A Functional Aqp1 Gene Product Localizes on The Contractile Vacuole Complex in Paramecium multimicronucleatum.

In a ciliate Paramecium, the presence of water channels on the membrane of contractile vacuole has long been predicted by both morphological and physiological data, however, to date either the biochemical or the molecular biological data have not been provided. In the present study, to examine the presence of aquaporin in Paramecium, we carried out RT-PCR with degenerated primers designed based on the ParameciumDB, and an aquaporin cDNA (aquaporin 1, aqp1) with a full-length ORF encoding 251 amino acids was obtained from Paramecium multimicronucleatum by using RACE. The deduced amino acid sequence of AQP1 had NPA-NPG motifs, and the prediction of protein secondary structure by CNR5000 and hydropathy plot showed the presence of six putative transmembrane domains and five connecting loops. Phylogenetic analysis results showed that the amino acid sequence of AQP1 was close to that of the Super-aquaporin group. The AQP1-GFP fusion protein clearly demonstrated the subcellular localization of AQP1 on the contractile vacuole complex, except for the decorated spongiome membrane. The functional analyses of aqp1 were done by RNA interference-based gene silencing, using an established feeding method. The aqp1 was found to be crucial for the total fluid output of the cell, the function of contractile vacuole membranes.

Structure-activity relationship studies on acremomannolipin A, the potent calcium signal modulator with a novel glycolipid structure 4: Role of acyl side chains on d-mannose.

As part of an ongoing study on the structure-activity relationship of acremomannolipin A (1)-the novel glycolipid isolated from Acremonium strictum possessing potent calcium signal-modulating activity-the role of acyl substituents on the d-mannose moiety was examined. Three partially deacylated homologs (2a-2c) and 20 homologs (2d-2w) bearing different acyloxy side chains were synthesized via the stereoselective ß-mannosylation of appropriately protected mannosyl sulfoxides (3) with d-mannitol derivatives (4), and their calcium signal-modulating activities were examined. The activities of 2a-2c were completely lost. Homologs bearing relatively short acyloxy groups at C-3, C-4, and C-6 positions (2t-2v) exhibited less activity than 1, whereas a heptanoyl homolog (2w: C7) maintained activity nearly equal to that of 1. When the acyl groups at these three positions were substituted by an octanoyl group (2i: C8), the activity was completely lost. On the other hand, of the 10 homologs in which the octanoyl at C-2 was substituted by other acyloxy moieties (2j-2s), three (2m: C7, 2n: C9, 2o: C10) maintained potent activity. These results suggested that peracylated mannose structure is critical for calcium signal-modulating activity, and this activity is precisely dependent on the length of four acyl side chains on d-mannose.