New types of metacaspases in phytoplankton reveal diverse origins of cell death proteases.

Metacaspases are evolutionarily distant homologs of caspases that are found outside the metazoan and are known to have key roles in programmed cell death (PCD). Two types of metacaspases (types I and II) have been defined in plants based on their domain structures; these have similarities to metazoan 'initiator' and 'executioner' caspases. However, we know little about metacaspases in unicellular organisms and even less about their roles in cell death. We identified a novel group of metacaspases in sequenced phytoplanktonic protists that show domain architectures distinct from either type I or II enzymes; we designate them as type III. Type III metacaspases exhibit a rearrangement of domain structures between N- and C-terminus. In addition, we found a group of metacaspase-like proteases in phytoplankton that show sequence homology with other metacaspases, but defy classification in conventional schemes. These metacaspase-like proteases exist in bacteria alongside a variant of type I metacaspases and we propose these bacterial metacaspases are the origins of eukaryotic metacaspases. Type II and III metacaspases were not detected in bacteria and they might be variants of bacterial type I metacaspases that evolved in plants and phytoplanktonic protists, respectively, during the establishment of plastids through the primary and secondary endosymbiotic events. A complete absence of metacaspases in protists that lost plastids, such as oömycetes and ciliates indicates the gene loss during the plastid-to-nucleus gene transfer. Taken together, our findings suggest endosymbiotic gene transfer (EGT) is a key mechanism resulting in the evolutionary diversity of cell death proteases.

Determining parameters of the numerical response.

The numerical response, the change in specific growth rate with food concentration, is a fundamental component of many aquatic microbial studies. Accurately and precisely determining the parameters of this response is essential to obtain useful data for both aut- and synec-ological studies. In this work we emphasize four points that are often ignored in designing numerical response experiments: (1) the inclusion of subthreshold concentrations (i.e., where growth rate is negative) in the experimental design; (2) an appropriate allocation of effort, i.e., the superiority of choosing more individual prey concentrations rather than replicating fewer; (3) the potential superiority of replicating experiments rather than simply replicating treatment in a single experiment; and (4) the placement of most measurements near the lower end of the concentration gradient, well below the asymptote, possibly following a geometric progression. We illustrate the first point by examining a small subset of published data on planktonic oligotrich ciliates and then, using a Monte Carlo simulation, rigorously evaluate the experimental design, supporting the remaining points.

Differential Effects of Nitrogen Limitation on Photosynthetic Efficiency of Photosystems I and II in Microalgae.

The effects of nitrogen starvation on photosynthetic efficiency were examined in three unicellular algae by measuring changes in the quantum yield of fluorescence with a pump-and-probe method and thermal efficiency (i.e. the percentage of trapped energy stored photochemically) with a pulsed photoacoustic method together with the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea to distinguish photosystems I and II (PSI and PSII). Measured at 620 nm, maximum thermal efficiency for both photosystems was 32% for the diatom Thalassiosira weissflogii (PSII:PSI ratio of 2:1), 39% for the green alga Dunaliella tertiolecta (PSII:PSI ratio of 1:1), and 29% for the cyanobacterium Synechococcus sp. PCC 7002 (PSII:PSI ratio of 1:2). Nitrogen starvation decreased total thermal efficiency by 56% for T. weissflogii and by 26% for D. tertiolecta but caused no change in Synechococcus. Decreases in the number of active PSII reaction centers (inferred from changes in variable fluorescence) were larger: 86% (T. weissflogii), 65% (D. tertiolecta), and 65% (Synechococcus). The selective inactivation of PSII under nitrogen starvation was confirmed by independent measurements of active PSII using oxygen flash yields and active PSI using P700 reduction. Relatively high thermal efficiencies were measured in all three species in the presence of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting the potential for significant cyclic electron flow around PSI. Fluorescence or photoacoustic data agreed well; in T. weissflogii, the functional cross-sectional area of PSII at 620 nm was estimated to be the same using both methods (approximately 1.8 x 102 A2). The effects of nitrogen starvation occur mainly in PSII and are well represented by variable fluorescence measurements.

Expanding the capabilities of laboratory instruments using built-in microprocessors and serial interfaces: adapting an LKB Ultrospec II UV spectrophotometer for scanning and enzyme kinetic analyses.

An IBM-compatible microcomputer was used to enable an LKB Ultrospec II UV-Visible spectrophotometer to perform two features which are not available on the basic unit: wavelength scanning and enzyme kinetic analyses. The spectrophotometer was controlled using a simple BASIC program, an RS232c interface and the instrument's own built-in microprocessor. This technique required no special analogue to digital conversion hardware or software and only a rudimentary knowledge of programming. User-designed software permits a greater degree of flexibility in routines and output formats than is offered in most pre-programmed instruments, while offering substantial savings.

Relationship Between Body Size, Growth Rate, and Maximal Enzyme Activities in the Brine Shrimp, Artemia franciscana.

Activity-body size relationships for eight enzymes (citrate synthase, CS; lactate dehydrogenase, LDH; pyruvate kinase, PK; alanine aminotransferase, ala AT; aspartate aminotransferase, asp AT; glutamate dehydrogenase, GDH; glucose-6-phosphate dehydrogenase, G6Pdh; and nucleoside diphosphate kinase, NDPK) were examined in the brine shrimp, Artemia franciscana. The animals were fed on the alga Dunaliella salina, which was provided in three concentrations representing a 25-fold range. Enzyme activities per animal (Y) were regressed against body size (M, expressed as dry mass or protein) in the form of the allometric equation, log Y = log a + b log M, where a and b are fitted constants. For all enzymes considered, the value of the scaling exponent (b) was significantly higher when dry mass was used, as a body size index, than when protein mass was used. Therefore, the index of body size chosen can influence the exponent obtained in allometric studies. Although specific growth rates of different cultures varied greatly, no significant differences in scaling relationships were found between cultures for any enzyme. For many enzymes, growth rate may not be a source of variation in scaling relationships. Unlike the other enzymes examined, the log-transformed NDPK activity versus log-transformed mass was not linear; NDPK activity reached a plateau. Variation in NDPK scaling relationships with growth may provide a means to predict growth rate in Artemia.