VCRPs,small cysteine-rich proteins,might be involved in extracellular signaling in the green alga Volvox

The sex-inducer of the spherical green alga Volvox carteri is one of the most potent biological effector molecules known: it is released into the medium by sexual males and triggers the switch to the sexual cleavage program in the reproductive cells of vegetatively grown males and females even at concentrations as low as 10^-16 M. In an adult Volvox alga, all cells are embedded in an extensive extracellular matrix (ECM), which constitutes >99% of the volume of the spheroid. There exist no cytoplasmic connections between the cells in an adult alga, so any signal transduction between different cells or from the organism''s environment to a reproductive cell must involve the ECM. Recently, a small cysteine-rich extracellular protein, VCRP, was identified in Volvox and shown to be quickly synthesized by somatic cells in response to the sex-inducer. Due to its characteristics, VCRP was speculated to be an extracellular second messenger from somatic cells to reproductive cells. Here a related protein, VCRP2, is presented, exhibiting a 56% amino acid sequence identity with VCRP. Two possible scenarios for signal transduction from the sex-inducer to the reproductive cell are discussed.Key words: cell wall, extracellular matrix, extracellular second messenger, green algae, sex-inducer, sex inducing pheromone, sexual development, stress response, Volvocaceae, wounding     

Comparability of Mixed IC50 Data – A Statistical Analysis

The biochemical half maximal inhibitory concentration (IC₅₀) is the most commonly used metric for on-target activity in lead optimization. It is used to guide lead optimization, build large-scale chemogenomics analysis, off-target activity and toxicity models based on public data. However, the use of public biochemical IC₅₀ data is problematic, because they are assay specific and comparable only under certain conditions. For large scale analysis it is not feasible to check each data entry manually and it is very tempting to mix all available IC₅₀ values from public database even if assay information is not reported. As previously reported for K_i database analysis, we first analyzed the types of errors, the redundancy and the variability that can be found in ChEMBL IC₅₀ database. For assessing the variability of IC₅₀ data independently measured in two different labs at least ten IC₅₀ data for identical protein-ligand systems against the same target were searched in ChEMBL. As a not sufficient number of cases of this type are available, the variability of IC₅₀ data was assessed by comparing all pairs of independent IC₅₀ measurements on identical protein-ligand systems. The standard deviation of IC₅₀ data is only 25% larger than the standard deviation of K_i data, suggesting that mixing IC₅₀ data from different assays, even not knowing assay conditions details, only adds a moderate amount of noise to the overall data. The standard deviation of public ChEMBL IC₅₀ data, as expected, resulted greater than the standard deviation of in-house intra-laboratory/inter-day IC₅₀ data. Augmenting mixed public IC₅₀ data by public K_i data does not deteriorate the quality of the mixed IC₅₀ data, if the K_i is corrected by an offset. For a broad dataset such as ChEMBL database a K_i- IC₅₀ conversion factor of 2 was found to be the most reasonable.     

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n‐octyl‐oligo‐oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N‐dimethyldodecylamine N‐oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size‐dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.     

Structure-function relationship studies of PTH(1-11) analogues containing sterically hindered dipeptide mimetics.

The N-terminal 1-34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and reproduces all biological responses characteristic of the native intact PTH. In order to develop safer and non-parenteral PTH-like bone anabolic agents, we have studied the effect of introducing conformationally constrained dipeptide mimetics into the N-terminal portion of PTH in an effort to generate miniaturized PTH-mimetics. To this end, we have synthesized and conformationally and biologically characterized PTH(1-11) analogues containing 3R-carboxy-6S-amino-7,5-bicyclic thiazolidinlactam (7,5-bTL), a rigidified dipeptide mimetic unit. The wild type sequence of PTH(1-11) is H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-NH(2). The following pseudo-undecapeptides were prepared: [Ala(1), 7,5-bTL(3, 4), Nle(8), Arg(11)]hPTH(1-11)NH(2) (I); [Ala(1), 7,5-bTL(6, 7), Nle(8), Arg(11)]hPTH(1-11)NH(2) (II); [Ala(1), Nle(8), 7,5-bTL(9, 10), Arg(11)]hPTH(1-11)NH(2) (III). In aqueous solution containing 20% TFE, only analogue I exhibited the typical CD pattern of the alpha-helical conformation. NMR experiments and molecular dynamics calculations located the alpha-helical stretch in the sequence Ile(5)-His(9). The dipeptide mimetic unit 7,5-bTL induces a type III beta-turn, occupying the positions i - 1 and i of the turn. Analogue II exhibited an equilibrium between a type I beta-turn and an alpha-helix, and analogue III did not show any ordered structure. Biological tests revealed poor activity for all analogues (EC(50) > 0.1 mM). Apparently, the relative side-chain orientation of Val(2), Ile(5) and Met(8) can be critical for effective analogue-receptor interaction. Considering helicity as an essential property to obtain active PTH agonists, one must decorate the correctly positioned dipeptide mimetic azabicycloalkane scaffold with substitutions corresponding to the displaced amino acids.     

Changes in carbohydrate composition and trehalase-activity during the budding cycle of Saccharomyces cerevisiae

Summary The carbohydrate composition and the specific activity of the trehalase of cyclic partially synchronised yeast populations have been investigated. Under glucose limitation and appropriate cultural conditions synchronous growth in a chemostat was achieved. The cells accumulated the reserve carbohydrates during the single cell phase between two buddings. The rapid degradation of part of these reserves began shortly before the swelling of the bud. The importance of the mobilisation of endogenous reserves for the development of the cell is discussed.The specific activity of the trehalase changed during the budding cycle. The result gives rise to the assumption that the synthesis of this enzyme is linked to the growth cycle.     

Higher-accuracy method for measuring minichromosome stability in Saccharomyces cerevisiae

The minichromosome maintenance assay isfrequently used to characterize mutations genetically that affect the initiation of DNA replication or to decipherfunctional components in autonomously replicating sequences. The assay determines minichromosome loss by measuring the percentage of plasmid-containing cells in cultures after a period of growth in nonselective medium. Here we analyze data acquisition errors that contribute to the low accuracy of the routine versions of the assay. We propose modifications that eliminate errors in the acquisition of two variables and significantly improve the accuracy of the assay.     

Identifying genes that contribute most to good classification in microarrays

Background  

The goal of most microarray studies is either the identification of genes that are most differentially expressed or the creation of a good classification rule. The disadvantage of the former is that it ignores the importance of gene interactions; the disadvantage of the latter is that it often does not provide a sufficient focus for further investigation because many genes may be included by chance. Our strategy is to search for classification rules that perform well with few genes and, if they are found, identify genes that occur relatively frequently under multiple random validation (random splits into training and test samples).     

Gene delivery with low molecular weight linear polyethylenimines

BACKGROUND: Linear polyethylenimine (LPEI) with a molecular weight (MW) of 22 kDa has been described as having a superior ability to induce gene transfer compared to its branched form. However, the transfection efficiency of the polymer cannot be enhanced beyond a certain limit due to cytotoxicity. We explored the potential of utilizing LPEIs with MWs ranging from 1.0 to 9.5 kDa to overcome this limitation. METHODS: Polyplexes of plasmid DNA encoding for the enhanced green fluorescent protein (EGFP) and various LPEIs were compared concerning their transfection efficiency and cytotoxicity in CHO-K1 and HeLa cells by flow cytometry. The involvement of endolysosomes in LPEI-mediated gene transfer was investigated by applying the proton pump inhibitor bafilomycin A1 and the lysosomotropic agent sucrose. Confocal laser scanning microscopy was applied to assess the size and shape of polyplexes under cell culture conditions, to detect their endolysosomal localization and to observe their translocation to the nucleus. RESULTS: The transfection efficiency could be altered by varying the MW and the amount of the polymer available for polyplex formation. The highest transfection efficiency (about 44%), i.e. the fraction of EGFP-positive cells, was obtained with LPEI 5.6 kDa, while the cytotoxicity remained low. The colocalization of polyplexes and endolysosomes was observed, and it appeared that the larger polyplexes escaped from the acidic organelles particularly quickly. For LPEI 5.0 and 9.0 kDa, the number of cells and nuclei that had taken up DNA after 6 hours was similar, as determined by flow cytometry. CONCLUSIONS: Our study suggests that LPEIs with low MWs are promising candidates for non-viral gene delivery, because they are more efficient and substantially less toxic than their higher MW counterparts.