Glycoprotein hypersecretion alters the cell wall in Trichoderma reesei strains expressing the Saccharomyces cerevisiae dolichylphosphate mannose synthase gene

Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble beta-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.     

The molecular and biochemical characteristics of proline iminopeptidase from rye seedlings (Secale cereale L.)

A proline iminopeptidase (EC. 3.4.11.5) was isolated from shoots of 3 day old seedlings. The purification procedure consisted of 5 steps: acid precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on Sepharose CL 6B, twice repeated hydrophoic chromatography on Phenyl-Sepharose HP. The enzyme was purified 404.8-fold, with the specific activity of 8.5 units mg⁻¹ of protein with recovery yield of 3%. The purified enzyme had a molecular mass of 225 kDa estimated by gel filtration and 55.4 kDa by SDS PAGE. This indicates that native enzyme is composed of four subunits. The enzyme was specific for proline β-naphtylamide among various amino acid β-naphtylamides. An optimal activity was observed at 37 °C at pH 7.75. The enzyme was thermostable up to 37 °C for 30 min. The enzyme was strongly inhibited by pHMB, E-64, heavy metal ions and partially by PMSF, DFP. The results suggest that cysteine and serine residues may participate in the enzyme activity.     

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.     

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.     

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.