Conservation of Brachyury, Mef2, and Snail in the myogenic lineage of jellyfish: a connection to the mesoderm of bilateria

One major difference between simple metazoans such as cnidarians and all the bilaterian animals is thought to involve the invention of mesoderm. The terms diploblasts and triploblasts are therefore, often used to group prebilaterian and bilaterian animals, respectively. However, jellyfish contain well developed striated and smooth muscle tissues that derive from the entocodon, a mesoderm-like tissue formed during medusa development. We investigated the hypothesis, that the entocodon could be homologous to the third germ layer of bilaterians by analyzing the structures and expression patterns of the homologues of Brachyury, Mef2, and Snail in the jellyfish Podocoryne carnea. These are regulatory genes from the T-box, MADS-box and zinc finger families known to play important roles in bilaterian mesoderm patterning and muscle differentiation. The sequence and expression data demonstrate that the genes are structurally and functionally conserved and even more similar to humans or other deuterostomes than to protostome model organisms such as Drosophila or Caenorhabditis elegans. Based on these data we conclude that the common ancestor of the cnidarians and bilaterians not only shared genes that play a role in regulating myogenesis but already used them to develop and differentiate muscle systems similar to those of triploblasts.     

Gene synthesis, expression in E. coli, and in vitro refolding of Pseudomonas sp. KWI 56 and Chromobacterium viscosum lipases and their chaperones

Pseudomonas lipases are industrially used as detergent additives, in the food industry, and in organic synthesis. Currently, these lipases are either isolated from wild-type strains or overexpressed in recombinant Pseudomonas host strains which may be subject to special safety regulations and thus be unsuitable for enzyme engineering via directed evolution. Here we describe the heterologous expression of two Pseudomonas lipases in Escherichia coli. The lipase genes of Pseudomonas sp. KWI 56 (recently reclassified as Burkholderia cepacia) and Chromobacterium viscosum and the genes of their specific chaperones, which are required for correct folding, were synthesized with an optimized nucleotide sequence and overexpressed (up to 50%) in E. coli. However, both lipases were inactively expressed inside inclusion bodies. Quantitative in vitro refolding of the lipases in the presence of their specific chaperones yielded 310,000 U/g (Pseudomonas sp. KWI 56) and 190,000 U/g (C. viscosum) wet cells. In addition, these lipases could be demonstrated to refold efficiently in the presence of chaperones of related lipases.     

Citrinin in fruit juices

Despite the wide distribution of citrinin-producingPenicillium spp. there are only rare reports about the occurence of this mycotoxin in foodstuffs. Particularly, the discrepancy between the common detection of the applerotting fungusP expansum and the complete lack of data about the occurrence of citrinin in apple-based foods is noteworthy. Based on an indirect enzyme immunoassay (EIA) a study was performed aiming at the sensitive detection of citrinin in apple and other fruit juices. The direct analysis of diluted apple juices by the EIA failed due to pronounced sample matrix effects. Though these problems could be resolved by the extraction of artificially contaminated apple juice with dichloromethane, a poor recovery rate (20–30%) for citrinin was observed. Astonishingly, similar results (mean recovery of 29.9%) were received when doted apple juices were directly purified on immunoaffinity columns despite the minimal sample treatment associated with this method. For the detection of citrinin in tomatoe juices samples were purified with a liquid-liquid partition step. Again, the mean recovery rate was very low (32.0%). Analyzing 55 fruit and vegetable juices purchased in local retail stores only traces of citrinin (maximum 0.2 μg/L) could be detected in the samples.     

X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif

Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 A resolution. rsJAM consists of two immunoglobulin-like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U-shaped JAM dimers are oriented in cis on the cell surface and form a two-dimensional network by trans-interactions of their N-terminal domains with JAM dimers from an opposite cell surface.     

Short-term effects of the 21-aminosteroid lazaroid tirilazad mesylate (PNU-74006F) and the pyrrolopyrimidine lazaroid PNU-101033E on energy metabolism of human peripheral blood mononuclear cells

Two groups of antioxidant compounds, the 21-aminosteroids and the pyrrolopyrimidines, have been found to act as neuroprotective drugs against lipid peroxidation in the injured CNS. Like glucocorticoids at high doses they are assumed to produce their effects at least in part by direct membrane stabilizing effects. In order to prove this hypothesis, we have investigated in this study the effects of these drugs on the energy metabolism of activated human peripheral blood mononuclear cells (PBMC) since these cells have been shown to serve as a suitable test system for substances affecting processes of ATP turnover. We compared the in vitro effects of (i) the 21-aminosteroid lazaroid tirilazad, (ii) the pyrrolopyrimidine lazaroid PNU-101033E and (iii) the glucocorticoid methylprednisolone on mitogen-induced respiration rate and ATP-consumption. We show that tirilazad inhibits concanavalin A-stimulated respiration rate and sodium cycling across the plasma membrane. The effect of methylprednisolone is similar indicating corresponding cellular mechanisms. However, unlike methylprednisolone, tirilazad produced no significant effect on calcium cycling across the plasma membrane. PNU-101033E in our test system caused cytotoxic effects on PBMC that did not allow us to quantify cellular actions on energy metabolism. Our results underline the view that tirilazad, first, is mimicking the high-dose immunosuppressive pharmacology of glucocorticoids such as methylprednisolone and, second, is likely to produce its therapeutic effects by direct physicochemical interactions with cellular membranes.     

Daily Torpor in Free-Ranging Gray Mouse Lemurs (Microcebus murinus) in Madagascar

I aimed to determine when and under which seasonal environmental conditions gray mouse lemurs (Microcebus murinus), a small nocturnal primate species endemic to Madagascar, utilize daily torpor. Using temperature-sensitive radio collars, I measured skin temperature (T _sk ) of free-ranging mouse lemurs under natural conditions. My results showed that male and female mouse lemurs in the wild enter torpor spontaneously over a wide range of ambient temperatures (T _a ) during the dry season, but not during the rainy season. Mouse lemurs that remained normothermic had significantly lower body masses (mean: 59.7 g) than individuals that used torpor (mean: 80.2 g). Skin temperatures dropped to 20.9°C and the mean torpor bout duration is 10.3 h. The use of torpor on a given night varied among individuals, whereas the propensity for torpor did not differ significantly between males and females. I found no evidence that T _a can be used to predict whether mouse lemurs will remain normothermic or enter torpor. It appears that the most reliable indicator for the occurrence of torpor in free-ranging Microcebus murinus is time of the year, i.e., photoperiod.