B. subtilis ykuD protein at 2.0 A resolution: insights into the structure and function of a novel, ubiquitous family of bacterial enzymes

The crystal structure of the product of the Bacillus subtilis ykuD gene was solved by the multiwavelength anomalous dispersion (MAD) method and refined using data to 2.0 A resolution. The ykuD protein is a representative of a distinctly prokaryotic and ubiquitous family found among both pathogenic and nonpathogenic Gram-positive and Gram-negative bacteria. The deduced amino acid sequence reveals the presence of an N-terminal LysM domain, which occurs among enzymes involved in cell wall metabolism, and a novel, putative catalytic domain with a highly conserved His/Cys-containing motif of hitherto unknown structure. As the wild-type protein did not crystallize, a double mutant was designed (Lys117Ala/Gln118Ala) to reduce excess surface conformational entropy. As expected, the structure of the LysM domain is similar to the NMR structure reported for an analogous domain from Escherichia coli murein transglycosylase MltD. The molecular model also shows that the 112-residue-long C-terminal domain has a novel tertiary fold consisting of a beta-sandwich with two mixed sheets, one containing five strands and the other, six strands. The two beta-sheets form a cradle capped by an alpha-helix. This domain contains a putative catalytic site with a tetrad of invariant His123, Gly124, Cys139, and Arg141. The stereochemistry of this active site shows similarities to peptidotransferases and sortases, and suggests that the enzymes of the ykuD family may play an important role in cell wall biology.     

Predicting a relapse of Graves' hyperthyroidism in adults during the early phase of treatment with anti-thyroid drugs

INTRODUCTION: The long-term effectiveness of anti-thyroid drugs (ATD) in the treatment of Graves' hyperthyroidism (GH) is still unsatisfactory and difficult to predict. The aim of this study was to evaluate the usefulness of a determination of serum level of thyrotropin-binding inhibiting immunoglobulins (second generation TBII assay) in predicting the possibility of relapse in the early phase of pharmacological treatment. MATERIAL AND METHODS: We investigated 37 patients within the 20-60 age range with the first occurrence of GH. All patients were treated with thiamazole for 12 months. Clinical assessment, ultrasound estimation of thyroid volume and determination of serum thyrotropin, free thyroxine, free triiodothyronine, thyroid autoantibodies and TBII levels were carried out at the onset and after 1, 3, 6, 9 and 12 months of ATD treatment. RESULTS: The mean follow-up period after ATD withdrawal was 27.24 +/- 5.81 months. Of 37 patients 12 (32%) had a relapse of hyperthyroidism (mean time 8.17 +/- 6.91 months after drug withdrawal). The difference in TBII levels between the relapse and the remission group was found to be significant after the first month of therapy until the end of ATD treatment. We observed that patients with TBII above 14 IU/L after 3 months and above 8 IU/L after 6 months of therapy relapsed more frequently than patients with lower levels (sensitivity 50% and specificity 92 and 96%, respectively). CONCLUSIONS: The study confirmed that TBII estimation in the early phase of ATD could be useful in the proper planning of GH therapy and early qualification to more radical treatment (radioiodine or surgery).     

Nitric oxide,hydrogen cyanide and ethylene are required in the control of germination and undisturbed development of young apple seedlings

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are emerging as important regulators of plant development (germination, flowering, senescence), acting as secondary messengers in cooperation with classical phytohormones. Apple seeds are dormant, unless they undergo a 3 month long cold stratification. Deep dormancy of isolated apple embryos can also be broken by short pre-treatment with HCN or NO with the effect associated with enhanced ethylene synthesis. Non-dormant embryos germinate well and young seedlings grown from non-dormant embryos do not exhibit any morphological anomalies, such as asymmetric growth and greening of cotyledons. One of the aims of this work was to investigate the correlation between RNS- mediated (HCN- and NO-dependent) dormancy removal and ROS (H₂O₂ and O₂^−•) accumulation in the embryos. The beneficial effect of NO and HCN on germination of dormant apple embryos has been associated with marked increases in H₂O₂ and O₂^−• concentration in the embryos at early germination stages. We also analyzed growth of young seedlings developed from embryos pre-treatment with HCN or NO or exposed to ethylene (ethephone) and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC). ACC and ethephone removed all morphological anomalies of the seedlings (asymmetric growth and greening of cotyledons) but the radicle growth was rather slight. We propose that accumulation of ROS provoked by HCN and NO pre-treatment is required for embryo germination “sensu stricto”, while ethylene is required for post-germination seedling growth.     

Cell shape and TGF‐β signaling define the choice of lineage during in vitro differentiation of mouse primary hepatic precursors

Cellular differentiation relies on both physical and chemical environmental cues. The bipotential mouse embryonic liver (BMEL) cells are early progenitors of liver epithelial cells with an apparently infinite proliferative potential. These cells, which remain undifferentiated in a monolayer culture, differentiate upon release from geometrical constraints imposed by growth on a stiff plastic plate. In a complex three dimensional environment of a Matrigel extracellular matrix, BMEL cells form two types of polarized organoids of distinct morphologies: cyst‐like structures suggesting cholangiocyte‐type organization or complex organoids, reminiscent of liver parenchyma and associated with acquisition of hepatocyte‐specific phenotypic markers. The choice of the in vitro differentiation lineage is governed by Transforming Growth Factor‐β (TGF‐β) signaling. Our results suggest that morphological cues initiate the differentiation of early hepatic precursors and confirm the inhibitory role of TGF‐β on hepatocytic lineage differentiation. J. Cell. Physiol. 225: 186–195, 2010. © 2010 Wiley‐Liss, Inc.     

CITRATE OXIDATION IN THE CYTOPLASMIC FRACTION OF RAT BRAIN

Abstract— The cytoplasmic fraction derived from rat brain was shown to possess the ability to oxidize citrate in the presence of NADP, but not in that of NAD. The rate of citrate oxidation is limited by the rate of the aconitase-catalysed isomerization. The dependence of the reaction rate on the protein, citrate and NADP concentrations, and on reaction time, was determined. It was found that 2-oxoglutarate and NADPH, both formed in the citrate oxidation reaction, do not appear in amounts equivalent to the losses of citrate. The possible ways of utilization of the above products in the rat brain cytoplasmic fraction are discussed.
It is suggested that the oxidation of citrate in rat brain cytoplasm may proceed–among other metabolic routes–through its conversion into isocitrate (aconitase) and then 2-oxoglutarate (NADP dehydrogenase) and oxaloacetate (aspartate aminotransferase). In addition it has been shown that hypoxia may markedly effect the activity of the cytoplasmic citrateoxidizing system.     

Lymphokines secreted from sodium periodate-treated lymphocytes

This study was designed to determine if sodium metaperiodate (NaIO₄)-treated lymphocytes secrete lymphokines and if these lymphokines are similar to those obtained from mitogen- or antigen-stimulated lymphocytes. A brief exposure of CBA spleen cells to NaIO₄ induced the secretion of significant amounts of migration inhibitory factor (MIF). This MIF had a molecular weight range between 30,000 and 58,000, and was stable when heated at 56 °C for 30 min, but unstable at 80 °C. These characteristics are similar to those previously reported for mitogen- and antigen-induced MIF. In addition, NaIO₄ induced the secretion of lymphotoxin (LT) from CBA and Balb/c spleen cells, as well as from guinea pig lymph node cells. NaIO₄ was compared to the other inducers in regard to the quantity of LT secreted. Supernatant derived from NaIO₄-treated mouse spleen cells contained less LT than supernatants derived from concanavalin A- or phytohemagglutinin-treated cells, but contained more activity than those supernatants derived from lipopolysaccharide-treated cells. CBA spleen cells secreted significantly more LT than Balb/c spleen cells after NaIO₄ stimulation. NaIO₄-stimulated CBA spleen cells secreted LT in cultures with or without serum, but stimulated Balb/c spleen cells secreted LT only in serum-containing cultures. The advantages of NaIO₄ as an inducer of lymphokines, as opposed to other mitogens or antigens, is the brief exposure of this agent to the cells after which the NaIO₄ is removed, and the lymphokines can be obtained free from the inducer.     

Antimicrobial activity and total content of polyphenols of <Emphasis Type="Italic">Rheum</Emphasis> L. species growing in Poland

In the crude ethanol extracts obtained from the rhizome and roots of Rheum palmatum L., Rheum undulatum L. and Rheum rhaponticum L. growing in Poland concentration of polyphenols ranged from 46.11 to 76.45 mg/g. Concentration of tannins ranged from 7.07% to 8.67%, while anthracene derivatives and anthraquinones varied by species - R. palmatum measured 36.3 and 34 mg/g, while R. undulatum or R. rhaponticum did not exceed 20.4 and 18.1 or 19.8 mg/g and 16.6 mg/g, respectively. Using a broth microdilution method it was found that all of the Rheum spp. extracts were more active against reference strains of Gram-positive bacteria (Staphylococcus spp.) than against those of Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis). The strongest inhibitory effect against Staphylococcus spp. was exerted by R. undulatum extract with MIC = 125–250 μg/mL. The moderate in vitro antibacterial activity of R. undulatum suggests that this plant, often used in the European cuisine to improve flavour, may be also important and useful as an alternative or auxiliary medicine remedy in the treatment of uncomplicated superficial infections caused especially by clinically important staphylococci, potentially pathogenic S. aureus or opportunistic S. epidermidis.     

Molecular data reveal high taxonomic diversity of allogromiid Foraminifera in Explorers Cove (McMurdo Sound,Antarctica)

Allogromiids are organic-walled or agglutinated, single-chambered Foraminifera, common in deep-sea and polar benthic communities. The simple forms and paucity of distinctive features make allogromiid identification difficult by traditional means. Molecular phylogenetic methods offer alternative tools for species identification and are used here to investigate allogromiid diversity. We obtained 135 partial small-subunit ribosomal DNA sequences of allogromiids collected in Explorers Cove, McMurdo Sound, Antarctica. In contrast to the 27 morphotypes identified, phylogenetic analysis revealed 49 molecular types (considered separate species) that differ by more than 5% of sequence divergence. The 49 genetic types form 28 molecular supra-groups that differ by more than 20% and probably represent distinct genera or families. Large genetic distances separating the molecular types indicate unexpectedly high taxonomic diversity. Comparison of our data with sequences of non-Antarctic allogromiids suggests that Explorers Cove species might be endemic and only distantly related to comparable northern hemisphere fauna.     

Parasitic microfungi of the Tatra Mountains: 5. <Emphasis Type="Italic">Plasmopara</Emphasis> representatives on the species of <Emphasis Type="Italic">Geranium</Emphasis>

A list of species and the distribution of the members of Plasmopara (Chromista, Peronosporales) on Geranium spp. in the Tatra National Park (Western Carpathians) biospheric reserve are given. Three fungal species parasitizing 3 plant species were recorded in the area. Plasmopara praetermissa is a species new to Poland and Slovakia.