Towards complete sets of farnesylated and geranylgeranylated proteins

Three different prenyltransferases attach isoprenyl anchors to C-terminal motifs in substrate proteins. These lipid anchors serve for membrane attachment or protein-protein interactions in many pathways. Although well-tolerated selective prenyltransferase inhibitors are clinically available, their mode of action remains unclear since the known substrate sets of the various prenyltransferases are incomplete. The Prenylation Prediction Suite (PrePS) has been applied for large-scale predictions of prenylated proteins. To prioritize targets for experimental verification, we rank the predictions by their functional importance estimated by evolutionary conservation of the prenylation motifs within protein families. The ranked lists of predictions are accessible as PRENbase (http://mendel.imp.univie.ac.at/sat/PrePS/PRENbase) and can be queried for verification status, type of modifying enzymes (anchor type), and taxonomic distribution. Our results highlight a large group of plant metal-binding chaperones as well as several newly predicted proteins involved in ubiquitin-mediated protein degradation, enriching the known functional repertoire of prenylated proteins. Furthermore, we identify two possibly prenylated proteins in Mimivirus. The section HumanPRENbase provides complete lists of predicted prenylated human proteins-for example, the list of farnesyltransferase targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs) used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domain-containing FLJ32421 (termed BROFTI), and Rab28 (short isoform) as exclusive farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is presented for the selective farnesylation of targets with an evolutionary conserved modification site.     

Oligopeptide Signaling through TbGPR89 Drives Trypanosome Quorum Sensing

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Maturity and fertility of rhesus monkey oocytes collected at different intervals after an ovulatory stimulus (human chorionic gonadotropin) in in vitro fertilization cycles

In rhesus monkeys undergoing ovarian stimulation for in vitro fertilization (IVF), a midcycle injection of human chorionic gonadotropin (hCG) substitutes for the LH surge and induces preovulatory oocyte maturation. The time interval between injection and oocyte collection, ideally, allows for the completion of oocyte maturation without ovulation, which would reduce the number of oocytes available for harvest. To evaluate the influence of this time interval on oocyte parameters following hCG administration, we conducted a series of gonadotropin treatment protocols in 51 animals in which the interval from hCG administration to follicular aspiration was systematically varied from 27 to 36 hr. Follicle number and size, evaluated prior to hCG administration by sonography, did not vary significantly or consistently with preovulatory maturation time. Oocytes were harvested by laparotomy or laparoscopy, and scored for maturity before insemination. The percentage of mature, metaphase II (MII) oocytes at recovery increased significantly with increasing preovulatory time and was inversely proportional to that of metaphase I (MI) oocytes. However, oocyte yield tended toward a progressive decrease with increasing preovulatory maturation times from a high of 27 oocytes at 27 hr to a low of 17 oocytes/animal at the 36 hr time interval. Fertilization levels declined significantly from a high of 50% at 27 hr to a low of 30% at 36 hr. Thus, although higher percentages of mature oocytes were recovered at the longer time intervals, optimal oocyte/embryo harvests were realized after the shorter time intervals (27 and 32 hr) and are most compatible with the goal of achieving high yields of fertile oocytes and embryos following gonadotropin stimulation in rhesus monkeys. © 1996 Wiley-Liss, Inc.     

Strain-specific Influence of Microcystis Aeruginosa on Food Ingestion and Assimilation of some Cladocerans and Copepods

Ingestion rates where estimated for daphnids, Cyclops spp. and Bosmina (Eubosmina) coregoni thersites fed hepatotoxic and non-toxic M. aeruginosa either separate or mixed with the readily available food alga Ankistrodesmus falcatus. The ingestion rates of hepatotoxic strains of M. aeruginosa are very low compared with those of A. falcatus or non-toxic M. aeruginosa HUB 5-3 fed to Daphnia magna or D. longispina. However, a close relationship between ingestion rate of different M. aeruginosa strains and their toxicity could not be observed. Addition of the toxic strain M. aeruginosa HUB 5-2-4 reduces the ingestion rates of A. falcatus progressively due to increased food rejection by D. magna. Additionally, the assimilation efficiency of M. aeruginosa HUB 5-2-4 is two times lower compared with A. falcatus and M. aeruginosa HUB 5-3 leading to strong starvation.     

Daphnia and Toxic Blooms of Microcystis aeruginosa in Bautzen Reservoir (GDR)

As a part of a whole-lake, long-term experiment in biomanipulation in. the hypertrophic Bautzen reservoir (G.D.R.), during three years (1984–1986) the dynamics of mouse-related LD 50 of Microcystis aeruginosa was compared with the biomass development of this blue-green and the grazing pressure exerted by Daphnia galeata. Since the three summer averages of the biomass of D. galeata revealed strong differences due to decreasing predation activity of fish from 1984 to 1986, the effects of different grazing pressure on Microcystis toxicity could be investigated under field conditions. Microcystis was nontoxic at the beginning of the growing season and developed high toxicity during its first strong biomass increase in summer in all three years. But this decrease of the LD 50 together with the first biomass increase of the season is found in quite different periods in different years (1984: August, 1985: July, 1986: June). It is obvious that the higher the mean effective filtration rate of D. galeata during summer is found the faster the toxicity of Microcystis is formed. If these observations are combined with findings of other authors, the conclusion can be drawn that the development of toxic Microcystis blooms seems to be promoted by a combination of five conditions: (1) Presence of a mixture of toxic and nontoxic Microcystis strains at the beginning of the growing season even if the portion of toxic strains is very low, (2) physical and chemical growth conditions which favour Microcystis over other phytoplankton, (3) high grazing pressure by zooplankton on edible food particles over a rather long period, (4) patchy distribution of the different Microcystis strains if nonselective filtrators such as Daphnia dominate the zooplankton, and (5) absence of defense mechanisms of Microcystis against grazing which are not coupled with toxicity (e.g. large colony size). These conclusions contribute to a better understanding of the possibilities and limits of in-lake eutrophication control by biomanipulation and emphasize the need to combine top-down and bottom-up control mechanisms in eutrophic and hypertrophic waters.     

The Usefulness of Various Numerical Methods for Assessing the Specific Effects of Pollution on Aquatic Biota

The data obtained during an extensive multidisciplinary investigation of a polluted harbor basin in Hamburg were analyzed in various ways in order to determine which statistical methods are easiest to interpret and permit the most accurate evaluations. The methods analyzed include the species deficit, index of species similarity, and saprobity index, as well as the more complex cluster and gradient analyses, which revealed a variety of relationships among the biotic communities at the various sampling sites and some ecological tolerance limits of various species. The shortcomings of numerical indices for characterizing water bodies are discussed. It is suggested that biostatistical methods are much more valuable for the empirical evaluation of data than for artificially classifying ecosystems.     

Prolyl hydroxylation of collagen type I is required for efficient binding to integrin alpha 1 beta 1 and platelet glycoprotein VI but not to alpha 2 beta 1

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.     

Vasopressin type 1A receptor up-regulation by cyclosporin A in vascular smooth muscle cells is mediated by superoxide

Based on our previous results, we investigated whether cyclosporin A (CsA)-induced vasopressin type 1A receptor up-regulation was mediated by free radicals. We report that CsA analogues with different affinities for cyclophilin and calcineurin were able to up-regulate vasopressin type 1A receptor and to generate free radicals in smooth muscle cells independently of calcineurin. Further, we demonstrate that the antioxidant N-acetyl-L-cysteine blocked the increase in vasopressin type 1A receptor mRNA and protein levels induced by CsA and that low concentrations of prooxidants were able to directly increase vasopressin type 1A receptor mRNA and protein levels. In addition, short exposure to CsA or pro-oxidants was sufficient to significantly increase vasopressin type 1A receptor mRNA and protein levels. Using cell-permeable forms of superoxide dismutase and catalase, we finally show that superoxide mediates the CsA-induced effects on vasopressin type 1A receptor. These results provide strong evidence that CsA-induced superoxide generation is causally involved in vasopressin type 1A receptor expression and demonstrate for the first time that low physiological concentrations of radicals, most probably superoxide, are able to directly affect cellular signaling to increase vasopressin type 1A receptor expression in rat aortic smooth muscle cells.     

Inference and Validation of Protein Identifications

Discovery or shotgun proteomics has emerged as the most powerful technique to comprehensively map out a proteome. Reconstruction of protein identities from the raw mass spectrometric data constitutes a cornerstone of any shotgun proteomics workflow. The inherent uncertainty of mass spectrometric data and the complexity of a proteome render protein inference and the statistical validation of protein identifications a non-trivial task, still being a subject of ongoing research. This review aims to survey the different conceptual approaches to the different tasks of inferring and statistically validating protein identifications and to discuss their implications on the scope of proteome exploration.