IFN-gamma and IL-10 mediate parasite-specific immune responses of cord blood cells induced by pregnancy-associated Plasmodium falciparum malaria

Available evidence suggests that immune cells from neonates born to mothers with placental Plasmodium falciparum (Pf) infection are sensitized to parasite Ag in utero but have reduced ability to generate protective Th1 responses. In this study, we detected Pf Ag-specific IFN-gamma(+) T cells in cord blood from human neonates whose mothers had received treatment for malaria or who had active placental Pf infection at delivery, with responses being significantly reduced in the latter group. Active placental malaria at delivery was also associated with reduced expression of monocyte MHC class I and II in vivo and following short term in vitro coculture with Pf Ag compared with levels seen in neonates whose mothers had received treatment during pregnancy. Given that APC activation and Th1 responses are driven in part by IFN-gamma and down-regulated by IL-10, we examined the role of these cytokines in modulating the Pf Ag-specific immune responses in cord blood samples. Exogenous recombinant human IFN-gamma and neutralizing anti-human IL-10 enhanced T cell IFN-gamma production, whereas recombinant human IFN-gamma also restored MHC class I and II expression on monocytes from cord blood mononuclear cells cocultured with Pf Ag. Accordingly, active placental malaria at delivery was associated with increased frequencies of Pf Ag-specific IL-10(+)CD4(+) T cells in cord blood mononuclear cell cultures from these neonates. Generation and maintenance of IL-10(+) T cells in utero may thus contribute to suppression of APC function and Pf Ag-induced Th1 responses in newborns born to mothers with placental malaria at delivery, which may increase susceptibility to infection later in life.     

The PDZ2 domain of syntenin at ultra-high resolution: bridging the gap between macromolecular and small molecule crystallography

The crystal structure of the second PDZ domain of the scaffolding protein syntenin was solved using data extending to 0.73 A resolution. The crystallographic model, including the hydrogen atoms and the anisotropic displacement parameters, was refined to a conventional R-factor of 7.5% and Rfree of 8.7%, making it the most precise crystallographic model of a protein molecule to date. The model reveals discrete disorder in several places in the molecule, and significant plasticity of the peptide bond, with some omega angles deviating by nearly 20 degrees from planarity. Most hydrogen atoms are easily identifiable in the electron density and weak hydrogen bonds of the C-H...O type are clearly visible between the beta-strands. The study sets a new standard for high-resolution protein crystallography.     

The structure and ligand binding properties of the B. subtilis YkoF gene product, a member of a novel family of thiamin/HMP-binding proteins

The crystal structure of the Bacillus subtilis YkoF gene product, a protein involved in the hydroxymethyl pyrimidine (HMP) salvage pathway, was solved by the multiwavelength anomalous dispersion (MAD) method and refined with data extending to 1.65 A resolution. The atomic model of the protein shows a homodimeric association of two polypeptide chains, each containing an internal repeat of a ferredoxin-like betaalphabetabetaalphabeta fold, as seen in the ACT and RAM-domains. Each repeat shows a remarkable similarity to two members of the COG0011 domain family, the MTH1187 and YBL001c proteins, the crystal structures of which were recently solved by the Northeast Structural Genomics Consortium. Two YkoF monomers form a tightly associated dimer, in which the amino acid residues forming the interface are conserved among family members. A putative small-ligand binding site was located within each repeat in a position analogous to the serine-binding site of the ACT-domain of the Escherichia coli phosphoglycerate dehydrogenase. Genetic data suggested that this could be a thiamin or HMP-binding site. Calorimetric data confirmed that YkoF binds two thiamin molecules with varying affinities and a thiamine-YkoF complex was obtained by co-crystallization. The atomic model of the complex was refined using data to 2.3 A resolution and revealed a unique H-bonding pattern that constitutes the molecular basis of specificity for the HMP moiety of thiamin.     

Genetic disregulation of TNF alpha and TNF alpha type II receptors in colon cancer at the II and III stage of disease

The expression of the genes coding TNFalpha and TNF RII receptors (TNF RII: TNFR2 membrane and soluble domain, TNFR2/R7 soluble domain) was analysed in colon cancer at the II and III stage of disease, by estimation of mRNA expression. The study included 80 patients with histopathologically confirmed adenocarcinoma. The number of TNFalpha mRNA, TNFR2 mRNA and TNFR2/R7 mRNA copies were estimated in tumour and healthy tissue. The highest number of mRNA TNF-alpha copies were investigated in all samples of tissue and independently of the stage of disease. Simultaneously, we noticed the largest number of mRNA copies for TNFalpha and TNF R2/R7 in healthy cells at stage III of the disease. It is possible to draw a hypothetical line separating the anti-cancer activity of TNFalpha and its influence on cancer progression.     

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.     

A Study on Selected Physiological Parameters of Plants Grown Under Lithium Supplementation

Exposure of sunflower and maize plants to increasing concentrations of lithium (0?C50?mg Li dm^?3) in a nutrient solution induced changes in biomass, leaf area and photosynthetic pigment accumulation, as well as levels of lipid peroxidation. The highest applied lithium dose (50?mg Li dm^?3) evoked a significant reduction in the shoot biomass for both examined species, as well as necrotic spots and a reduction of the leaf area in sunflower plants. An enrichment of a nutrient solution with 5?C50?mg Li dm^?3 did not significantly affect chlorophylls a and b and the carotenoid content in sunflower plants. However, in maize, a significant decrease in all pigment content under highest used lithium concentration was noted. The levels of lipid peroxidation of the cell membranes in leaves of sunflower plants and the roots of maize increased significantly in the presence of 50?mg Li dm^?3, which suggests disturbances of the membrane integrity and pro-oxidant properties of the excess lithium ions. Nonetheless, in maize, an increase of shoot biomass and leaf area in the presence of 5?mg Li dm^?3 was found. An analysis of the metal content indicated that lithium accumulated significantly in sunflower and maize shoots in a dose-dependent manner, but differences occurred between species. The sunflower plants accumulated considerably greater amounts of this metal than maize. The potassium content in shoots remained unchanged under lithium treatments, except for a significant increase in the potassium levels for sunflower plants grown in the presence of 50?mg Li dm^?3. These results suggest that lithium at 50?mg Li dm^?3 is toxic to both plant species, but the symptoms of toxicity are species-specific. Moreover, the lithium influence on plants is dose-dependent and its ions can exert toxicity at high concentrations (50?mg Li dm^?3) or stimulate growth at low concentrations (5?mg Li dm^?3).     

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.