SmMAK16, the Schistosoma mansoni homologue of MAK16 from yeast, targets protein transport to the nucleolus.

The SmMAK16 gene from Schistosoma mansoni was cloned by chance when an adult worm cDNA library was probed with antiserum to affinity-purified S. mansoni GSH S-transferases. SmMAK16 encodes a hydrophilic protein of 259 amino acids with a molecular mass of 31 kDa. The protein shares 43% sequence identity and 66% similarity to the nuclear protein MAK16 of Saccharomyces cerevisiae that has been implicated both in cell cycle progression and biogenesis of 60S ribosomal subunits. Both proteins display a similar degree of sequence similar to the hypothetical protein CeMAK16 from Caenorhabditis elegans. These proteins share a number of apparent protein motifs, including two nuclear localization signals (NLS), multiple sites for phosphorylation by protein kinase CK2 and four conserved cysteine residues that resemble a zinc binding domain. SmMAK16 mRNA is more highly expressed in adult female worm than males. Recombinant SmMAK16 was phosphorylated by human protein kinase CK2. When chimeric constructs containing SmMAK16 fused the green fluorescent protein (GFP) were transiently transfected into COS-7s cells, the reporter was localized not in nuclei, but exclusively in nucleoli. The yeast and nematode homologues were likewise able to direct nucleolar accumulation of the fluorescent reporter. The high degree of sequence conservation together with the ability to direct nucleolar protein transport supports the hypothesis that MAK16 proteins play a key role in the biogenesis of 60S subunits.

Induction of Schistosoma mansoni glutathione S-transferase by xenobiotics.

The effects of 3-methylcholanthrene, butylated hydroxyanisole, and phenobarbital on the expression of glutathione S-transferase (GST, EC 2.5.1.18) were examined in the human parasite Schistosoma mansoni. GST specific activity toward 1-chloro-2,4-dinitrobenzene increased by 170% in parasites recovered from mice injected with 3-methylcholanthrene and 230% in parasites recovered from mice maintained on a diet containing butylated hydroxyanisole. These increases in specific enzyme activity were paralleled by accumulation of mRNA hybridizing to pGT16.4, a cDNA clone that encodes the most abundant GST subunit, SmGST-3. Northern hybridization analysis showed a 5-fold increase in mRNA hybridizing to pGT16.4 72 h after exposure to 3-methylcholanthrene, a 10-fold increase after 12 days exposure to butylated hydroxyanisole, and a 6-fold increase 16 h after treatment with phenobarbital. In contrast, no accumulation of mRNA hybridizing to either of two other cDNA clones that encode the SmGST-4 and SmGST-6 subunits was detected. Hybrid select translation using pGT16.4 combined with reverse-phase high-pressure liquid chromatographic analysis demonstrated that in addition to SmGST-3 mRNA, the clone also hybridized to mRNA species encoding the SmGST-1 subunit, a member of the same isoenzyme family. High-pressure liquid chromatographic analysis of GST affinity purified from butylated hydroxyanisole-exposed parasites revealed a 2.5-fold increase in the concentration of SmGST-1 and SmGST-3 present compared with an equivalent amount of tissue from control organisms. There was no change, however, in the SmGST-1 to SmGST-3 ratio (1:6), indicating that both subunits were induced to the same extent by this agent. The results of these studies suggest that alterations in GST expression may influence the parasite's survival within the host environment.

Glutamine-supported motility of adult filarial parasites in vitro and the effect of glutamine antimetabolites.

The survival in culture of adult female Brugia pahangi, Acanthocheilonema viteae, and Onchocerca volvulus and adult male Onchocerca gibsoni was assessed by measuring parasite motility. Survival of all species was maximal in a nutritionally complex medium (RPMI-1640). All species survived for up to 48 hr in a simpler medium in which the only energy source was 10 mM glutamine; motility in this medium was dependent upon pH. For the species of Onchocerca, motility was maintained better in the presence of glutamine as the sole energy source than in glucose-only medium. Motility of B. pahangi incubated in 10 mM succinate was equivalent to that seen with 10 mM glutamine, but no other tricarboxylic acid intermediate supported this parasite in vitro. Antimycin A (1 microM) and potassium cyanide (KCN, 100 microM) paralyzed B. pahangi incubated in 10 mM glutamine, an effect antagonized by glucose. KCN at 10 or 100 microM was effective also against Onchocerca gutturosa in glutamine-only medium. Several glutamine antimetabolites reduced motility of B. pahangi by 72 hr. This inhibition was prevented by 2 mM glutamine. However, the inhibition of motility in the species of Onchocerca caused by these compounds was attenuated only partially by glutamine. These data demonstrate that, under certain conditions, filarial nematodes can utilize non-sugar substrates as energy sources. The differential sensitivity seen among these organisms to mitochondrial toxins and glutamine antimetabolites may be related to the extent to which they can use these alternative substrates to generate energy.

Chemotherapy of filariases.

The filarial parasites that affect humans most seriously include Onchocerca volvulus, Wuchereria bancrofti, Brugia malayi and Loa loa. In general, these species cause disease that is debilitating long before it is fatal, producing clinical manifestations such as general malaise, pruritus and lichenification of the skin, lymphangitis, elephantiasis and blindness(1). As a result, infection with any one of these organisms is physically, as well as economically, devastating. Currently, the pharmacological armamentarium with which to treat filarial infections is very limited and many of the drugs that are known to be efficacious against these worms may produce side effects that cause extreme discomfort. Here, Elizabeth Vande Waa describes the quest for new chemotherapeutic approaches for the treatment of filarial infections.

Effects of steroids and steroid synthesis inhibitors on fecundity ofSchistosoma mansoni in vitro.

In vitro egg production bySchistosoma mansoni was examined following a 72-hr incubation period in the presence of various steroids, steroid precursors, or steroid synthesis inhibitors. Mevinolin, an inhibitor of cholesterol biosynthesis, significantly decreased egg production at 10(-6) M. However, neither cholesterol (at 10(-5) M) nor any of its hydroxylated derivatives (at 10(-4) M) affectedS. mansoni fecundity. Dianisidine, an inhibitor of cholesterol metabolism to hormonal products, was also ineffective in influencing egg production. The steroid hormones progesterone, 17α-hydroxyprogesterone and medroxyprogesterone acetate, all significantly decreased egg production; however, parasite muscle tension was also significantly reduced by the concentrations of these steroids needed to produce an effect on egg laying. Various androgenic hormones (androsterone, androstenedione, testosterone) and estrogenic hormones (17ß-estradiol and estrone) demonstrated no effect on in vitro egg production. Significant elevation in the rate of parasite oviposition was seen in the presence of the corticosteroid prednisolone (at 10(-5) M), but not by dexamethasone, a corticosteroid analog.