In vitro lipofection with novel asymmetric series of 1,2-dialkoylamidopropane-based cytofectins containing single symmetric bis-(2-dimethylaminoethane) polar headgroups

Novel N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl[bis-(2-dimethylaminoethane)] bivalent cationic lipids were synthesized and evaluated for in vitro transfection activity against a murine melanoma cell line. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the dioleoyl derivative 22 (1,2lb5) elicited transfection activity. The transfection activity of this lipid was reduced when formulated with DOPE. Contrary to that, the dimyristoyl derivative 19 (1,2lb2) mediated no activity when used alone but induced the highest levels of marker gene expression in the presence of DOPE. In an effort to correlate the transfection activity with cationic lipid structures, the physicochemical properties of cationic lipids in isolation and of lipoplexes were studied with surface tensiometry, photon correlation spectroscopy, gel electrophoresis mobility shift assay, and fluorescence techniques. In regard to the lipoplex properties, gel electrophoresis mobility shift assay and EtBr exclusion fluorescence assay revealed that the 1,2lb5 was the only lipid to associate and condense plasmid DNA, respectively. Photon correlation spectroscopy analysis found that 1,2lb5/DNA complexes were of relatively small size compared to all other lipoplexes. With respect to the properties of isolated lipids, Langmuir monolayer studies and fluorescence anisotropy on cationic lipid dispersions verified high two-plane elasticity and increased fluidity of the transfection competent dioleoyl derivative 1,2lb5, respectively. The results indicate that high transfection activity is mediated by cationic lipids characterized by an expanded mean molecular area, high molecular elasticity, and increased fluidity.     

A novel caspase-2 complex containing TRAF2 and RIP1

The enzymatic activity of caspases is implicated in the execution of apoptosis and inflammation. Here we demonstrate a novel nonenzymatic function for caspase-2 other than its reported proteolytic role in apoptosis. Caspase-2, unlike caspase-3, -6, -7, -9, -11, -12, and -14, is a potent inducer of NF-kappaB and p38 MAPK activation in a TRAF2-mediated way. Caspase-2 interacts with TRAF1, TRAF2, and RIP1. Furthermore, we demonstrate that endogenous caspase-2 is recruited into a large and inducible protein complex, together with TRAF2 and RIP1. Structure-function analysis shows that NF-kappaB activation occurs independent of enzymatic activity of the protease and that the caspase recruitment domain of caspase-2 is sufficient for the activation of NF-kappaB and p38 MAPK. These results demonstrate the inducible assembly of a novel protein complex consisting of caspase-2, TRAF2, and RIP1 that activates NF-kappaB and p38 MAPK through the caspase recruitment domain of caspase-2 independently of its proteolytic activity.     

Binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid R6K

Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the gamma origin of plasmid R6K, the Rep protein, pi, is distinctive in that it can bind the seven 22-bp iterons in two forms; pi monomers activate replication, whereas pi dimers act as inhibitors. In this work, we used wild type and variants of the pi protein with altered monomer/dimer ratios to study iteron/pi interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by pi monomers and dimers. We also isolated iteron mutants that affected the binding of pi monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, pi monomers interact with nucleotides spanning the entire length of the iteron. In contrast, pi dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.     

Low genetic diversity and contrasting patterns of differentiation in the two monotypic genera Halacsya and Paramoltkia (Boraginaceae) endemic to the Balkan serpentines

Halacsya and Paramoltkia are two monotypic genera of the Balkan ultramafic outcrops, both classified as paleoendemics. They show close phylogenetic relationship, similar ecology and partially sympatric range, but contrasting life-history traits, patterns of distribution and chromosomal features. We performed a study to compare their infraspecific genetic structure using AFLP fingerprinting. Within-population heterozygosity (H_S) and total heterozygosity (H_T) were lower in diploid H. sendtneri than in polyploid P. doerfleri (0.121 and 0.142 vs. 0.165 and 0.178, respectively). Number of polymorphic loci was also higher in the latter species. Halacsya showed significant levels of among-population variation (14.5%) and population differentiation (F_ST = 0.145), as well as a structuring in four main geographic groups connected by low gene flow. Combined with inability for long-distance dispersal, these data suggest that present-day range fragmentation in this species is the result of historical losses of intervening populations and support its status of paleoendemic surviving in refugial serpentine “islands”. Populations of P. doerfleri were instead genetically very close (F_ST = 0.049), and variation was partitioned almost exclusively among individuals (95.1%). Lack of population groupings and higher levels of gene flow suggested a single, panmictic group, fitting more a metapopulation than a “classical” paleoendemic model.     

Latitudinal variation in seeds characteristics of Acer platanoides and A. pseudoplatanus

Climate change will likely affect population dynamics of numerous plant species by modifying several aspects of the life cycle. Because plant regeneration from seeds may be particularly vulnerable, here we assess the possible effects of climate change on seed characteristics and present an integrated analysis of seven seed traits (nutrient concentrations, samara mass, seed mass, wing length, seed viability, germination percentage, and seedling biomass) of Acer platanoides and A. pseudoplatanus seeds collected along a wide latitudinal gradient from Italy to Norway. Seed traits were analyzed in relation to the environmental conditions experienced by the mother trees along the latitudinal gradient. We found that seed traits of A. platanoides were more influenced by the climatic conditions than those of A. pseudoplatanus. Additionally, seed viability, germination percentage, and seedling biomass of A. platanoides were strongly related to the seed mass and nutrient concentration. While A. platanoides seeds were more influenced by the environmental conditions (generally negatively affected by rising temperatures), compared to A. pseudoplatanus, A. platanoides still showed higher germination percentage and seedling biomass than A. pseudoplatanus. Thus, further research on subsequent life-history stages of both species is needed. The variation in seed quality observed along the climatic gradient highlights the importance of studying the possible impact of climate change on seed production and species demography.     

Marbleseeds are gromwells – Systematics and evolution of Lithospermum and allies (Boraginaceae tribe Lithospermeae) based on molecular and morphological data

Phylogenetic relationships are complex within the Lithospermeae, a large subgroup of the Boraginaceae s.str. The relationships of New World Lasiarrhenum, Macromeria, Nomosa, Onosmodium, Perittostoma, and Psilolaemus to subcosmopolitan and much larger Lithospermum have not been critically investigated in the recent past. No molecular data on the phylogeny of these genera and Lithospermum have so far been published. We investigated the relationships within Lithospermeae using three loci (nuclear ITS plus 5.8S rRNA, chloroplast trnL-F-spacer, and trnS-G-spacer) and micromorphological character traits (pollen, nutlets). Lithospermum s.l. constitutes the sistergroup of Asian Ulugbekia and is monophyletic only when its American segregates “Macromeria”, monotypic Nomosa, and Onosmodium are included. Both the African and the South American species groups of Lithospermum are monophyletic, but North American representatives are not resolved in a single clade. Morphological characters that have been considered as important for generic delimitation in the past (such as large, yellow corollas without faucal scales, particular pollen types, coarsely veined leaves, shrubby habit) have evolved in at least two only distantly related lineages within Lithospermum s.l. The reduction of American “Macromeria”, Nomosa, and Onosmodium as well as Asian Ulugbekia under Lithospermum is proposed to render the latter monophyletic. This redefined Lithospermum s.l. appears to have undergone a type of recent “island radiation” in the Americas, reflected in a morphological diversity far exceeding that found in the Old World.     

A novel SOD-mimetic permeability transition inhibitor agent protects ischemic heart by inhibiting both apoptotic and necrotic cell death

In ischemia-reperfusion injuries, elevated calcium and reactive oxygen species (ROS) induce mitochondrial permeability transition (mPT), which plays a pivotal role in mediating damages and cell death. Inhibition of mPT decreases necrotic cell death; however, during reperfusion, the continuous production of ROS may contribute to the temporary opening of the pore and thus the onset of the delayed apoptotic cell death. Based on amiodarone structure, we developed the first SOD-mimetic mPT inhibitor (HO-3538) that can eliminate ROS in the microenvironment of the permeability pore. In isolated mitochondria, HO-3538 inhibited mPT and the release of proapoptotic mitochondrial proteins. It had a ROS scavenging effect and antiapoptotic effect in a cardiomyocyte line and it diminished release of mitochondrial proapoptotic proteins. Furthermore, HO-3538 significantly enhanced the recovery of mitochondrial energy metabolism and functional cardiac parameters; decreased infarct size, lipid peroxidation, and protein oxidation; and suppressed necrotic as well as apoptotic cell death pathways in Langendorff-perfused hearts. In these respects it was somewhat superior to its two constituents, amiodarone and a pyrrol-derivative free radical scavenger. These data suggest that the SOD-mimetic mPT inhibitors are ideal candidates for drug development for the alleviation of postinfarct myocardial injuries.