Comparative genome analysis of the yellow fever mosquito Aedes aegypti with Drosophila melanogaster and the malaria vector mosquito Anopheles gambiae.

An in silico comparative genomics approach was used to identify putative orthologs to genetically mapped genes from the mosquito, Aedes aegypti, in the Drosophila melanogaster and Anopheles gambiae genome databases. Comparative chromosome positions of 73 D. melanogaster orthologs indicated significant deviations from a random distribution across each of the five A. aegypti chromosomal regions, suggesting that some ancestral chromosome elements have been conserved. However, the two genomes also reflect extensive reshuffling within and between chromosomal regions. Comparative chromosome positions of A. gambiae orthologs indicate unequivocally that A. aegypti chromosome regions share extensive homology to the five A. gambiae chromosome arms. Whole-arm or near-whole-arm homology was contradicted with only two genes among the 75 A. aegypti genes for which orthologs to A. gambiae were identified. The two genomes contain large conserved chromosome segments that generally correspond to break/fusion events and a reciprocal translocation with extensive paracentric inversions evident within. Only very tightly linked genes are likely to retain conserved linear orders within chromosome segments. The D. melanogaster and A. gambiae genome databases therefore offer limited potential for comparative positional gene determinations among even closely related dipterans, indicating the necessity for additional genome sequencing projects with other dipteran species.

Characterization of an Aedes aegypti bacterial artificial chromosome (BAC) library and chromosomal assignment of BAC clones for physical mapping quantitative trait loci that influence Plasmodium susceptibility.

Previous studies have confirmed a genetic basis for susceptibility of mosquitoes to Plasmodium parasites. Here we describe our efforts to characterize a bacterial artificial chromosome genomic library for the yellow fever mosquito, Aedes aegypti, and to identify BAC clones containing genetic markers that define quantitative trait loci (QTL) for Plasmodium gallinaceum susceptibility. This library (NDL) was prepared from the Ae. aegypti Liverpool strain and consists of 50 304 clones arrayed in 384-well microplates. We used PCR analysis with oligonucleotide primer pairs specific to 106 genetic markers (as sequence-tagged sites or STS) to screen the NDL library. Each STS identified between one and thirteen independent clones with an average of 3.3 clones. The average insert size was 122 kb and therefore the NDL library provides approximately 7.87-fold genome coverage. The availability of the NDL library should greatly facilitate physical mapping efforts, including positional cloning of QTL for traits of interest such as Plasmodium susceptibility and for whole genome sequence determination and assembly.

Global motion processing is not tuned for binocular disparity.

An important goal of the visual system is the segmentation of image features into objects and their backgrounds. A primary cue for this is motion: when a region shares the same pattern of motion it is segregated from its surround. Three experiments were carried out to investigate whether the segmentation of image features on the basis of motion information is facilitated by the addition of binocular disparity. Coherence thresholds were measured for the discrimination of the global direction of motion of random dot kinematograms (RDKs) in which the relative disparity of the signal and noise dots was manipulated. When the signal dots were embedded in a three dimensional cloud of noise dots, coherence thresholds were similar to those measured when signal and noise dots were both presented with zero disparity. However, when the signal dots were separated from the noise dots in depth, global motion processing was strongly facilitated. These results were considered in terms of two models, one in which global motion is processed by disparity tuned mechanisms, the other in which the discrimination of the direction of motion is mediated by an attention-based system. It was concluded that global motion processing is not tuned for binocular disparity and that the facilitation of the discrimination of direction provided by binocular disparity in certain circumstances reflects the rôle of an attention-based system.

Acetyl-CoA hydrolase activity and function in Ascaris suum muscle mitochondria.

Acyl-CoA compounds are stable in adult Ascaris suum mitochondrial preparations. However, when incubated in the presence of 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), acetyl-CoA or propionyl-CoA are hydrolyzed to form free coenzyme A. This acetyl-CoA hydrolase activity has been partially purified and found to be specific for the above CoA derivatives. Gel filtration indicates an apparent molecular weight of 232,000. The hydrolase activity has been purified free from acyl-CoA transferase activities and appears not to be accounted for on the basis of a thiolase. Because Ascaris is an intestinal parasite that metabolizes primarily anaerobically and accumulates a large number of volatile fatty acids that are formed as the coenzyme A derivatives, the hydrolase would be expected to function in the regeneration of free CoA. However, how the hydrolase reaction would be pulled in the absence of the nonphysiologic DTNB is not known.

Acyl-CoA transferase activities in homogenates of Fasciola hepatica adults.

Succinyl-CoA is an intermediate in the formation of the fermentation product, propionate, by Fasciola hepatica adults. Acyl-CoA transferase activities are present in crude homogenates of Fasciola, which could account for the synthesis of succinyl-CoA from succinate by the transfer of CoA from either propionyl-CoA or acetyl-CoA. No transferase activity was apparent from 2-methylbutyryl-CoA or 2-methylvaleryl-CoA as was previously reported for the nematode, Ascaris suum. Heat denaturation experiments indicate that all of the Fasciola transferase activities may result from a single protein.

2-Methylbutyryl-CoA: succinate acyl-CoA transferase activity and function in Ascaris suum muscle.

A branched-chain acyl-CoA transferase activity which transfers coenzyme A from either 2-methylbutyryl or 2-methylvaleryl-CoA to succinate is present in the muscle mitochondria from the intestinal nematode, Ascaris suum. Its physiological function is discussed. This activity appears to differ from the previously described acetyl-CoA: propionate and propionyl-CoA:succinate acyl-CoA transferases on the basis of heat stability, substrate specificity and the requirement of a "factor" from boiled Ascaris mitochondria for optimal activity of only the branched-chain acyl-CoA transferase. The "factor" has been recovered from HPLC and some of its properties examined. It could not be replaced by a crude soluble fraction from rat liver mitochondria, or by adenine, guanine or inosine di- or triphosphates. Activity was lost upon ashing, but was not affected by treatment with either pepsin or chymotrypsin.

Illusory, motion, and luminance-defined contours interact in the human visual system.

Psychophysical studies of interactions between contours defined by different image attributes report that luminance-defined and illusory contours show little if any interaction. Because the contours defined by these attributes may vary in perceptual saliency, we employed the tilt aftereffect (TAE) and a cross-adaptation procedure to evaluate interaction effects between luminance-defined and illusory contours under varying saliency conditions as well as to explore the interaction between illusory and motion-defined contours. When contour salience of the adaptation or test stimuli was modified by the addition of various amounts of static noise, we observed a TAE for all combinations of contour types including the novel motion-illusory and illusory-motion pairs. The interactions demonstrated between the contour classes in this as well as other studies suggests contour invariance in the orientation domain.

Biochemical analyses of secretory and excretory products of adult Dipetalonema viteae in culture.

Radioisotopically labeled glucose and pyruvate were employed to elucidate biochemical mechanisms utilized by the filariid Dipetalonema viteae during cultivation. Adults isolated from amicrofilaremic hamsters were incubated at 37 C in a mixture of NCTC135:IMDM (NI), with either D-[14C-(U)]glucose or [1-14C]pyruvate, under a gas phase of 5% CO2/N2 for 3 days. Labeled organic acids were separated and quantified by ion exchange chromatography. High performance liquid chromatography (HPLC) was used for separation and quantification of the 23 free amino acids in the NI medium. Ion exchange chromatography revealed that lactate was the major glycolytic end product, accounting for 90-97% of the original carbon utilized. Small amounts of radioactivity were recovered in succinate and variably in acetate fractions. HPLC analysis demonstrated that some amino acids increased, some decreased, and some remained at the initial concentration. Alanine exhibited the greatest change, consistently increasing from 2 to 4 times the original concentration. Analyses of purified amino acid peaks revealed radioactivity only in the alanine peak, accounting for 2-4% of the original carbon utilized.

Comparisons of glucose and amino acid use in adults and microfilariae of Brugia pahangi.

With [U-14C]glucose we confirmed that essentially all of the glucose carbon used aerobically by intact Brugia pahangi adults was accounted for in the recovered lactate, in spite of the presence of cristate mitochondria in these parasites. Approximately 0.2% of the glucose carbon that disappeared was recovered in CO2. However, aerobiosis was required for the incorporation of alanine, proline, and glutamate into both respiratory CO2 and protein, but the glucose was used on the order of 1000 times faster than the amino acids. Homogenate fractions were examined for amino acid use, and all except the 100,000 g soluble fraction showed activity. However, all of the active fractions also contained living microfilariae and other developmental stages that arise from the adult uterus, since the females give birth to living larvae. Sonicates of the microfilariae formed CO2 from proline 30 times more rapidly than crude homogenates of the adult. Similarly, CO2 was formed from glucose 14 times more rapidly by intact microfilariae than by intact adults. The possibility arises that the low rates of CO2 formation from both the amino acids and glucose by intact adults may arise from the metabolism of the aerobic microfilariae and other developmental stages present in utero.

Separation and functions of two acyl CoA transferases from Ascaris lumbricoides mitochondria.

Many invertebrates accumulate propionate, or products derived from propionate, as products of fermentation. Evidence has been reported that the nematode, Ascaris suum, the cestode, Spirometra mansonoides, and the trematode, Fasciola hepatica, accumulate propionate by means of an adenosine triphosphate (ATP)-generating decarboxylation of succinate. To generate energy, an acyl coenzyme A (CoA) transferase that would transfer CoA to succinate is required as one component of the sequence of reactions. Recently, an acyl CoA transferase was isolated from Ascaris mitochondria and purified to electrophoretic homogeneity. However, upon examination of the substrate specificities of this enzyme, it was found essentially to lack the ability to use succinate or succinyl CoA as an acceptor or donor of CoA, respectively. Therefore, this transferase could not serve to activate succinate. This article describes the isolation of an additional acyl CoA transferase from Ascaris mitochondria that appears to be unique in its substrate specificity and that could easily account not only for the activation of succinate but also for the regulation of succinate metabolism primarily in the direction of decarboxylation to propionate. This is in contrast with mammalian tissues, which act in the opposite direction by catalyzing the fixation of CO2 into propionate, thereby forming succinate and accounting for the glycogenic nature of dietary propionate. Possible functions of the two acyl CoA transferases are discussed.

Purification and properties of an acyl CoA transferase from Ascaris suum muscle mitochondria.

An acyl CoA transferase has been purified to electrophoretic homogeneity from the soluble compartment of Ascaris suum muscle mitochondria. From SDS-PAGE, isoelectric focusing and molecular exclusion chromatography, homogeneity was confirmed and the enzyme appears to be composed of two similar or identical subunits of apparent mol. wts of 50,000 resulting in an apparent mol. wt of 100,000 for the holoenzyme. The apparent isoelectric point was 5.6 +/- 0.1 by both chromatofocusing columns and slab gel isoelectric focusing. The transferase was relatively specific for the short, straight-chain acyl CoA donors as well as the CoA acceptors, being active on acetyl CoA, propionyl CoA, butyryl CoA, valeryl CoA and hexanoyl CoA as donors to acetate and propionate. Neither succinyl CoA nor succinate were appreciably active as CoA donor or acceptor, respectively. This enzyme cannot serve physiologically to activate succinate for decarboxylation to propionate, but may serve to ensure a supply of propionyl CoA which appears to be required in catalytic amounts for the decarboxylation of succinate.