Polynuclear water-soluble dinitrosyl iron complexes with cysteine or glutathione ligands: Electron paramagnetic resonance and optical studies

Electron paramagnetic resonance and optical spectrophotometric studies have demonstrated that low-molecular dinitrosyl iron complexes (DNICs) with cysteine or glutathione exist in aqueous solutions in the form of paramagnetic mononuclear (М-DNICs) and diamagnetic binuclear complexes (B-DNICs). The latter represent Roussin’s red salt esters and can be prepared by treatment of aqueous solutions of Fe²⁺ and thiols (рН 7.4) with gaseous nitric oxide (NO) at the thiol:Fe²⁺ ratio 1:1. М-DNICs are synthesized under identical conditions at the thiol:Fe²⁺ ratios above 20 and produce an EPR signal with an electronic configuration {Fe(NO)₂}⁷ at g_aver. = 2.03. At neutral pH, aqueous solutions contain both M-DNICs and B-DNICs (the content of the latter makes up to 50% of the total DNIC pool). The concentration of B-DNICs decreases with a rise in pH; at рН 9–10, the solutions contain predominantly M-DNICs. The addition of thiol excess to aqueous solutions of B-DNICs synthesized at the thiol:Fe²⁺ ratio 1:2 results in their conversion into М-DNICs, the total amount of iron incorporated into M-DNICs not exceeding 50% of the total iron pool in B-DNICs. Air bubbling of cys-М-DNIC solutions results in cysteine oxidation-controlled conversion of М-DNICs first into cys-B-DNICs and then into the EPR-silent compound Х able to generate a strong absorption band at 278 nm. In the presence of glutathione or cysteine excess, compound Х is converted into B-DNIC/M-DNIC and is completely decomposed under effect of the Fe²⁺ chelator о-phenanthroline or N-methyl-d-glucamine dithiocarbamate (MGD). Moreover, MGD initiates the synthesis of paramagnetic mononitrosyl iron complexes with MGD. It is hypothesized that compound Х represents a polynuclear DNIC with cysteine, most probably, an appropriate Roussin’s black salt thioesters and cannot be prepared by simple substitution of М-DNIC cysteine for glutathione. Treatment of М-DNIC with sodium dithionite attenuates the EPR signal at g_aver. = 2.03 and stimulates the appearance of an EPR signal at g_aver. = 2.0 with a hypothetical electronic configuration {Fe(NO)₂}⁹. These changes can be reversed by storage of DNIC solutions in atmospheric air. The EPR signal at g_aver. = 2.0 generated upon treatment of B-DNICs with dithionite also disappears after incubation of B-DNIC solutions in air. In all probability, the center responsible for this EPR signal represents М-DNIC formed in a small amount during dithionite-induced decomposition of B-DNIC.     

Crystal Structure of the N-Acetylmannosamine Kinase Domain of GNE

Background

UDP-GlcNAc 2-epimerase/ManNAc 6-kinase, GNE, is a bi-functional enzyme that plays a key role in sialic acid biosynthesis. Mutations of the GNE protein cause sialurea or autosomal recessive inclusion body myopathy/Nonaka myopathy. GNE is the only human protein that contains a kinase domain belonging to the ROK (repressor, ORF, kinase) family.

Principal Findings

We solved the structure of the GNE kinase domain in the ligand-free state. The protein exists predominantly as a dimer in solution, with small populations of monomer and higher-order oligomer in equilibrium with the dimer. Crystal packing analysis reveals the existence of a crystallographic hexamer, and that the kinase domain dimerizes through the C-lobe subdomain. Mapping of disease-related missense mutations onto the kinase domain structure revealed that the mutation sites could be classified into four different groups based on the location – dimer interface, interlobar helices, protein surface, or within other secondary structural elements.

Conclusions

The crystal structure of the kinase domain of GNE provides a structural basis for understanding disease-causing mutations and a model of hexameric wild type full length enzyme.

Enhanced Version

This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.     

A novel mannitol teichoic acid with side phosphate groups of Brevibacterium sp. VKM Ac-2118.

The cell wall of Brevibacterium sp. VKM Ac-2118 isolated from a frozen (mean annual temperature -12 degrees C) late Pliocene layer, 1.8-3 Myr, Kolyma lowland, Russia, contains mannitol teichoic acid with a previously unknown structure. This is 1,6-poly(mannitol phosphate) with the majority of the mannitol residues bearing side phosphate groups at O-4(3). The structure of the polymer was established by chemical methods, NMR spectroscopy, and MALDI-TOF mass spectrometry.     

Azospirillum brasilense glutamine synthetase: influence of the activating metal ions on the enzyme properties

The kinetic properties of the Mg²⁺-activated and Mn²⁺-activated glutamine synthetase (GS) of Azospirillum brasilense in the biosynthetic reaction were studied. The Mg²⁺-supported and Mn²⁺-supported GSs in an average state of adenylylation varied in pH optimum, maximum activity, saturation functions for ammonium and glutamate, affinity to substrates, and in the Me²⁺-ATP ratio required for the optimal enzyme activity. Seventeen other cations were tested for the maintenance of GS activity. The level of the latter and the kinetic behavior of the GS in A.brasilense is suggested to depend essentially on the concentrations of Mg²⁺, Mn²⁺ and Co²⁺, as well as on their ratio     

Cytokine profiles of HIV patients with pulmonary tuberculosis resulting from adjunct immunotherapy with herbal phytoconcentrates Dzherelo and Anemin

Dzherelo (Immunoxel) and Anemin when combined with standard anti-tuberculosis therapy (ATT) were shown to produce better clinical outcome than chemotherapy alone. Sixty HIV-positive patients with active pulmonary TB were equally divided into three matched groups to receive either ATT, ATT + Dzherelo, or ATT + Dzherelo + Anemin. Peripheral blood samples were measured by ELISA for plasma levels of IL-2, IL-6, TNF-α, IFN-γ, and IFN-α. After 6 months of follow-up Dzherelo and Dzherelo + Anemin combinations produced 61% (P = 0.005) and 44.4% (P = 0.06) higher levels of IL-2, whereas in ATT group they were reduced by 33.1% (P = 0.002). The levels of IL-6 increased by 17% (P = 0.15) in ATT group, but declined in both immune intervention groups by 26.2% (P = 0.007) and 21.3% (P = 0.22). TNF-α was suppressed in two immunotherapy groups by 19.1% (P = 0.06) and 76.3% (P = 0.02), respectively, but had risen by 14% (P = 0.42) in ATT patients. The pattern of production of IFN-γ was opposite to that of TNF-α, but statistical significance was stronger in patients receiving ATT and Dzherelo + Anemin than in Dzherelo group: −34% (P = 0.004), +31.9% (P = 0.008), and +17.3% (P = 0.33), respectively. Moderately decreased levels of IFN-α were observed in all treatment arms (range 0.9–16.6%) but differences were not significant. Despite considerable intra-group variation in cytokine production, the baseline inter-group averages were not statistically different indicating that the results were not biased by sample heterogeneity. Immunomodulators used in this study possibly act by enhancing natural immune response against TB. Expanded study of other cytokines and correlates relevant to control and protection from TB and HIV is needed in order to identify biomarkers of favorable treatment outcome, which may aid design of better immune interventions and vaccines.     

Law of the Minimum Paradoxes

The “Law of the Minimum” states that growth is controlled by the scarcest resource (limiting factor). This concept was originally applied to plant or crop growth (Justus von Liebig, 1840, Salisbury, Plant physiology, 4th edn., Wadsworth, Belmont, 1992) and quantitatively supported by many experiments. Some generalizations based on more complicated “dose-response” curves were proposed. Violations of this law in natural and experimental ecosystems were also reported. We study models of adaptation in ensembles of similar organisms under load of environmental factors and prove that violation of Liebig’s law follows from adaptation effects. If the fitness of an organism in a fixed environment satisfies the Law of the Minimum then adaptation equalizes the pressure of essential factors and, therefore, acts against the Liebig’s law. This is the the Law of the Minimum paradox: if for a randomly chosen pair “organism–environment” the Law of the Minimum typically holds, then in a well-adapted system, we have to expect violations of this law.     

BIOLUMBASE--the database of natural and transgenic bioluminescent organisms.

The Institute of Biophysics SB RAS hosts and maintains a specialized collection of luminous bacteria (CCIBSO 836) containing over 700 strains isolated in various regions of the world's oceans. The culture collection is a source of lux genes and biologically active substances. The wide application of bioluminescence in medicine and ecology has given importance to analysing information on the structure and functioning of bioluminescence systems in natural and transgenic microorganisms, as well as on their features that are closely interrelated with bioluminescence. The aims of our BIOLUMBASE database are: gathering information on microorganisms with lux genes, their analysis and free access, and distribution of this data throughout the global network. The database includes two sections, natural and transgenic luminous microorganisms, and is updated by our own experimental results, the published literature and internet resources. For the future, a publicly available internet site for BIOLUMBASE is planned. This will list the strains and provide comprehensive information on the properties and functions of luminous bacteria, the mechanisms of regulation of bioluminescence systems, constructs with lux genes, and applications of bioluminescence in microbiology, ecology, medicine and biotechnology. It is noteworthy that this database will also be useful for evaluation of biological hazards of transgenic strains. Users will be able to carry out bibliographic and strain searches starting from any feature of interest.     

Phenotypic differences in behavior, physiology and neurochemistry between rats selected for tameness and for defensive aggression towards humans

To better understand the biology of tameness, i.e. tolerance of human presence and handling, we analyzed two lines of wild-derived rats (Rattus norvegicus) artificially selected for tameness and defensive aggression towards humans. In response to a gloved human hand, tame rats tolerated handling, whereas aggressive rats attacked. Cross-fostering showed that these behavioral differences are not caused by postnatal maternal effects. Tame rats were more active and explorative and exhibited fewer anxiety-related behaviors. They also had smaller adrenal glands, larger spleens and lower levels of serum corticosterone. Blood glucose levels were lower in tame rats, whereas the concentrations of nine amino acids were higher. In the brain, tame rats had lower serotonin and higher taurine levels than aggressive rats. Our findings reinforce the notion that tameness is correlated with differences in stress response and will facilitate future efforts to uncover the genetic basis for animal tameness.     

Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+

The nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a beta beta alpha-metal topology. Here we report the crystal structures of a Zn2+-bound N-ColE7 (H545E mutant) in complex with a 12-bp duplex DNA and a Ni2+-bound N-ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 A and 2.0 A, respectively. Metal-dependent cleavage assays showed that N-ColE7 cleaves double-stranded DNA with a single metal ion cofactor, Ni2+, Mg2+, Mn2+, and Zn2+. ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg2+ at concentrations of <25 mM, indicating that zinc is the physiologically relevant metal ion in N-ColE7 in host E. coli. In the crystal structure of N-ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni2+ is bound in N-ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His-metal finger motif can be coordinated to six ligands, such as Mg2+ in I-PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni2+ or Zn2+ in ColE7. Universally, the metal ion in the His-metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P-O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.