Non-muscle alpha-actinin-4 interacts with plasminogen activator inhibitor type-1 (PAI-1)

PAI-1 modulates many biological processes involving fibrinolysis, cell migration or tissue remodelling. In addition to inhibiting serine proteases (mainly tPA and uPA), PAI-1 interacts with vitronectin (Vn), fibrin or alpha(1)-acid glycoprotein, interactions which are important for PAI-1-mediated effects in inflammation, tumor invasion and metastasis. To further identify proteins interacting with PAI-1, the yeast two-hybrid strategy was employed. Screening of a human placenta cDNA library identified--in addition to the C-terminal region of cytokeratin 18 (CK18(182-430))--a large C-terminal fragment of alpha-actinin-4 (Act-4) as a binding partner for PAI-1. Two different cDNA clones encoding Act-4(287-911) and Act-4(330-911) respectively, were isolated. An Act-4(330-911)/GST-fusion protein, but not GST alone, was immunoprecipitated together with active PAI-1. In solid phase binding assays, active wild-type PAI-1 as well as the PAI-1 variant Q123K (which does not interact with multimeric Vn) was found to bind to Act-4(330-911)/GST. Latent PAI-1, latent Q123K, and the inactive PAI-1 variant Q55P did not display any binding activity. Act-4 is mainly present intracellularly and is involved in cellular motility via interaction with the actin cytoskeleton, thus probably affecting the metastatic potential of tumor cells. However, an extracellular Act-4-derived fragment (mactinin) has previously been identified, which (i) is generated by proteolytic action of uPA, (ii) displays significant chemotactic activity for monocytes, and (iii) promotes monocyte/macrophage maturation. We suggest that PAI-1, via interaction with both Act-4 and uPA, may function as a modulator of this mononuclear phagocyte response, not only in inflammation but also in tumor invasion and metastasis.     

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.     

Carbon and energy fluxes during haloadaptation of<Emphasis Type="Italic"> Halomonas</Emphasis> sp. EF11 growing on phenol

The haloalkaliphile Halomonas sp. EF11 can grow on phenol as sole source for carbon and energy, while maintaining an osmotic equilibrium predominantly by adjusting levels of a certain compatible solute. To determine the energy costs of haloadaptation and the fate of substrate-carbon, the strain was grown continuously in an isothermal compensation calorimeter, keeping all conditions constant except salinity. As salinity increased, slight linear reductions in exothermic heat flow and biomass formation occurred, and 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) synthesis increased linearly. However, beyond a certain salinity threshold the stationary phenol concentration increased exponentially, while heat flow fell sharply, indicating intoxication or wash-out. The clear transition point between the phases, where ectoine formation peaked, suggests that calorimetric measurements could be used to control the conversion of growth-inhibiting substrates (like phenol) into ectoine and to optimize the process. Enthalpy balance and chemical determinations revealed acetate and formate were formed as side products when the C/N ratio in the feed was low, while 2-muconic acid semialdehyde and formate were produced when the ratio was high. These findings indicate that phenol assimilation occurs via the meta pathway. However, enzyme assays implied that assimilation occurs via the ortho and meta pathways at a low C/N ratio and exclusively via the meta pathway at a high C/N ratio.Communicated by W.D. Grant     

Neogenin mediates the action of repulsive guidance molecule

Repulsive guidance molecule (RGM) is a recently identified protein implicated in both axonal guidance and neural tube closure. The avoidance of chick RGM in the posterior optic tectum by growing temporal, but not nasal, retinal ganglion cell axons is thought to contribute to visual map formation. In contrast to ephrins, semaphorins, netrins and slits, no receptor mechanism for RGM action has been defined. Here, an expression cloning strategy identified neogenin as a binding site for RGM, with a sub-nanomolar affinity. Consistent with selective axonal responsiveness to RGM, neogenin is expressed in a gradient across the chick retina. Neogenin is known to be one of several netrin-binding proteins but only neogenin interacts with RGM. The avoidance of RGM by temporal retinal axons is blocked by the anti-neogenin antibody and the soluble neogenin ectodomain. Dorsal root ganglion axons are unresponsive to RGM but are converted to a responsive state by neogenin expression. Thus, neogenin functions as an RGM receptor.     

Antigens up the nose: identification of putative biomarkers for nasal tolerance induction functional studies combined with proteomics

Intranasal autoantigen delivery is the most effective means of inducing mucosal tolerance and suppression of autoimmune disease. In an effort to identify markers of the "tolerant state", we employed proteomics technology at the level of the cervical lymph node. The analysis revealed that nasal antigen administration (without adiuvant) led to modulation of various proteins among which the most prominent were haptoglobin, nonintegrin 67 kDa laminin receptor, and MRP8. The immunoregulatory haptoglobin may qualify as (bio)marker for effective immunotherapy.     

Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

Mitochondrial gene diversity in the common vole Microtus arvalis shaped by historical divergence and local adaptations

The phylogeography of the common vole (Microtus arvalis) was examined by analysing mitochondrial DNA (mtDNA) sequence variation in 1044 base pairs (bp) of the cytochrome b (cytb) gene and in 322 bp of the control region (ctr) among 106 individuals from 58 locations. The geographical distribution of four previously recognized cytb evolutionary lineages in Europe was refined and a new lineage was found in southern Germany. All lineages were distributed allopatrically, except in one sample that was probably located in a contact zone. The occurrence of several lineages in the Alps is in keeping with their recent recolonization from distinct sources. The translation of 84 cytb DNA sequences produced 33 distinct proteins with relationships that differed from those of the DNA haplotypes, suggesting that the mtDNA lineages did not diverge in response to selection. In comparison with M. agrestis, a neutrality test detected no overall evidence for selection in the cytb gene, but a closer examination of a structural model showed that evolutionarily conserved and functionally important positions were often affected. A new phylogeographical test of random accumulation of nonsynonymous mutations generated significant results in three lineages. We therefore conclude that the molecular diversity of cytb in M. arvalis is overall the result of the demographic history of the populations, but that there have been several episodes of local adaptation to peculiar environments.     

Fibroblast growth factors in epithelial repair and cytoprotection

Growth factors are polypeptides that stimulate the division of certain cell types at low concentrations. Fibroblast growth factor (FGF) 7 (FGF-7) and its homologue FGF-10 act specifically on various types of epithelial cells including keratinocytes of the skin, intestinal epithelial cells and hepatocytes. In addition, FGF-7 and FGF-10 have been shown to be more than growth factors: they can protect epithelial cells from damaging effects induced, for example, by radiation and oxidative stress. Therefore, they are currently in clinical trials for the treatment of oral mucositis, a severe side-effect of cancer therapy characterized by painful inflammation and ulceration of the oral epithelium. To gain insight into the mechanisms of FGF-7/FGF-10 action in epithelial cells, we searched for genes that are regulated by these growth factors. Indeed, we identified genes that help us to explain the mechanisms that underlie the effects of FGF-7. Most interestingly, several genes were identified that are likely to mediate the cytoprotective effect of FGF-7 for epithelial cells in vitro and possibly also in injured and diseased tissues in vivo.     

A molecular dissection of the glycoprotein hormone receptors

In glycoprotein hormone receptors, a subfamily of rhodopsin-like G protein-coupled receptors, the recognition and activation steps are carried out by separate domains of the proteins. Specificity of recognition of the hormones thyrotropin (TSH), lutropin (LH), human chorionic gonadotropin (hCG) and follitropin (FSH) involves leucine-rich repeats (LRRs) present in an N-terminal ectodomain, and can be associated with a limited number of residues at key positions of the LRRs. The mechanism by which binding of the hormones results in activation is proposed to involve switching of the ectodomain from a tethered inverse agonist to a full agonist of the serpentine, rhodopsin-like region of the receptor. Unexpectedly, the picture is complicated by the observation that promiscuous activation of one of the receptors (FSHr) by hCG or TSH can result from activating mutations affecting the serpentine region of the receptors.     

Morphologic and genetic variability in the marine planktonic ciliate Laboea strobila Lohmann, 1908 (Ciliophora, Oligotrichia), with notes on its ontogenesis

Laboea strobila Lohmann, 1908 is a conspicuous oligotrich ciliate in the marine plankton. In order to compare different populations, the morphology of specimens from the Mediterranean Sea, North Sea, and Irish Sea was investigated using live observation, protargol impregnation, and scanning electron microscopy. Furthermore, the PCR-amplified products of the SSrRNA gene from a monoclonal culture of L. strobila from the Mediterranean Sea were sequenced and aligned with sequences of other oligotrichs, including a population of L. strobila from the Atlantic coast of the USA. Finally, the data from the ecological literature were summarized and the cultivation methods were described. The SSrRNA gene sequences of the two distantly located L. strobila populations from the North Atlantic are identical. Likewise, the morphometrics of most populations so far investigated after protargol impregnation (i.e. from the North Atlantic) do not show obvious differences. In all computed phylogenetic trees, L. strobila groups with Strombidium species, forming a monophyletic taxon corresponding to the subclass Oligotrichia. These results are corroborated by the ontogenetic comparison. Since no type species was fixed for Laboea Lohmann, 1908, L. strobila was designated in the present paper.