Specificity of the human frequency following response for carrier and modulation frequency assessed using adaptation

The frequency following response (FFR) is a scalp-recorded measure of phase-locked brainstem activity to stimulus-related periodicities. Three experiments investigated the specificity of the FFR for carrier and modulation frequency using adaptation. FFR waveforms evoked by alternating-polarity stimuli were averaged for each polarity and added, to enhance envelope, or subtracted, to enhance temporal fine structure information. The first experiment investigated peristimulus adaptation of the FFR for pure and complex tones as a function of stimulus frequency and fundamental frequency (F0). It showed more adaptation of the FFR in response to sounds with higher frequencies or F0s than to sounds with lower frequency or F0s. The second experiment investigated tuning to modulation rate in the FFR. The FFR to a complex tone with a modulation rate of 213 Hz was not reduced more by an adaptor that had the same modulation rate than by an adaptor with a different modulation rate (90 or 504 Hz), thus providing no evidence that the FFR originates mainly from neurons that respond selectively to the modulation rate of the stimulus. The third experiment investigated tuning to audio frequency in the FFR using pure tones. An adaptor that had the same frequency as the target (213 or 504 Hz) did not generally reduce the FFR to the target more than an adaptor that differed in frequency (by 1.24 octaves). Thus, there was no evidence that the FFR originated mainly from neurons tuned to the frequency of the target. Instead, the results are consistent with the suggestion that the FFR for low-frequency pure tones at medium to high levels mainly originates from neurons tuned to higher frequencies. Implications for the use and interpretation of the FFR are discussed

Oleic Acid Biosynthesis in Plasmodium falciparum: Characterization of the Stearoyl-CoA Desaturase and Investigation as a Potential Therapeutic Target

BACKGROUND:Plasmodium falciparum parasitization of erythrocytes causes a substantial increase in the levels of intracellular fatty acids, notably oleic acid. How parasites acquire this monounsaturated fatty acid has remained enigmatic. Here, we report on the biochemical and enzymatic characterization of stearoyl-CoA desaturase (SCD) in P. falciparum. METHODOLOGY/PRINCIPAL FINDINGS:Metabolic labeling experiments allowed us to demonstrate the production of oleic acid from stearic acid both in lysates of parasites incubated with [(14)C]-stearoyl-CoA and in parasite-infected erythrocytes labeled with [(14)C]-stearic acid. Optimal SCD activity was detected in schizonts, the stage of maximal membrane synthesis. This activity correlated with a late trophozoite stage-specific induction of PFE0555w transcripts. PFE0555w harbors a typical SCD signature. Similar to mammalian SCDs, this protein was found to be associated with the endoplasmic reticulum, as determined with PFE0555w-GFP tagged transgenic P. falciparum. Importantly, these parasites exhibited increased rates of stearic to oleic acid conversion, providing additional evidence that PFE0555w encodes the plasmodial SCD (PfSCD). These findings prompted us to assess the activity of sterculic acid analogues, known to be specific Delta9-desaturase inhibitors. Methyl sterculate inhibited the synthesis of oleic acid both with parasite lysates and infected erythrocytes, most likely by targeting PfSCD. This compound exhibited significant, rapid and irreversible antimalarial activity against asexual blood stages. This parasiticidal effect was antagonized by oleic acid. CONCLUSION/SIGNIFICANCE:Our study provides evidence that parasite-mediated fatty acid modification is important for blood-stage survival and provides a new strategy to develop a novel antimalarial therapeutic based on the inhibition of PfSCD

Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development

Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs