The standard dilution technique can provide unbiased estimates of phytoplankton growth and microzooplankton grazing rates only when certain restrictive assumptions are met. The most important of these assumptions -that grazing impact varies in direct proportion to the dilution of grazer population density -can be easily violated when clearance rate of individual grazers and/or growth response of the grazer population vary significantly with food concentration over the course of the incubation. We have developed a modified protocol which now allows the dilution technique to be applied unambiguously, even when its original assumptions may be violated. The new protocol uses flow-cytometry measured disappearance of fluorescently labeled tracer cells (FLB or FLA) as an internal measure of 'relative grazing activity' in each dilution treatment. Coefficients of phytoplankton growth and mortality rates are determined from Model 11 regression analyses of 'net growth' versus 'relative grazing', rather than the usual Model I regressions of 'net growth' versus 'dilution factor'. Tests of this hybrid experimental design in the central equatorial Pacific during an EQPAC cruise in August 1992 gave results essentially identical to the standard dilution interpretation.
Trypanothione reductase (TR) is both a valid and an attractive target for the design of new trypanocidal drugs. Starting from menadione, plumbagin, and juglone, three distinct series of 1,4-naphthoquinones (NQ) were synthesized as potential inhibitors of TR from Trypanosoma cruzi (TcTR). The three parent molecules were functionalized at carbons 2 and/or 3 by various polyamine chains. Optimization of TcTR inhibition and TcTR specificity versus human disulfide reductases was achieved with the 3,3'-[polyaminobis(carbonylalkyl)]bis(1,4-NQ) series 19-20, in which an optimum chain length was determined for inhibition of the trypanothione disulfide reduction. The most active derivatives against trypanosomes in cultures were also studied as subversive substrates of TcTR and lipoamide dehydrogenase (TcLipDH). The activities were measured by following NAD(P)H oxidation as well as coupling the reactions to the reduction of cytochrome c which permits the detection of one-electron transfer. For TcTR, 20(4-c) proved to be a potent subversive substrate and an effective uncompetitive inhibitor versus trypanothione disulfide and NADPH. Molecular modeling studies based on the known X-ray structures of TcTR and hGR were conducted in order to compare the structural features, dimensions, and accessibility of the cavity at the dimer interface of TcTR with that of hGR, as one of the putative NQ binding sites. TcLipDH reduced the plumbagin derivatives by an order of magnitude faster than the corresponding menadione derivatives. Such differences were not observed with the pig heart enzyme. The most efficient and specific subversive substrates of TcTR and TcLipDH exhibited potent antitrypanosomal activity in in vitro T. brucei and T. cruzi cultures. The results obtained here confirm that reduction of NQs by parasitic flavoenzymes is a promising strategy for the development of new trypanocidal drugs.
The dynamics of the microbial plankton community of Kaneohe Bay, Hawaii were investigated in September 1982 using in situ diffusion chambers and dilution manipulations. Total community carbon at the time of the experiments was estimated at 86 pg C 1-' of which Chlorella sp. accounted for 47 %, autotrophic microflagellates 14 %, chroococcoid cyanobacteria 11 %, and heterotrophic microflagellates and bacteria each 9 %. Instantaneous growth rates ranged from 1.2 to 1.9 d-' and 1.4 to 2.0 d-l, and mortality rates varied from 0.5 to 1
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