Structure of the membrane reconstituted transmembrane-juxtamembrane peptide EGFR(622-660) and its interaction with Ca2+/calmodulin

The transmembrane (TM) and juxtamembrane (JM) regions of the epidermal growth factor receptor (EGFR) couple ligand binding in the extracellular domain to activation of the kinase domain. Solid-state NMR and polarized FTIR measurements of peptides corresponding to the TM plus JM regions of EGFR (residues 622-660) reconstituted in model phospholipid membranes are presented to address the role of the short cytoplasmic JM sequence (residues 645-660) in regulating EGFR activity. We show that the TM domain is helical with a transition to non-helical structure at the TM-JM boundary. Fluorescence measurements indicate that the JM region of EGFR(622-660) binds to the membrane surface and that binding can be reversed by the addition of the complex of Ca2+ and calmodulin. Together these data support models suggesting the cytoplasmic JM region of EGFR plays an active role in regulating receptor activity.     

Complete mitochondrial DNA analysis of eastern Eurasian haplogroups rarely found in populations of northern Asia and eastern Europe

With the aim of uncovering all of the most basal variation in the northern Asian mitochondrial DNA (mtDNA) haplogroups, we have analyzed mtDNA control region and coding region sequence variation in 98 Altaian Kazakhs from southern Siberia and 149 Barghuts from Inner Mongolia, China. Both populations exhibit the prevalence of eastern Eurasian lineages accounting for 91.9% in Barghuts and 60.2% in Altaian Kazakhs. The strong affinity of Altaian Kazakhs and populations of northern and central Asia has been revealed, reflecting both influences of central Asian inhabitants and essential genetic interaction with the Altai region indigenous populations. Statistical analyses data demonstrate a close positioning of all Mongolic-speaking populations (Mongolians, Buryats, Khamnigans, Kalmyks as well as Barghuts studied here) and Turkic-speaking Sojots, thus suggesting their origin from a common maternal ancestral gene pool. In order to achieve a thorough coverage of DNA lineages revealed in the northern Asian matrilineal gene pool, we have completely sequenced the mtDNA of 55 samples representing haplogroups R11b, B4, B5, F2, M9, M10, M11, M13, N9a and R9c1, which were pinpointed from a massive collection (over 5000 individuals) of northern and eastern Asian, as well as European control region mtDNA sequences. Applying the newly updated mtDNA tree to the previously reported northern Asian and eastern Asian mtDNA data sets has resolved the status of the poorly classified mtDNA types and allowed us to obtain the coalescence age estimates of the nodes of interest using different calibrated rates. Our findings confirm our previous conclusion that northern Asian maternal gene pool consists of predominantly post-LGM components of eastern Asian ancestry, though some genetic lineages may have a pre-LGM/LGM origin.     

Microbotryum heliospermae, a new anther smut fungus parasitic on Heliosperma pusillum in the mountains of the European Alpine System

The members of the smut genus Microbotryum are pathogens of a wide range of host plant species from nine dicotyledonous families. Within the genus, the species sporulating in anthers of Caryophyllaceae form a monophyletic group that in recent years attracted much interest in various biological studies. The phylogenetic framework developed for species delimitation within Microbotryum revealed that high level host-specificity is a major feature of most caryophyllaceous anther smuts. However, the great number of anther smut specimens on diverse host plant species reported worldwide has still not been included in phylogenetic analyses due to the inaccessibility of recently collected specimens, and thus many species remain still undiscovered. In this study, anther smut specimens on Heliosperma pusillum originating from all main mountain ranges of the European Alpine System were examined using partial rDNA sequence and/or morphological analyses. The investigation revealed that all specimens are morphologically uniform and phylogenetically represent a monophyletic lineage, sister to Microbotryum lagerheimii complex on Atocion rupestre/Silene lacera/Silene vulgaris/Viscaria vulgaris. This lineage cannot be attributed to any of the previously described species, and therefore the smut in anthers of H. pusillum is described and illustrated here as a new species, Microbotryum heliospermae. The species is known from subalpine zone of the Alps, the Carpathians, the Dinaric Alps, and the Pyrenees, inhabiting host plants growing in open spring communities or semihumid mountain meadows.     

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate ^1-3. We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich''s ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged ^4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich''s ataxia patients and control individuals ⁶, human newborn fibroblasts, as well as human keratinocytes.     

Connexin 43 and metabolic effect of fatty acids in stressed endothelial cells

Changes in the inner mitochondrial membrane potential (∆ψ) may lead either to apoptosis or to protective autophagy. Connexin 43 (Cx43), a gap junction protein, is suggested to affect mitochondrial membrane permeability. The aim of our study was to analyze Cx43 gene expression, Cx43 protein localization and mitochondrial function in the human endothelial cells stressed by dietary-free fatty acids (FFA) and TNFα. Human endothelial cells (HUVECs) were incubated with (10–30 uM) palmitic (PA), oleic (OA), eicosapentaenoic (EPA) or arachidonic (AA) acids for 24 h. TNFα (5 ng/ml) was added at the last 4 h of incubation. The Cx43 gene expression was analyzed by the quantitative real-time PCR. The Cx43 protein concentrations in whole cells and in the isolated mitochondria were measured. Changes in ∆ψ and Cx43 localization were analyzed by flow cytometry or fluorescence microscopy. Generated ATP was measured by a luminescence assay. TNFα, PA and OA significantly decreased ∆ψ, while AA (P = 0.047) and EPA (P = 0.004) increased ∆ψ value. Preincubation with EPA or AA partially prevented the TNFα-induced decrease of ∆ψ. Incubation with AA resulted in up-regulation of the Cx43 gene expression. AA or PA significantly increased Cx43 protein content; however, presence of TNFα in general aggravated the negative effect of FFA. Only EPA was found to increase ATP generation in HUVECs. The fatty acid-specific induction of changes in Cx43 expression and protein concentration as well as the normalization of ∆ψ and increase of ATP generation seem to be the separate, independent mechanisms of FFA-mediated modulatory effect in the human endothelial cells pathology.     

Identification and molecular characterization of <Emphasis Type="Italic">Theileria</Emphasis> sp. infecting red deer (<Emphasis Type="Italic">Cervus elaphus</Emphasis>) in northwestern Poland

Piroplasms from Theileria genus were detected in blood and spleen of red deer Cervus elaphus culled during the months of September 2004–January 2005 in northwestern Poland. The polymerase chain reaction revealed the presence of Theileria deoxyribonucleic acid in 88% (36 of 41) of the animals examined. Molecular characterization of the parasites based on large piece of 18S ribosomal ribonucleic acid gene containing hypervariable region V4 showed 99.9% similarity to two Theileria spp. sequences: Theileria sp. 3185/02 and Theileria capreoli BAB1158. Phylogenetic analysis confirmed that the three isolates cluster together with high bootstrap support. It is supposed that those pathogens can be classified as one group characteristic for the Eurasian continent, contrary to protozoon of Theileria from the T. cervi group, which are often found on the North American continent and can also infect the representatives of Cervidae. In conclusion, this study suggested that free-living C. elaphus in northwestern Poland are a competent reservoir of Theileria sp. ZS T04 C.e. parasites, although the vector of the piroplasms is still unknown.     

Changes in the initial phase of lipid peroxidation induced by elicitor from Phytophthora infestans in Solanum species

The initial phase of the lipid peroxidation process in leaves of Solanum nigrum var. gigantea, Solanum tuberosum cv Bzura and clone H-8105, which represent non-host resistance, field resistance and susceptibility, respectively, against Phytophthora infestans, was investigated. Based on quantitative and qualitative high-performance liquid chromatography (HPLC) analyses of free and esterified fatty acid hydroperoxides (FAHs), we characterized the lipid peroxidation process induced by the pathogen-derived elicitor, culture filtrate (CF), in leaves of the studied genotypes. In all plants, FAHs generated due to 13-lipoxygenase (LOX) action dominated over those from the non-enzymatic pathway. The FAHs derived from 9-LOX activity were found only in CF-treated leaves of the non-host resistant S. nigrum. However, experiments in vitro and in planta with exogenous linoleic acid (LA) as a substrate for LOX revealed high constitutive activity of 9-LOX in all genotypes, which increased in response to CF treatment. The time course changes in polyunsaturated fatty acid (PUFA) pools in the total lipid fractions as well as the degree of their oxidation suggested that CF-induced PUFA peroxidation was enhanced mostly in S. nigrum, less so in Bzura and least in the susceptible clone H-8105. The obtained results are discussed in light of the overall biochemical cell status of plants in the studied interactions.     

Jasmonates Inhibit Flowering in Short-Day Plant Pharbitis nil

The role of jasmonates in the photoperiodic flower induction of short-day plant Pharbitis nil was investigated. The plants were grown in a special cycle: 72 h of darkness, 24 h of white light with lowered intensity, 24-h long inductive night, 14 days of continuous light. At 4 h of inductive night the cotyledons of non-induced plants contained about two times the amount of endogenous jasmonates (JA/JA-Me) compared to those induced. A 15-min long pulse of far red light (FR) applied at the end of a 24-h long white light phase inhibited flowering of P. nil. The concentration of jasmonates at 2 and 4 h of inductive night in the cotyledons of the plants treated with FR was similar. Red light (R) could reverse the effect of FR. R light applied after FR light decreased the content of jasmonates by about 50%. Methyl jasmonate (JA-Me) applied to cotyledons, shoot apices and cotyledon petioles of P. nil inhibited the formation of flower buds during the first half of a 24-h long inductive or 14-h long subinductive night. Application of JA-Me to the cotyledons was the most effective. None of the plants treated with JA-Me on the cotyledons in the middle of the inductive night formed terminal flower buds. The aspirin, ibuprofen and phenidone, jasmonates biosynthesis inhibitors partially reversed the effect of FR, stimulating the formation of axillary and terminal flower buds. Thus, the results obtained suggests that phytochrome system control both the photoperiodic flower induction and jasmonates metabolism. Jasmonates inhibit flowering in P. nil.     

The Dynamics of Filamentous Structures in the Apical Band, Oral Crescent, Fission Line and the Postoral Meridional Filament in Tetrahymena thermophila Revealed by Monoclonal Antibody 12G9

The ciliate Tetrahymena thermophila possesses a multitude of cytoskeletal structures whose differentiation is related to the basal bodies the main mediators of the cortical pattern. This investigation deals with immunolocalization using light and electron microscopy of filaments labeled by the monoclonal antibody 12G9, which in other ciliates identifies filaments involved in transmission of cellular polarities and marks cell meridians with the highest morphogenetic potential. In Tetrahymena interphase cells, mAb 12G9 localizes to the sites of basal bodies and to the striated ciliary rootlets, to the apical band of filaments and to the fine fibrillar oral crescent. We followed the sequence of development of these structures during divisional morphogenesis. The labeling of the maternal oral crescent disappears in pre-metaphase cells and reappears during anaphase, concomitantly with differentiation of the new structure in the posterior daughter cell. In the posterior daughter cell, the new apical band originates as small clusters of filaments located at the base of the anterior basal bodies of the apical basal body couplets during early anaphase. The differentiation of the band is completed in the final stages of cytokinesis and in the young post-dividing cell. The maternal band is reorganized earlier, simultaneously with the oral structure.The mAb 12G9 identifies two transient structures present only in dividing cells. One is a medial structure demarcating the two daughter cells during metaphase and anaphase, and defining the new anterior border of the posterior daughter cell. The other is a post-oral meridional filament marking the stomatogenic meridian in postmetaphase cells. Comparative analysis of immunolocalization of transient filaments labeled with mAb12G9 in Tetrahymena and other ciliates indicates that this antibody identifies a protein bound to filamentous structures, which might play a role in relying polarities of cortical domains and could be a part of a mechanism which governs the positioning of cortical organelles in ciliates.