Proteomic Analysis of Trichomonas vaginalis Phagolysosome,Lysosomal Targeting,and Unconventional Secretion of Cysteine Peptidases

The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.     

Non-marine Ostracoda (Crustacea) of Banat district in Serbia

In order to enrich the knowledge on ostracods in Serbia, field work was carried out in the central and southern part of the Banat district during 2002 and 2003. Samples from the collection of the Institute of Zoology, Faculty of Biology in Belgrade were also examined. Altogether, 133 samples were investigated. In this research, 23 ostracod species were found. Most of them have a Palaearctic distribution. Ilyocypris decipiens, Bradleycypris obliqua and Tanycypris pellucida are new to the faunas of Serbia and Montenegro, as is the subfamily Cypricercinae. Three species were found again after several decades, including a new and stabile population of Candona natronophila Petkovski, 1969 . The most frequent species were Cypridopsis vidua and Physocypria kraepelini. Statistical analysis of ostracod communities and their relations to measured ecological variables is also given.     

Is the catalytic activity of triosephosphate isomerase fully optimized? An investigation based on maximization of entropy production

Triosephosphate isomerase (TIM) is often described as a fully evolved housekeeping enzyme with near-maximal possible reaction rate. The assumption that an enzyme is perfectly evolved has not been easy to confirm or refute. In this paper, we use maximization of entropy production within known constraints to examine this assumption by calculating steady-state cyclic flux, corresponding entropy production, and catalytic activity in a reversible four-state scheme of TIM functional states. The maximal entropy production (MaxEP) requirement for any of the first three transitions between TIM functional states leads to decreased total entropy production. Only the MaxEP requirement for the product (R-glyceraldehyde-3-phosphate) release step led to a 30% increase in enzyme activity, specificity constant k_cat/K_M, and overall entropy production. The product release step, due to the TIM molecular machine working in the physiological direction of glycolysis, has not been identified before as the rate-limiting step by using irreversible thermodynamics. Together with structural studies, our results open the possibility for finding amino acid substitutions leading to an increased frequency of loop six opening and product release.     

Photoperiodic induction of flowering in green and photobleachedChenopodium rubrum L. ecotype 184 - a short- day plant

Chenopodium rubrum L. ecotype 184 is a qualitative short-day plant with critical length of the night of eight hours that must be exceeded in order to flower: Five days after sowing, the plants were exposed to a various number of inductive cycles (14/10 h of däy/night cycle) to test the optimal photoperiodic conditions for flowering. In our experimental conditions the plants flowered with high percentage after more than four received inductive cycles, but there was no flowering below that. The plants grown on the herbicide Norflurazon (photobleached plants) showed different photoperiodic characteristics. There was negligible flowering of photobleached plants in the same experimental conditions as for the green ones.     

Plant composition and diversity at edges in a semi-natural forest–grassland mosaic

Plant Ecology - As key components of landscapes, edges have received considerable scientific attention in anthropogenic ecosystems. However, edges in natural and semi-natural forest–grassland...