Gatifloxacin (0.3%), Amikacin (0.3%) and Capreomycin (0.3%). There was no second line drug monoresistance among HIV positive TB patients. Multi/poly drug resistance were noted among HIV-negative patients in, INH + AMK (0.7%), INH + PZA (1%), INH + GFX (0.7%, INH + ETO (0.7%, STY + ETO (1%), ETH + ETO (1.0%), INH + KAN (0.7%) and INH + CAP (0.7%) strains/cases at 95% confidence interval. Among HIV positive patients INH + GFX (1.1%), INH + ETO (0.4%) and INH + KAN (0.4%) strains of M. tuberculosis were identified with a confidence interval of 95%. Geographical distribution patterns analysis of M. tuberculosis drug polyresistant strains across the 10 counties were recorded. Among HIV TB patients, resistant strains were identified in Nyamira (INH + GFX, INH + KAN), Bungoma ((ETO + STY), Busia (ETH + ETO and STY + ETO) Homabay (RIF + AMK. ETO + ETH and ETO + STY), Kisumu (ETH + ETO and PZA + ETO) and in Kakamega, Kisii and Vihiga (INH + KAN and RIF + AMK). There was no M. tuberculosis polyresistant strain identified in Migori and Siaya counties. Among HIV positive TB patients, M. tuberculosis resistant strains were identified in three counties, Nyamira (INH + KAN) Homabay (INH + GFX and INH + AMK) and Kakamega (INH + GFX). There was no polyresistant M. tuberculosis strain identified in Migori, Bungoma, Kisii, Vihiga, Busia, Siaya and Kisumu Counties. Discussion The distribution patterns of M. tuberculosis drug resistance among HIV negative and positive TB patients could be as a result of reported high prevalence of HIV in Western Kenya counties especially the area under study. Tuberculosis is one of the opportunistic diseases that have been shown to be the major cause of AIDS among HIV infected patients. Resent reports by National AIDS Control Council shows that Kisumu, Siaya, Homabay, Migori, Busia have the overall leading in HIV prevalence in Kenya. The low prevalence of drug resistant strains among HIV tuberculosis patients could be as a result of drug adherence attitude adopted by HIV patients, availability of continuous counselling and close follow up and notification by healthcare workers and community health volunteers. Conclusion Drug resistant M. tuberculosis strains prevalence is still high among HIV negative and positive patients in Western Kenya with the most affected being HIV negative TB patients. It is therefore probable that the existing control measures are not adequate to control transmission of drug resistant strains. Further, miss diagnosis or delayed diagnosis of TB patients could be contributing to the emergence of M. tuberculosis drug polyresistant strains. Recommendation Based on the result of this study, regular TB drug resistance surveillance should be conducted to ensure targeted interventions aimed at controlling increased transmission of the tuberculosis drug resistant strains among HIV/AIDS and HIV negative patients. There is also need for improved drug resistant infection control measures, timely and rapid diagnosis and enhanced and active screening strategies of tuberculosis among suspected TB patients need to be put in place. Further, studies using a larger patient cohort and from counties across the country would shed much needed insights on the true national prevalence of different variants of M. tuberculosis drug resistance.

Vector-borne diseases (VBDs) cause enormous health burden worldwide, as they account for more than 17% of all infectious diseases and over 700,000 deaths each year. A significant number of these VBDs are caused by RNA virus pathogens. Here, we used metagenomics and metabarcoding analysis to characterize RNA viruses and their insect hosts among biting midges from Kenya. We identified a total of 15 phylogenetically distinct insect-specific viruses. These viruses fall into six families, with one virus falling in the recently proposed negevirus taxon. The six virus families include Partitiviridae, Iflaviridae, Tombusviridae, Solemoviridae, Totiviridae, and Chuviridae. In addition, we identified many insect species that were possibly associated with the identified viruses. Ceratopogonidae was the most common family of midges identified. Others included Chironomidae and Cecidomyiidae. Our findings reveal a diverse RNA virome among Kenyan midges that includes previously unknown viruses. Further, metabarcoding analysis based on COI (cytochrome c oxidase subunit 1 mitochondrial gene) barcodes reveal a diverse array of midge species among the insects used in the study. Successful application of metagenomics and metabarcoding methods to characterize RNA viruses and their insect hosts in this study highlights a possible simultaneous application of these two methods as cost-effective approaches to virus surveillance and host characterization. IMPORTANCE The majority of the viruses that currently cause diseases in humans and animals are RNA viruses, and more specifically arthropod-transmitted viruses. They cause diseases such as dengue, West Nile infection, bluetongue disease, Schmallenberg disease, and yellow fever, among others. Several sequencing investigations have shown us that a diverse array of RNA viruses among insect vectors remain unknown. Some of these could be ancient lineages that could aid in comprehensive studies on RNA virus evolution. Such studies may provide us with insights into the evolution of the currently pathogenic viruses. Here, we applied metagenomics to field-collected midges and we managed to characterize several RNA viruses, where we recovered complete and nearly complete genomes of these viruses. We also characterized the insect host species that are associated with these viruses. These results add to the currently known diversity of RNA viruses among biting midges as well as their associated insect hosts.

This study aimed at molecular and biochemical characterization of low-density polyethene (LDPE) degrading fungi and bacteria from Dandora dumpsite, Nairobi. Twenty bacterial and 10 fungal isolates were identified using 16S rDNA and 18S rDNA sequences for bacteria and fungi, respectively. The highest fungal degradation was attributed to Aspergillus oryzae strain A5,1 while the highest bacterial degradation was attributed to Bacillus cereus strain A5,a and Brevibacillus borstelensis strain B2,2, respectively. Isolates were screened for their ability to produce extracellular laccase and esterase; Aspergillus fumigatus strain B2,2 exhibited the highest presence of laccase (15.67 mm) while Aspergillus oryzae strain A5,1 exhibited the highest presence of esterase (14.33 mm). Alkane hydroxylase-encoding genes were screened for using primer AlkB 1 which amplified the fragment of size 870 bp. Four bacterial samples were positive for the gene. Optimum growth temperature of the fungal isolates was 30°C. The possession of laccase, esterase, and alkane hydroxylase activities is suggested as key molecular basis for LDPE degrading capacity. Knowledge of optimum growth conditions will serve to better utilize microbes in the bioremediation of LDPE. The application of Aspergillus oryzae strain A5,1 and Bacillus cereus strain A5,a in polyethene degradation is a promising option in this kind of bioremediation as they exhibited significantly high levels of biodegradation. Further investigation of more alkane degrading genes in biodegrading microbes will inform the choice of the right microbial consortia for bioaugmentation strategies.

This study aimed at isolating and identifying bacteria and fungi with the capacity to degrade low density polyethylene (LDPE). The level of biodegradation of LDPE sheets with bacterial and fungal inoculums from different sampling points of Dandora dumpsite was evaluated under laboratory conditions. Incubation of the LDPE sheets was done for sixteen weeks at 37°C and 28°C for bacteria and fungi respectively in a shaker incubator. Isolation of effective candidates for biodegradation was done based on the recorded biodegradation outcomes. The extent of biodegradation on the polyethylene sheets was assessed by various techniques including weight loss analysis, Fourier Transform Infrared Spectroscopy (FTIR) and GC-MS. Fourier Transform Infra-Red spectroscopy (FTIR) analysis revealed the appearance of new functional groups attributed to hydrocarbon degradation after incubation with the bacteria and fungi. Analysis of the 16S rDNA and 18S rDNA sequences for bacteria and fungi respectively showed that bacteria belonging to genera Pseudomonas, Bacillus, Brevibacillus, Cellulosimicrobium, Lysinibacillus and fungi of genus Aspergillus were implicated as polyethylene degraders. An overall analysis confirmed that fungi are generally better degraders of polyethylene than bacteria. The highest fungal degradation activity was a mean weight reduction of 36.4±5.53% attributed to Aspergillus oryzae strain A5, 1 (MG779508). The highest degradation activity for bacteria was a mean of 35.72± 4.01% and 20.28± 2.30% attributed to Bacillus cereus strain A5,a (MG645264) and Brevibacillus borstelensis strain B2,2 (MG645267) respectively. Genus Aspergillus, Bacillus and Brevibacillus were confirmed to be good candidates for Low Density Poly Ethene bio-degradation. This was further confirmed by the appearance of the aldehyde, ether and carboxyl functional groups after FTIR analysis of the polythene sheets and the appearance of a ketone which is also an intermediary product in the culture media. To improve this degrading capacity through assessment of optimum conditions for microbial activity and enzyme production will enable these findings to be applied commercially and on a larger scale.

Scientific and technological progress envisages the solving the most important problems in the main areas of mineral processing, leading to the perfection of technological processes, improving quality indicators and reducing the cost of production. Rational use of minerals at the stages of their extraction and enrichment is a single indivisible problem, the main task of which is the fullest use of basic and rare minerals. Geo-resources include mineral deposits and subsurface materials (solid, liquid, gaseous, mixtures), underground space (cavity), subsurface energy (static, geo-dynamic, geomagnetic). Geo-resources are provisionally recognized as geoinformation, characterized by various attributes and properties of subsurface material (minerals, overburden, etc.), and the other geo-resources, including streams migrating into the depths (fluids, magma, etc.). It is clear that mineral resources are part of the subsurface material. Therefore, the most important aspect of geo-resources development today is the initiation of recycling of their industrial processing.

Trans-generational transfer of gregarious-phase traits in the desert locustSchistocerca gregaria(Forskål, 1775) is mediated by primer gregarizing pheromonal signals produced by ovipositing females that experience crowding. We monitored time-course proteomic events in eggs from solitary-reared locusts that had been exposed for 1, 3, 5, 7, 10 and 12 days to different levels of the sand-associated gregarizing signal originating from 0, 3, 5 or 10 ovipositions by crowd-reared females. Evidence for the phase transition was sought by comparing the protein patterns of embryos thus exposed with those from crowd-reared (gregarious) controls; this comparison was continued until the stage of the first instars. Expressed proteins were analysed by two-dimensional protein gel electrophoresis, and patterns from the different treatments within stages were compared by profile matching andχ2analyses. Eggs derived from crowd- and solitary-reared females showed essentially similar protein patterns at early stages of embryogenesis; however, mature stages (particularly, days 10 and 12) and hatchlings demonstrated significantly different patterns. Protein patterns of eggs from solitary-reared females that were incubated in sand contaminated with the pheromonal signal and of the hatchlings that emerged were similar to those derived from gregarious females and dependent on the level of the pheromone to which the embryos had been exposed. The results confirm the gregarizing effect of the signal and constitute a useful basis for unravelling the mechanism of the signalling cascades associated with gene expressions triggered by the pheromone.

The prevalence of a genetic polymorphism(s) at codon 268 in the cytochrome b gene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227 Plasmodium falciparum parasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC 50 ) and parasite genetic polymorphism; mutant alleles had higher IC 50 s than the wild type.

Midgut trypsin and lectin levels were determined in three tsetse species, namely Glossina morsitans morsitans, G. longipennis and G. fuscipes fuscipes. In addition, the abilities of midgut homogenates prepared from these flies to transform bloodstream-form Trypanosoma brucei brucei and T. congolense were compared in vitro. In all the species examined, trypsin levels did not differ significantly up to 24 h post-bloodmeal. There were similar rates of transformation of the bloodstream-form trypanosomes into procyclic (midgut) forms in vivo, so that all species had similar levels of infection in the midgut. However, trypsin levels continued to increase beyond 24 h, reaching a peak between 48 and 72 h. The peak was lowest in G. m. morsitans. The midgut homogenates in this species also had the lowest levels of lectin. The species had the highest levels of mature T. congolense and T. brucei infections. We propose that the lower levels of peak midgut tryspin and lectin in G. m. morsitans is important in the establishment of trypanosome infections in this species of tsetse.

Summary The midgut of the human body louse Pediculus humanus humanus contains a thermally stable leucine aminopeptidase, which was detected by agarose gel electrophoresis using l-amino oxidase. Midgut extracts were homogenized in saline or in 1% Triton X-100 and the aminopeptidase was purified by Superose 6 gel filtration chromatography. A peak with enzyme activity that was extracted with or without Triton X-100 was eluted at a molecular weight 67–69 kDa. Non-denaturing polyacrylamide gel electrophoresis resolved one band of molecular weight of 69 kDa for samples that were extracted in a saline buffer. Two closely linked bands of molecular weight 67 kDa and 69 kDa were observed in samples that were extracted in 1% Triton X-100.

The concentrations of catechins in a Kenyan tea germplasm collection of 102 accessions were determined by HPLC. Total green leaf catechin concentrations and the ratio of dihydroxylated to trihydroxylated catechins were used to establish genetic differentiation in the germplasm. Upon multivariate analysis, accumulation of the various catechins separated the tea clones into 3 major and 5 minor groups according to their phylogenetic origins. The Cambod teas had the highest ratio (7:10) followed by China teas (3:5) while Assam teas had the lowest ratio (1:4). This biochemical differentiation indicates that there is potential for broadening the genetic base of the mainly Assam teas in Kenya (90%) with the putative China and Cambod teas.

A leucine aminopeptidase was found in the midgut of the human body louse, Pediculus humanus humanus L. (Anoplura: Pediculidae). The enzyme is activated by the bloodmeal with a pH optimum at 8. The enzyme is soluble in both aqueous and detergent-containing solutions. The two forms of the enzyme had the same Km but exhibited different catalytic activities with regard to Vmax values in these solutions. The enzyme is inhibited competitively by a substrate analogue 1,10-phenanthroline and by Mn2+ ions in the presence and absence of detergent.

Midgut homogenates prepared from Glossina morsitans morsitans, that had previously been fed on different host blood samples, were tested for their abilities to transform bloodstream Trypanosoma brucei into procyclic (midgut) forms in vitro. Compared to rat and goat blood samples, eland blood had the least capacity to support trypanosome transformation, whereas buffalo blood showed intermediate capacity. Fractionation of rat blood showed the importance of the cellular portion since both rat and eland red blood cells (RBCs) supported the process. Virtually no transformation was observed in rat and eland plasma or serum fractions. Suspending rat blood cells in eland plasma led to a reduction in parasite transformation rates. Further experiments showed that the RBC membranes were also capable of supporting the process. These results clearly show the important role played by blood, especially the red blood cells, in the transformation of bloodstream trypanosomes. In addition, the low transformation rates observed in eland blood is due to an inhibitory factor(s) present in the plasma fraction.

Genetically determined artemisinin resistance in Plasmodium falciparum has been described in Southeast Asia. The relevance of recently described Kelch 13-propeller mutations for artemisinin resistance in Sub-Saharan Africa parasites is still unknown. Southeast Asia parasites have low genetic diversity compared to Sub-Saharan Africa, where parasites are highly genetically diverse. This study attempted to elucidate whether genetics provides a basis for discovering molecular markers in response to artemisinin drug treatment in P. falciparum in Kenya. The genetic diversity of parasites collected pre- and post- introduction of artemisinin combination therapy (ACT) in western Kenya was determined. A panel of 12 microsatellites and 91 single nucleotide polymorphisms (SNPs) distributed across the P. falciparum genome were genotyped. Parasite clearance rates were obtained for the post-ACT parasites. The 12 microsatellites were highly polymorphic with post-ACT parasites being significantly more diverse compared to pre-ACT (p < 0.0001). The median clearance half-life was 2.55 hours for the post-ACT parasites. Based on SNP analysis, 15 of 90 post-ACT parasites were single-clone infections. Analysis revealed 3 SNPs that might have some causal association with parasite clearance rates. Further, genetic analysis using Bayesian tree revealed parasites with similar clearance phenotypes were more closely genetically related. With further studies, SNPs described here and genetically determined response to artemisinin treatment might be useful in tracking artemisinin resistance in Kenya.

The properties of a blood-meal-induced lectin (agglutinin) from the midgut of Glossina morsitans capable of agglutinating Trypanosoma brucei were studied in vitro. The midgut homogenate from flies that had been fed twice had the highest agglutination activity, followed by that from the once-fed flies and that from the unfed insects. As compared with the bloodstream-form trypanosomes, a much lower concentration of the midgut homogenate was required for agglutination of the procyclic parasites. Furthermore, the agglutination process was specifically inhibited by D-glucosamine. Soybean trypsin inhibitor abrogated agglutination of the bloodstream-form parasites, whereas the procyclics were unaffected. The agglutination process was temperature-sensitive, with little activity being evident between 4 degrees and 15 degrees C. Similarly, heating the midguts to 60 degrees-100 degrees C led to loss of activity. When the midgut homogenate was separated by anion-exchange chromatography, the agglutination activity co-eluted with trypsin activity at approximately 50% NaCl. These results suggest a very close relationship between midgut trypsin-like enzyme and the agglutinin. Since successful agglutination of bloodstream-form trypanosomes requires protease activity, it may be that the enzyme cleaves off some surface molecules on the parasite surface, thus exposing the lectin-binding sites.

The stem extract of Tephrosia purpurea showed antiplasmodial activity against the D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) strains of Plasmodium falciparum with IC(50) values of 10.47 +/- 2.22 mu g/ml and 12.06 +/- 2.54 mu g/ml, respectively. A new prenylated flavone, named terpurinflavone, along with the known compounds lanceolatin A, (-)-semiglabrin and lanceolatin B have been isolated from this extract. The new compound, terpurinflavone, showed the highest antiplasmodial activity with IC(50) values of 3.12 +/- 0.28 mu M (D6) and 6.26 +/- 2.66 mu M (W2). The structures were determined on the basis of spectroscopic evidence.

The effect of the amino sugar D-glucosamine on trypsin in crude midgut homogenates of Glossina morsitans morsitans was studied in vitro. The results showed that the midgut trypsin was specifically and competitively inhibited by D-Glucosamine. Glucose, fructose, mannose, inositol, galactose, galactosamine, N-acetyl-D-glucosamine, and methyl-alpha-D-glucosamine were ineffective as inhibitors, even at concentrations exceeding 600 mM. D-glucosamine also had a similar inhibitory effect on bovine pancreatic trypsin. In both cases, the inhibition was incomplete as shown by nonlinear Dixon plots. The Michaelis and inhibition constants estimated for the midgut trypsin were 41 +/- 2 microM and 68 +/- 3 mM, respectively. These results suggest that the susceptibility of tsetse flies to trypanosome infection, which is associated with high midgut glucosamine levels, could be due to inhibition of trypsin or trypsin-like enzymes by this sugar.

Certain breeds of domestic ruminants show remarkable resistance to the effects of African trypanosomosis. Unlike susceptible animals, trypanotolerant animals control parasitemia and do not show severe anaemia or production loss. Identification of trypanotolerance genes in cattle is hampered by cost and breeding time. Marked differences between inbred strains of mice in their response to T. congolense infection can be exploited in the analysis of the genetic basis of the infection. Murine trypanotolerance QTLs have been identified on chromosome 17, 5 and 1, and designated as Tir1, 2 and 3, respectively. Tir1 and 2 have been fine mapped to a confidence interval of 1 cM. In order to find the mouse homologous region on the bovine genome, nucleotide sequence across 95% CI of Tir2 and 3 were used in the selection of candidate genes. Homologous sequences were used in the definition of synteny relationships and subsequent identification of the shared disease response genes. The homologous genes within the human genome were then identified and aligned to the bovine radiation hybrid map in order to identify the mouse/bovine homologous regions. This revealed homology between murine and bovine QTL on Tir3 while the region on Tir2 is linked to innate immune response.

The dichloromethane extracts of the root and leaf of Tephrosia vogelii Hook F. (Family: Papilionaceae) grown in Kenya, were subjected to antimicrobial assays against Staphylococcus aureus, Escherichia coli and Fusarium phoseolida . The minimum inhibition concentrations (MIC) ranged between 0.25 to 6.4 μg/ml for all the microorganisms tested. The crude root and leaf extracts were also toxic to the brine shrimps, organisms used as indicators of toxicity (LC50: 0.960; 0.958 μg/ml, respectively). Chromatographic separation led to the identification of (6as, 12as, 5's)-6a, 12a, 4',5'-tetra hydro-2,3-dimethoxy 5'-s- isopropenyl–furano (3',2': 8,9)-6H-rotoxen-12-one, (rotenone) in 9.75% yield, (roots), and 11.75% (leaves). Characterisation was achieved by use of spectroscopic techniques and comparison with rotenone standard and literature data. Novel HPLC conditions for purification of rotenoids were established. The MIC of rotenone against the tested microbes was found to be 5.2 and 1.0 μg/ml for Staphylococcus aureus and Escherichia col i respectively, while the LC50 against the brine shrimps was found to be 3.20 μg/ml. The findings indicate that the extracts of T. vogelii contain metabolites that have antimicrobial activity comparable to other medicinal plants. Keywords : Tephrosia vogelii , Rotenone, Staphylococcus aureus, Escherichiacoli, Fusarium phoseolida Journal of Agriculture, Science and Technology Vol. 8 (1) 2006: pp. 1-14

A blood-meal-induced lectin (agglutinin) with proteolytic activity was isolated from midgut extracts of Glossina longipennis by a two-step procedure involving anion-exchange chromatography. It is a glycoprotein [native molecular weight (M(r) 61,000 +/- 3000 da) composed of two noncovalently-linked subunits designated alpha (M(r), approximately 27,000 da) and beta (M(r), approximately 33,000 da). The trypsin activity and the glycosyl residues were present on the alpha- and beta-subunits, respectively. The native protein was capable of agglutinating both bloodstream-form and procyclic trypanosomes as well as rabbit red blood cells. This activity was strongly inhibited by D-glucosamine and weakly inhibited by N-acetyl-D-glucosamine. Similarly, soybean trypsin inhibitor abrogated agglutination of bloodstream-form parasites, whereas the procyclics were unaffected. The agglutination activity was sensitive to temperatures above 40 degrees C but was unaffected by chelators of metal ions. Antibodies raised against the protein were used in immunoblotting experiments to show the presence of a similar protein in several members of the Glossina species. However, no cross-reactivity was detected with midgut extracts prepared from sandflies, mosquitoes, or stable flies. It is proposed that this molecule might play an important role in differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms.

The ability of Trypanosoma brucei brucei to inhibit trypsin or trypsin-like enzymes in crude midgut homogenates of Glossina morsitans morsitans was studied in vitro. The isolated parasites caused a concentration-dependent decrease in midgut trypsin activity. Furthermore, trypanosomes lysed by repeated freeze-thawing had a similar effect on trypsin activity. In both cases, the inhibition by either intact or lysed parasites was partial as revealed by Dixon plots. Similarly, trypanosome membrane proteins stoichiometrically inhibited trypsin activity, suggesting that the enzyme interacts specifically with a moiety on the parasite surface. The Km and Ki values obtained in this case were 35 microM and 0.18 mg/ml, respectively. These results suggest that one of the ways in which trypanosomes overcome the hostile tsetse-fly midgut barrier involves the inhibition of enzyme activity.

An in vitro system for studying the transformation of bloodstream forms of Trypanosoma brucei brucei into procylic (midgut) forms is described. In this system, transformation of the parasites was stimulated by Glossina morsitans morsitans midgut homogenates at 27 degrees C but not at 4 degrees C. The transformation-stimulating capacity was irreversibly destroyed by heating the midgut homogenates at 60 degrees C for 1 h. A correlation was established between the transformation activity of the midgut homogenates and trypsin activity. The protease inhibitors (soybean trypsin inhibitor and N-p-tosyl-L-lysine-chloromethyl-ketone) inhibited trypsin activity and completely blocked the transformation of the parasites. Furthermore, the midgut homogenates could induce transformation only in the presence of blood. These results provide evidence for the involvement of trypsin or trypsin-like enzymes within the tsetse midgut in stimulation of the transformation of bloodstream trypanosomes.

Nad-linked malic enzyme ( L -malate: NAD + oxidoreductase (decarboxylating), E.C. 1.1.1.38) was purified from muscle of the dung beetle Catharsius to a specific activity of 28 μmol/min/mg of protein. Its molecular weight was estimated as 250,000–260,000 by Sephadex gel filtration. Activity was dependent on Mg 2+ or Mn 2+ , for which K a values of 0.18 and 0.019 mM were obtained respectively. With Mg 2+ , at pH 7.2, apparent K m values obtained for L -malate (with 0.76 mM NAD + ) and NAD + (with 2.5 mM L -malate) were 1.40 and 0.54 mM respectively. Plots of activity versus substrate concentration tended to be sigmoidal, but became hyperbolic in the presence of succinate or fumarate. These dicarboxylic acids lowered apparent K m values, but did not affect maximum velocity. With succinate present, the apparent K m for NAD + decreased with increasing L -malate concentration towards a limiting value of 0.12 mM; the apparent K m for L -malate decreased with increasing NAD + concentration towards a limiting value of zero. The maximum velocity decreased sharply below pH 7.0; apparent K m values increased with increasing pH. With Mn 2+ , plots of activity versus substrate concentration were more nearly hyperbolic and apparent K m values were lower than with Mg 2+ . With NADP, maximum activities 30 and 40% those with NAD were obtained with Mg 2+ and Mn 2+ respectively. The reductive carboxylation of pyruvate occurred in the presence of Mn 2+ , and with high concentrations of bicarbonate and pyruvate, at up to 5% the maximum rate of the forward reaction. A Ca 2+ stimulated oxaloacetate decarboxylase activity was associated with the enzyme.

Relatively high activities of NAD-linked ‘malic’ enzyme (E.C.1.1.1.39) and alanine aminotransferase (E.C.2.6.1.2), predominantly or entirely mitochondrial, were found in the flight and coxal muscle of three species of dung beetle (Scarabaeidae: Coprinae). In this respect these muscles resembled the flight muscle of the tsetse fly Glossina morsitans , but contrasted with flight muscle from six other non-Coleopteran insect species examined. Flight and coxal muscle from the dung beetle Helicopris dilloni also contained high proline dehydrogenase and glutamate dyhydrogenase (E.C.1.4.1.2) activities, but particularly low activities of hexokinase (E.C.2.7.1.1), phosphofructokinase (E.C.2.7.1.11), aldolase (E.C.4.1.2.7) and lactate dehydrogenase (E.C.1.1.1.27). Mitochondrial preparations isolated from flight and leg muscle of H. dilloni respired more rapidly in the presence of proline than other added substrates. These results suggest the use of proline as a major direct energy source for both flight and non-flight muscle in Coprine beetles.

Indigenous fruit trees are widely used by humans in southern Africa. Little information is however available on regional genetic variation in these species, knowledge essential for their proper use and conservation. Here, we begin to address this gap by assessing RAPD and chloroplast variation in Sclerocarya birrea, a fruit tree that has been important to humans in the region for millennia. A strong overall positive correlation between genetic (RAPD) and geographic distances was observed for 12 populations of S. birrea subsp. caffra sampled from seven countries (standardised Mantel statistic, rM=0.857, Pb0.001), supporting a ‘structuring-by-distance’ model in devising genetic management strategies. Cluster analysis indicated, however, that genetic distances between geographically proximate stands were high on occasions, suggesting that inclusion of multiple stands nationally can sometimes be useful in rangewide management. Overall, an analysis of molecular variance (AMOVA) indicated that 19% of RAPD (assumed nuclear) variation partitioned among S. birrea subsp. caffra stands. Consistent with other nuclear– organellar comparisons for tree species, a much greater proportion of chloroplast variation (70%) partitioned among stands, suggesting a rather limited role for seed compared to pollen in mediating gene flow. Further analysis of S. birrea involved additional sampling from Tanzania of two other recognised subspecies (subsp. birrea and subsp. multifoliolata) that are not otherwise found in the southern Africa region. AMOVA indicated that more RAPD (29%) and chloroplast (75%) variation partitioned among relatively proximate subspecies stands (4 populations, 1 subsp. caffra, 2 subsp. birrea and 1 subsp. multifoliolata) in Tanzania than among subsp. caffra stands sampled extensively across southern Africa, suggesting Tanzania should be a focus for genetic management activities. © 2006 SAAB. Published by Elsevier B.V. All rights reserved.

High primary doses ofTrichinella spiralisadministered orally to Kenyan baboons (Papio anubis) induced a marked but unpredictable eosinophilia which started 2–3 weeks after infection and persisted as erratic waves for at least 6 months. Low primary oral doses induced no eosinophilia but a later, high challenge gave an accelerated eosino-philic response, although the peak was lower than in high primary infection. Intravenous injection of infectiveT. spiralislarvae resulted in a predictable, rapid eosinophilic response which persisted for several weeks. Intravenous injection of a particulate material, Sepharose, gave no oesinophilic response.PercutaneousSchistosoma mansoniinfection of baboons resulted in a two-stage eosinophilic response: an initial rise occurred about ⅔ of the way through the pre-patent period and was followed by a second rise at the onset of patency. After peaking, the eosinophilia waned erratically over the next 3 or 4 weeks.A strong antibody response, measured by countercurrent imnruno-electrophoresis, was given in oral infections withT. spiralis, but intravenous injections elicited little or no antibody formation. Parasitological evidence indicated no cross-resistance toS. mansoniin baboons injected withT. spiralis9 days previously. Thus, the intravenous injection of infectiveT. spiralislarvae appears to be a suitable method of inducing experimentally a non-specific eosinophilia to investigate possible immune mechanisms toS. mansoniin the baboon.

This study aimed to investigate the antimitotic and antiproliferation activities of crude acetone-methanol and aqueous extracts of two marine molluscs commonly found in the Niger Delta region of Nigeria; T.fuscatus and P.aurita, against human cancerous cell lines (DU145, Hep-2, and HCC1395) cell lines in vitro. The antimitotic activity of the extracts was evaluated using Allium cepa root meristematic cells. Antiproliferative activity of the plant extracts against the cancerous cell lines was compared with normal cell line (VeroE6). Doxorubicin was used as a positive control. Gene expression studies using qPCR for the proapoptotic genes, CASP3, CASP8 and P53 were also carried out. The alcohol extract of T.fuscatus (TFAC) exhibited the most promising activity against all the cancer cell lines tested (DU145 IC(50) = 96.48 ± 1.36 μg/ml, HCC 1395 IC(50) = 61.44 ± 2.45 μg/ml, Hep2 IC(50) = 0.52 ± 0.36 μg/ml) and also had the highest selectivity index of 4.94, 7.78 and 921.97 for DU145, HCC 1395 and Hep-2 cells respectively. Furthermore, TFAC was the only extract that significantly upregulated the expression of caspase 3, caspase 8 and P53. Thus, these findings suggest potential exploitation of TFAC as an anticancer agent.