Plasmodium chabaudi: Genetics of resistance to chloroquine

A high level of chloroquine resistance was developed in the rodent malaria parasite, Plasmodium chabaudi. This resistance was stable and its inheritance was shown to be multigenic; intermediate levels of resistance were obtained from a cross between highly resistant and sensitive parasites. Chloroquine resistance was shown to segregate independently of pyrimethamine resistance and enzyme markers.     

Schistosomatium douthitti: carbohydrate metabolism in the adults.

Pathways of carbohydrate metabolism in the adults of Schistosomatium douthitti: were investigated. Histochemical reactions for adenosinetriphosphatase (EC 3.6.1.3) glucose 6-phosphate dehydrogenase (EC 1.1.1.49), phosphogluconate dehydrogenase (EC 1.1.1.43), glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), lactate dehydrogenase (EC 1.1.1.27, 1.1.2.3) isocitrate dehydrogenase (EC 1.1.1.41), succinate dehydrogenase (EC 1.3.99.1), malate dehydrogenase (EC 1.1.1.37), cytochrome oxidase (EC 1.9.3.1), and adenosine triphosphatase (EC 3.6.1.3) were found in the adult worms. Glycogen deposits occurred in the parenchyma.Low oxygen tension immobilized the worms. Tartar emetic, sodium cyanide reduced adult motility in vitro. Manometric experiments demonstrated a respiratory quotient of approximately one. Oxygen uptake was completely inhibited by tartar emetic and partially inhibited by sodium fluoracetate and sodium cyanide. Inhibition by sodium fluoroacetate was partially counteracted by citric acid in the medium.Adults demonstrated an oxygen debt following anaerobic incubation. A maximum of 52% of the glucose consumed under aerobic conditions was excreted as lactic acid. Under anaerobic conditions the amount of lactic acid excreted increased. Acids other than lactic acid were also released. Results indicate that although glycolysis is the major pathway, two additional aerobic pathways also exist, one which is cyanide sensitive and the other cyanide insensitive.     

Schistosoma mansoni: Purification and characterization of malate dehydrogenases

Isoelectric focusing of a homogenate of Schistosoma mansoni, followed by malate dehydrogenase-specific staining, showed the presence of two major and five minor malate dehydrogenase isoenzymes (EC 1.1.1.37), with isoelectric points ranging from 7.3 to 9.5. The malate dehydrogenase isoenzymes were purified by gel filtration, followed by ion-exchange chromatography on DEAE- and CM-cellulose. The isoenzymes could be differentiated by their susceptibility to substrate inhibition. No differences in the Michaelis-Menten constants for substrate were found. One of the isoenzymes is inhibited by 5′-AMP. Further purification of this particular isoenzyme was achieved by affinity chromatography on 5′-AMP-Sepharose 4B. Analysis after subcellular fractionation indicated a mitochondrial origin for this isoenzyme. The mitochondrial isoenzyme (at a recovery of 80%) was purified 218-fold compared to the crude soluble extract, and contained about 40% of the total malate dehydrogenase activity. The enzyme has a molecular weight of 65,500 and showed absolute specificity for l-malic acid, NAD, and NADH. The final preparation has a specific activity of 451 U/mg protein. Physicochemical studies, including binding constants, substrate inhibition, thermostability, and pH optima, demonstrated differences between the mitochondrial and cytoplasmic enzymes. A role for malate dehydrogenase in Schistosoma mansoni metabolism is discussed.     

Anisakis,Phocanema, Contracaecum, and Sulcascaris Spp.: Electrophoresis and thermostability of alcohol and malate dehydrogenases from larvae

Electrophoretic surveys were conducted on individual larvae of four anisakine nematode genera: Anisakis, Phocanema, Contracaecum, and Sulcascaris. The larval worms were obtained from a variety of fish and molluscan hosts from widely dispersed geographic regions. Of several enzymes detected, constant and apparently species-specific electrophoretic patterns were obtained for alcohol dehydrogenase (ADH, alcohol:NAD oxidoreductase, EC 1.1.1.1) and malate dehydrogenase (MDH, l-malate: NAD oxidoreductase, EC 1.1.1.37). ADH, in all but Sulcascaris sp., possessed two isozymes, the slower of which was sensitive to temperature and inhibitors. Failure of preelectrophoretic treatment with NAD to cause interconversion of these isozymes suggests that they are products of separate genetic loci. Both isozymes were maximally active with isopropanol, sec-butanol, and amyl alcohol. Within a given species, ADH showed negligible variation (i.e., apparent genetic polymorphism) with respect to individual larvae, site of larvae in the host, or geographical origin of the host. MDH from Anisakis, Sulcascaris, and Phocanema spp. possessed one, two, and three bands of activity, respectively; MDH is highly thermostable in Anisakis sp. but not in the other species.     

Schistosoma mansoni: Mechanisms in regulation of glycolysis

Adult pairs of Schistosoma mansoni convert glucose to lactate rapidly and almost quantitatively under aerobic and anaerobic conditions E. Bueding, 1950, Journal of General Physiology33, 475–495). Glycolysis is the principal source of energy of schistosomes and its inhibition by trivalent organic antimonials, at the phosphofructokinase step [EC 2.7.1.11], may be the basis for the chemotherapeutic effects of these agents E. Bueding and J. M. Mansour, 1957, British Journal of Pharmacology and Chemotherapy12, 159–165). We have developed standardized conditions for the comparison of rates of glucose consumption and lactate production by intact schistosomes in vitro and by centrifuged homogenates of worms. The rates of glycolysis of homogenates prepared from freshly isolated worms, and from worms that have been lyophilized immediately after harvesting and stored for prolonged periods at ?80 C were identical, when measured in media containing appropriate concentrations of glucose, NAD, ATP, MgCl₂, KCl, and phosphate. The specific activities of the 11 glycolytic enzymes and of 3 related enzymes (fructose-biphosphatase [EC 3.1.3.11], glycerol-3-phosphate dehydrogenase [EC 1.1.1.8], and malate dehydrogenase [EC 1.1.1.37]) were measured in homogenates under optimal conditions. The profile of the relative activities of glycolytic enzymes of S. mansoni resembles closely that of Ehrlich ascites tumor cells, and differs markedly from that observed in erythrocytes or skeletal muscle. As is the case in many animal tissues, hexokinase [EC 2.7.1.1] was the enzyme of lowest specific activity, and the rate of glycolysis of homogenates was almost the same as the hexokinase activity. Several other lines of evidence support the view that the hexokinase reaction is the rate-limiting step in the glycolysis of worm homogenates. Hexokinase activity was not particulate in schistosome homogenates, and there was no detectable high K_m glucokinase-like activity. The rate of glycolysis by homogenates exceeded that of intact worms by a factor of nearly 5. The contributions of glucose transport, availability of ADP and inorganic phosphate, regulatory enzymes, and a substrate cycle catalyzed by fructose-bisphosphatase are considered as possible mechanisms for the restraint of glycolysis in intact worms. The mechanisms contributing to the rapid rates of glycolysis of adult S. mansoni have not been identified, although several can be excluded (unusually high capacity of the glycolytic enzymes, the presence of mitochondrial hexokinase, the occurrence of glycosomes, and the operation of defective mitochondrial shuttles). In view of the regulatory role of hexokinase in the glycolysis of S. mansoni, inhibition of this enzyme is a potentially important target for the development of new antischistosomal drugs.     

Temperature and metabolism in Leishmania. 3. Some dehydrogenases of L. donovani, L. mexicana, and L. tarentolae

The effects of temperature on four dehydrogenases in homogenates of promastigotes of Leishmania donovani (several strains), L. mexicana, and L. tarentolae were studied.     

Apparent unbalance between the activities of 6-phosphogluconate and glucose-6-phosphate dehydrogenases in rat liver

The ratio of activities of 6-phosphogluconate dehydrogenase/glucose-6-phosphate dehydrogenase measured in liver extracts of rats in lipogenic nutritional conditions is only 0.2, suggesting an apparent physiological unbalance between the two dehydrogenases of the hexosemonophosphate shunt. This potential unbalance is enhanced by the fact that TPNH is a more powerful competitive inhibitor of 6-phosphogluconate dehydrogenase than of glucose-6-phosphate dehydrogenase. Accordingly, a strong activation of 6-phosphogluconate dehydrogenase would be required for efficient functioning of this pathway, unless there is an alternative outlet for 6-phosphogluconate so far unrecognized in animal tissues.     

Schistosoma mansoni: Inhibition of glucosephosphate isomerase and glycolysis by sugar phosphates

Glucosephosphate isomerase (EC 5.3.1.9) of Schistosoma mansoni is inhibited competitively by a number of tetrose, pentose, and hexose phosphates with inhibitor constant (K_i) values in the range of 0.5 to 400 μM. The most potent inhibitor is 5-phospho-d-arabinonate which resembles the cis-enediolate transition state intermediate of the reaction. These analogs were also found to be effective inhibitors of the production of lactate from glucose by suitably supplemented worm homogenates. The rank order of potency of inhibition of glycolysis was inversely related to the magnitudes of the K_i values for glucosephosphate isomerase. These K_i values were similar to those previously reported for mammalian glucosephosphate isomerase, suggesting similarities in the steric and electronic characteristics of the active sites of these isofunctional enzymes. This conclusion was further supported by the observed pH dependence of the inhibition by 5-phospho-d-arabinonate. Although glucosephosphate isomerase is not a rate-limiting enzyme of glycolysis, in the conventional sense, its selective inhibition could be of chemotherapeutic importance, in part because of the accumulation in glycolyzing systems of glucose 6-phosphate which is a potent feedback inhibitor of hexokinase.     

Caenorhabditis elegans: Decay of isocitrate lyase during larval development

Changes in the levels of isocitrate lyase, malate synthase, catalase, fumarase, and NADP⁺-isocitrate dehydrogenase have been investigated during larval development of the free-living soil nematode Caenorhabditis elegans in the presence and absence of Escherichia coli. The specific activities of isocitrate lyase, malate synthase, and catalase are maximal at the time of egg hatching and, thereafter, decline during larval development when larvae feed on E. coli, whereas in the absence of E. coli specific activities of the same enzymes increase for 12 hr and subsequently remain constant. There is, however, no change in specific activity of fumarase or NADP⁺-isocitrate dehydrogenase during the same developmental period, in either case. Cycloheximide at 100 μM arrests the decline of isocitrate lyase during development of feeding larvae but has no effect upon the appearance of isocitrate lyase during starvation. The latter is true also for 15 mM itaconate. There is inactivation of isocitrate lyase in crude extracts of frozen worms in comparison to that in analogous extracts prepared from freshly harvested nematodes.     

Investigation of hydrogen peroxide formation in plants

A convenient and simple electrophoretic procedure was used to study the NAD(P)H-dependent generation of the hydrogen peroxide needed for the polymerization of coniferyl alcohol by peroxidases from the wood of Ailanthus glandulosa. The results showed that an NAD(P)H-dependent generation of hydrogen peroxide could be brought about by either: a FMN or riboflavin-dependent system; or a Mn²⁺ -dependent system. The most active system was the one incorporating Mn²⁺, followed closely by that incorporating riboflavin. In nature it appears that the method of hydrogen peroxide formation is determined by the amounts of cofactors present in the lignifying tissue. Because no quantitative data are available in the literature, further studies of the concentrations of these cofactors in the plant cell-wall are needed.     

Trichinella spiralis: Alterations of blood chemistry in the mouse

Alterations in host blood chemistry during mouse trichinosis were studied. Mice infected with Trichinella spiralis showed depressions in serum glucose, serum pyruvate, serum total cholesterol, serum bilirubin, serum alkaline phosphatase, serum total proteins, serum total globulin, uric acid, albumin, and A/G ratio below levels found in uninfected animals. The concentrations of serum lactic dehydrogenase, serum glutamic-oxaloacetic transaminase, and serum creatine phosphokinase in infected mice were above that of uninfected mice. Hemoglobin, packed-cell volume (Hematocrit), creatinine, and calcium were at similar levels in infected and uninfected mice. These changes are discussed in terms of primary pathophysiologic lesions occurring in the gut and muscles of the trichinous host.     

Glossina morsitans morsitansandGlossina palpalis palpalis:Dosage Compensation Raises Questions about the Milligan Model for Control of Trypanosome Development

Gooding, R. H., and McIntyre, G. S. 1998.Glossina morsitans morsitansandGlossina palpalis palpalis: Dosage compensation raises questions about the Milligan model for control of trypanosome development.Experimental Parasitology90, 244–249. Evidence that dosage compensation occurs in tsetse flies was obtained by comparing the activities of X chromosome-linked enzymes, arginine phosphokinase and glucose-6-phosphate dehydrogenase inGlossina m. morsitansand hexokinase and phosphoglucomutase inGlossina p. palpalis, with the activity of an autosome-linked enzyme, malate dehydrogenase, in each species. The shortcomings of the X chromosome model for the control ofTrypanozoonmaturation in tsetse are discussed in light of these findings and previously published reports on the lack of fitness effects of matureTrypanozooninfections in tsetse and on published results on antitrypanosomal factors in male and female tsetse flies.     

Hymenolepis microstoma: Ultrastructural localization of adenyl cyclase in the tegument

Peroxidase and phosphatase activities have been reported to be localized in the tegument of adult hymenolepidid tapeworms. In order to localize adenyl cyclase (EC 4.6.1.1) activity in the tegument of mature and gravid sections of Hymenolepis microstoma, 5-adenyl-imidodiphosphate was used as a substrate, and lead was used as a capturing agent. Results indicate that adenyl cyclase activity is present in the crypts between the microtriches of the mature sections and that activity is absent from the gravid sections.     

Leishmania mexicana: Conversion of dihydroorotate to orotate in amastigotes and promastigotes

The specific activity of dihydroorotate dehydrogenase, catalysing the conversion of l-5,6-dihydroorotate (l-DHO) to orotate, in Leishmania mexicana mexicana was found to be 22.1 ± 3.5 nmole/hr/mg protein in the amastigote, and 28.7 ± 4.6 nmole/hr/mg protein in the promastigote. The enzyme was found to be soluble and to require molecular O₂ for activity in both forms of the parasite. Oxygen utilisation was not mediated through the mitochondrial cytochrome-containing respiratory chain, and phenazine methosulphate and ferricyanide could be used as electron acceptors by the enzyme in both aerobic and anaerobic conditions. The enzyme from both amastigote and promastigote had a pH optimum of 7.0, and the K_m values for l-DHO were 11.8 ± 4.9 and 2.3 ± 0.4 μM, respectively. The pyrimidine analogs 5-methylorotate (K_i = 8.8 μM) and 5-aminoorotate (K_i = 57 μM) were shown to be competitive inhibitors of the promastigote enzyme, as was the reaction product orotate (K_i = 10 μM).     

Trypanosoma cruzi: Lactate dehydrogenase isoenzymes and infections in mice

Total plasma LDH isoenzyme (EC 1.1.1.27) levels increased significantly over the normal level in mice infected with strains of Trypanosoma cruzi from three different geographic locations, but some strain differences were observed. The most rapid increase was exhibited by the blood-induced Tulahuen strain, but this strain, unlike the House 510 or House 11, did not elicit an increase during the early period of infection. Overall increases in LDH-1 and LDH-2, heart isoenzymes, were most marked in vector-derived House 510 infections, but, as in the Tulahuen strain, a considerable increase was also observed in blood-induced infections. The House 510 strain also elicited significant increases in LDH-4; these were particularly high during the early period of the blood-induced infection. By contrast, the vector-derived Tulahuen strain elicited a higher increase in LDH-4 during the early period than the House 510 or House 11 strains. Comparable similarites and differences were also observed in regard to LDH-3, 5, and “X.” The most marked isoenzyme increases were those of “LDH-X” exhibited by the blood-induced House 510 and vector-derived Tulahuen strains.Parallel histopathologic studies of liver, heart, and skeletal muscle disclosed significant pathology in all the infections. Animals with blood-induced Tulahuen strain infections characteristically showed extensive necrosis with marked multiplication of parasites throughout the liver, but little or no evident damage to the heart and skeleal muscle. Animals infected with House 510 and House 11 strains exhibited minimal pathology in the liver but severe damage to the heart and skeletal muscle. Increases in LDH-4 and LDH-5, isoenzymes which represent both liver and skeletal muscle, in blood-induced Tulahuen infections were attributed largely to liver damage, but in the House 510 and House 11 infections were related more to skeletal muscle.     

Hymenolepis diminuta: Partial characterization of membrane-bound nucleotidase activities (ATPase and 5′-nucleotidase) in the isolated brush border membrane

Adenosine triphosphatase (ATPase; EC 3.6.1.3) and 5′-nucleotidase (5′-NTase; EC 3.1.3.5) activities of the isolated brush border membrane of Hymenolepis diminuta have been studied. The pH optimum for ATPase activity is 7.4, and divalent cations are necessary for maximum activity; no Na⁺-K⁺ activated ATPase is present in the isolated brush border membrane. ATPase activity is inhibited by molybdate and phosphorylated monosaccharides, but not by N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB), or fluoride. The pH optimum for 5′-NTase activity is 9.6–10.2, and divalent cations are necessary for maximum activity. 5′-NTase activity is inhibited by molybdate at pH 9.6 and 7.4, and activated by NEM and pCMB and pH 9.6 and 7.4, respectively; fluoride has no effect on 5′-NTase activity. Solubilization of the brush border membrane fraction in 1% sodium dodecyl sulfate has no inhibitory action on either enzyme activity.     

Schistosoma mansoni: Electrophoretic characterization of strains selected for different levels of infectivity to snails

Individual adult Schistosoma mansoni from strains selected for high or low infectivity to specific strains of the snail intermediate host, Biomphalaria glabrata, were subjected to enzyme electrophoresis on starch gels. Fourteen enzyme systems were analyzed in an attempt to find electrophoretic markers associated with genes for infectivity to snails. The S. mansoni strains were selected from different isolates from Puerto Rico in several strains of B. glabrata. Of an estimated 18 loci, 3 were polymorphic and the remainder monomorphic. For 1 of the 3 polymorphic enzyme loci, lactate dehydrogenase (Ldh, EC 1.1.1.27), phenotype frequencies were correlated with infectivity to snails. In schistosome strains of low infectivity, frequencies of the Ldh-N phenotype ranged between 0.56 and 0.69, while in strains of high infectivity, Ldh-N frequencies were typically 0.91 to 1.00. Whether the correlation is accidental or due to some form of association, such as chromosomal linkage, between the locus responsible for variation in lactate dehydrogenase and a gene for infectivity to snails remains to be determined.     

Identification of a deletion in the NDUFS4 gene using array-comparative genomic hybridization in a patient with suspected mitochondrial respiratory disease

We evaluated a patient, born after a normal 38-week pregnancy, with psychomotor retardation, poor coordination of ocular movements, recurrent vomiting and severe lactic acidosis. The patient was admitted to hospital at 2 months of age because of a mitochondrial-like syndrome and died at the age of 4.5 months. Array-comparative genomic hybridization (a-CGH) analysis revealed a homozygous deletion in 5q11.2 involving NADH dehydrogenase (ubiquinone) Fe–S protein 4, 18 kDa (NADH-coenzyme Q reductase; NDUFS4). Both parents were heterozygous for the mutation. The array revealed a deletion of ~ 32 kb that includes exon 2 of NDUFS4 subsequently confirmed by real time-PCR and multiplex PCR. NDUFS4 was previously correlated to Leigh syndrome since mutations in this gene block the assembly of complex I. This result demonstrates the relevance of a-CGH screening in patients affected by metabolic disorders of unknown etiology.     

Schistosoma mansoni: Characterization of two circulating polysaccharide antigens and the immunological response to these antigens in mouse,hamster, and human infections

In this study the nature and occurrence of two circulating polysaccharide antigens of Schistosoma mansoni, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), and the immunological response to these antigens in mouse, hamster, and human infections were investigated. Both CAA and CCA showed a large molecular weight range, less than 50,000 to over 300,000 for CAA and 50,000 to over 300,000 for CCA, possibly representing monomers and polymers. CAA and CCA could be purified from the trichloroacetic acid-soluble fraction of adult worm antigen (AWA-TCA) by means of DEAE ion exchange chromatography. The presence of at least two other components in AWA-TCA was shown. Both CAA and CCA were found to be gut associated, and could be demonstrated in the vomitus and in the excretory and secretory antigens of adult worms. Both antigens were present in the kidney eluates of infected hamsters, while CCA could normally be detected in the urine of these hamsters and CAA only occasionally. CAA was demonstrated in the Kupffer cells of the livers of infected mice and hamsters. Antibodies against CAA and CCA were shown in mouse, hamster, and human infections. In human infections specific IgM titers against these antigens were especially elevated in children and in recent infections of adults.     

The phylogeny of Ephemeroptera in Pterygota revealed by the mitochondrial genome of Siphluriscus chinensis (Hexapoda: Insecta)

The mayfly species Siphluriscus chinensis (Siphluriscidae) has valuable structures useful for phylogeny reconstruction, given its putative basal position within the Ephemeroptera. Here its nearly complete mitochondrial genome is sequenced. We built phylogenetic trees through multiple analytical strategies with some other insect mitogenomes. Structurally, the obtained mitochondrial genome of S. chinensis is 16,616 bp in length, ¹ containing 37 genes and an extra trnK-like (trnK2 (AAA)) gene. The 12 PCGs start with typical ATN codons, except the nad1 gene which starts with an unnormalized TTG. Like other known mayfly mitogenomes, the strand bias has negative AT-skew and negative GC-skew. Phylogenetically, our topologies suggest that Odonata is the basally diverged clade in Pterygota; Ephemeroptera is the sister group of the Neoptera; and S. chinensis is indeed the most basal mayfly branch.