Measurement of Inositol 1,4,5-Trisphosphate in Living Cells Using an Improved Set of Resonance Energy Transfer-Based Biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP₃) sensors were created to follow intracellular InsP₃ changes in single living cells and in cell populations. Similar to previous InsP₃ sensors the new sensors are based on the ligand binding domain of the human type-I InsP₃ receptor (InsP₃R-LBD), but contain a mutation of either R265K or R269K to lower their InsP₃ binding affinity. Tagging the InsP₃R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP₃ in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP₃ concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP₃ sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP₃ after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP₃ and Ca²⁺ levels in BRET experiments, the Cameleon D3 Ca²⁺ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P₂ resulted in the fall of InsP₃ level, followed by the decrease of the Ca²⁺-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca²⁺-signal preceded the fall of InsP₃ level indicating an InsP₃-, independent, direct regulation of capacitative Ca²⁺ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP₃ sensor can be used to monitor both the increase and decrease of InsP₃ levels in live cells suitable for high-throughput BRET applications.     

Genetic Heterogeneity Reveals On-Going Speciation and Cryptic Taxonomic Diversity of Stream-Dwelling Gudgeons (Teleostei,Cyprinidae) in the Middle Danubian Hydrosystem (Hungary)

Although stream-dwelling gudgeons (Cyprinidae, genus: Gobio) are widespread in Central Europe, the taxonomy of this group and the distribution of its species are still unexplored in detail. The aims of our study are to ascertain taxonomic composition and distribution of the former Gobio gobio superspecies in the inner area of the Carpathian Basin. Since the presence of cryptic species is suspected in this area, we examined the taxonomic and phylogenetic relationships of Central European Gobio taxa by sequencing the mitochondrial DNA control region (mtCR). Additionally, we characterized the genetic structure of 27 stream-dwelling gudgeon populations of this area by Amplified Fragment Length Polymorphism (AFLP). Results of mtCR analysis proved the presence of three species already known as G. obtusirostris (dominant in NW-Hungary), G. gobio (sporadic) and G. carpathicus (sporadic). Additionally, the analysis revealed the existence of one doubtful taxon, G. sp1 (dominant in NE-Hungary), and a new isolated haplogroup (dominant in SW-Hungary). Although Network analysis showed significant detachment among haplogroups, their genetic distances were quite small. Therefore Bayesian phylogenetic analysis showed weak nodal support for the branching pattern both for newly described haplotypes, and for the already accepted species. AFLP data showed distinct population structure and a clear pattern of isolation was revealed by distance of stocks. At the same time, level of separation was not affected by the altitudinal position of sites. Moreover we found three major clusters of populations which were separated according to hydrographic regions, and corresponded to the findings of mtCR analysis. Our results suggest the on-going speciation of gudgeons in the Carpathian Basin, however the separation of haplogroups seems to only be an intermediate phase. The discovered natural pattern seems to be only slightly influenced by anthropogenic impacts. Additionally our results put into question the suitability of the recently accepted within Gobio genus taxonomy.     

The power of regeneration and the stem-cell kingdom: freshwater planarians (Platyhelminthes)

The great powers of regeneration shown by freshwater planarians, capable of regenerating a complete organism from any tiny body fragment, have attracted the interest of scientists throughout history. In 1814, Dalyell concluded that planarians could "almost be called immortal under the edge of the knife". Equally impressive is the developmental plasticity of these platyhelminthes, including continuous growth and fission (asexual reproduction) in well-fed organisms, and shrinkage (degrowth) during prolonged starvation. The source of their morphological plasticity and regenerative capability is a stable population of totipotent stem cells--"neoblasts"; this is the only cell type in the adult that has mitotic activity and differentiates into all cell types. This cellular feature is unique to planarians in the Bilateria clade. Over the last fifteen years, molecular studies have begun to reveal the role of developmental genes in regeneration, although it would be premature to propose a molecular model for planarian regeneration. Genomic and proteomic data are essential in answering some of the fundamental questions concerning this remarkable morphological plasticity. Such information should also pave the way to understanding the genetic pathways associated with metazoan somatic stem-cell regulation and pattern formation.     

Receptor-like properties of the 26 kDa transmembrane form of TNF

Most members of the TNF family of proteins exist as transmembrane proteins with relatively long intracellular domains, and a number of them are involved in the ill-defined phenomenon of "reverse signaling". We have identified a putative nuclear localization signal in the cytoplasmic domain of TNF which proved to be functional in two assays. Western analysis identified an approximately 10 kDa peptide corresponding to the transmembrane and cytoplasmic domains of TNF after the proteolytic liberation of the 17 kDa, soluble form of TNF. This 10 kDa peptide was enriched in internal membranes and nuclear fractions of disrupted cells. Immune electron-microscopic studies proved its localization in transport vesicles and the nucleus. The nuclear transport of the intracellular segment of TNF resembles the signaling process through the Notch-type of receptors. Indeed, the presence of the 10 kDa peptide seems to influence the expression of another inflammatory cytokine, interleukin-1 beta. These findings suggest that the transmembrane form of TNF has receptor-like properties and its interaction with the receptors initiates a bidirectional signaling.     

NHE blockade inhibits chemokine production and NF-kappaB activation in immunostimulated endothelial cells

Na⁺/H⁺exchanger (NHE) activation has been documented to contribute toendothelial cell injury caused by inflammatory states. However, therole of NHEs in regulation of the endothelial cell inflammatoryresponse has not been investigated. The present study tested thehypothesis that NHEs contribute to endothelial cell inflammationinduced by endotoxin or interleukin (IL)-1. NHE inhibition usingamiloride, 5-(N-ethyl-N-isopropyl)-amiloride, and5-(N-methyl-N-isobutyl)amiloride as well as thenon-amiloride NHE inhibitors cimetidine, clonidine, and harmalinesuppressed endotoxin-induced IL-8 and monocyte chemoattractant protein(MCP)-1 production by human umbilical endothelial vein cells (HUVECs). The suppressive effect of amiloride on endotoxin-induced IL-8 production was associated with a decreased accumulation of IL-8 mRNA.NHE inhibitors suppressed both inhibitory (I)B degradation andnuclear factor (NF)-B DNA binding, suggesting that a decrease inactivation of the IB-NF-B system contributed to the suppression of HUVEC inflammatory response by NHE blockade. NHE inhibition decreased also the IL-1-induced HUVEC inflammatory response, becauseamiloride suppressed IL-1-induced E-selectin expression on HUVECs.These results demonstrate that maximal activation of the HUVECinflammatory response requires a functional NHE.

     

Thioredoxin motif of Caenorhabditis elegans PDI-3 provides Cys and His catalytic residues for transglutaminase activity

Previous reports have suggested that protein disulfide isomerases (PDIs) have transglutaminase (TGase) activity. The structural basis of this reaction has not been revealed. We demonstrate here that Caenorhabditis elegans PDI-3 can function as a Ca(2+)-dependent TGase in assays based on modification of protein- and peptide-bound glutamine residues. By site-directed mutagenesis the second cysteine residue of the -CysGlyHisCys- motif in the thioredoxin domain of the enzyme protein was found to be the active site of the transamidation reaction and chemical modification of histidine in their motif blocked TGase activity.