Biogenic nanosized iron oxides obtained from cultivation of iron bacteria from the genus Leptothrix.

A detailed investigation of nanostructured iron oxides/(oxy)hydroxides gathered after cultivation of bacteria from the genus Leptothrix as iron (II) oxidizers is presented. A specific type of medium is selected for the cultivation of the bacteria. Results for sediment powder and bio-film on glass substrate samples from the same media are discussed. XRD, Raman spectroscopy, SEM, and TEM images and PPMS measurements are used to prove the exact composition of the biogenic products and to interpret the oxidation process. Analysis of the data collected shows that around 80 % of the iron (II) from the growth medium has been transformed into iron (III) in the form of different (oxy)hydroxides, with the rest found to be in a mixed 2,5 valence in magnetite. Our investigation shows that the bio-film sample has a phase content different from that of the powdered biomass and that lepidocrocite (γ-FeOOH) is the predominant and the initial biogenic phase in both samples. Magnetite nanoparticles are a secondary product in the bio-film, part of which possesses a defective quasi-maghemite surface layer. In the powdered biomass, the oxidation steps are not fully completed. The initial products are non-stoichiometric and due to the mixed ferric and ferrous ions present, they develop into: (i) lepidocrocite (γ-FeOOH) as a basic sediment, (ii) magnetite (Fe3O4) and (iii) goethite (α-FeOOH) in small quantities. The average size of all iron-bearing particles is found to be below 30 nm. The magnetic measurements performed show a superparamagnetic behavior of the material at room temperature.

MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3.

The ubiquitin ligase MDM2 is best known for balancing the activity of the tumor suppressor p53. We have previously shown that MDM2 is vital for adipocyte conversion through controlling Cebpd expression in a p53-independent manner. Here, we show that the proadipogenic effect of MDM2 relies on activation of the STAT family of transcription factors. Their activation was required for the cAMP-mediated induction of target genes. Interestingly, rather than influencing all cAMP-stimulated genes, inhibition of the kinases directly responsible for STAT activation, namely JAKs, or ablation of MDM2, each resulted in abolished induction of a subset of cAMP-stimulated genes, with Cebpd being among the most affected. Moreover, STATs were able to interact with the transcriptional cofactors CRTC2 and CRTC3, hitherto only reported to associate with the cAMP-responsive transcription factor CREB. Last but not least, the binding of CRTC2 to a transcriptional enhancer that interacts with the Cebpd promoter was dramatically decreased upon JAK inhibition. Our data reveal the existence of an unusual functional interplay between STATs and CREB at the onset of adipogenesis through shared CRTC cofactors.

Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation.

The role of the E3 ubiquitin ligase murine double minute 2 (Mdm2) in regulating the stability of the p53 tumor suppressor is well documented. By contrast, relatively little is known about p53-independent activities of Mdm2 and the role of Mdm2 in cellular differentiation. Here we report a novel role for Mdm2 in the initiation of adipocyte differentiation that is independent of its ability to regulate p53. We show that Mdm2 is required for cAMP-mediated induction of CCAAT/enhancer-binding protein δ (C/EBPδ) expression by facilitating recruitment of the cAMP regulatory element-binding protein (CREB) coactivator, CREB-regulated transcription coactivator (Crtc2)/TORC2, to the c/ebpδ promoter. Our findings reveal an unexpected role for Mdm2 in the regulation of CREB-dependent transactivation during the initiation of adipogenesis. As Mdm2 is able to promote adipogenesis in the myoblast cell line C2C12, it is conceivable that Mdm2 acts as a switch in cell fate determination.

Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways.

In an effort to clone novel tyrosine-phosphorylated substrates of the epidermal growth factor receptor, we have initiated an approach coupling affinity purification using anti-phosphotyrosine antibodies to mass spectrometry-based identification. Here, we report the identification of a signaling molecule containing a Src homology 3 domain as well as an immunoreceptor tyrosine-based activation motif (ITAM). This molecule is 55% identical to a previously isolated molecule designated signal transducing adaptor molecule (STAM) that was identified as an interleukin (IL)-2-induced phosphoprotein and is therefore designated STAM2. Tyrosine phosphorylation of STAM2 is induced by growth factors such as epidermal growth factor and platelet-derived growth factor as well as by cytokines like IL-3. Several of the deletion mutants tested except the one containing only the amino-terminal region underwent tyrosine phosphorylation upon growth factor stimulation, implying that STAM2 is phosphorylated on several tyrosine residues. STAM2 is downstream of the Jak family of kinases since coexpression of STAM2 with Jak1 or Jak2 but not an unrelated Tec family kinase, Etk, resulted in its tyrosine phosphorylation. In contrast to epidermal growth factor receptor-induced phosphorylation, this required the ITAM domain since mutants lacking this region did not undergo tyrosine phosphorylation. Finally, overexpression of wild type STAM2 led to an increase in IL-2-mediated induction of c-Myc promoter activation indicating that it potentiates cytokine receptor signaling.

Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors.

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

Hydrolysis of short-chain phosphatidylcholines by bee venom phospholipase A2.

In order to find out the aggregation state of the substrate, preferred by bee venom phospholipase A2 (EC 3.1.1.4), its action on short-chain phosphatidylcholines with two identical (C6-C10) fatty acids has been tested. The rate of hydrolysis as a function of acyl chain length showed a maximum at dioctanoylphosphatidylcholine. The effects of alcohols, NaCl and Triton X-100, which affect the aggregation state of phospholipids in water, were also studied. The addition of n-alcohol led to a significant inhibition of the hydrolysis of the substrates present in micellar form and activated the hydrolysis of substrates which form liposomes. The inhibitory effect increased with increasing length of the aliphatic carbon chain of the alcohol. Triton X-100 at low Triton/phospholipid molar ratios enhanced enzyme activity. These results do not agree with the accepted idea that bee venom phospholipase A2 hydrolyzes short-chain lecithins in their molecularly dispersed form and that micelles cannot act as substrates. The data indicate that short-chain lecithins in the aggregated state are hydrolyzed and that the requirements of bee venom phospholipase A2 for the aggregation state of the substrate are not strict.

Kinetic study of the effect of heparin on the amidase activity of trypsin, plasmin and urokinase.

It was found that the effect of heparin on the amidase activity of urokinase (E C 3.4.21.31), plasmin (E C 3.4.21.7) and trypsin (E C 3.4.21.4) depended on the substrate used. No effect of heparin on the amidase activity of urokinase and trypsin was observed when Pyro Glu-Gly-Arg-p-nitroanilide (S-2444) and alpha-N-acetyl-L-lysine-p-nitroanilide (ALNA) were used as substrates. Heparin acted as a uncompetitive inhibitor of trypsin (Ki = 1.2 X 10(-6) M), plasmin (Ki = 4.9 X 10(-6) M) and urokinase (Ki = 1.0 X 10(-7) M) when Bz-Phe-Val-Arg-p-nitroanilide (S-2160), H-D-Val-Leu-Lys-p-nitroanilide (S-2251) and plasminogen, respectively, were used as substrates. These results, as well as the data obtained by studying the effect of the simultaneous presence of heparin and competitive inhibitors suggest that although heparin is not bound at the active center of these enzymes, it may influence the effectivity of catalysis.

Inhibitory effect of metal ions on alkaline mesentericopeptidase.

The effect of AG+, Cu2+, Cd2+, Co2+ and Ni2+ on the activity of alkaline mesentericopeptidase (EC 3.4.21.-) has been studied. Ag+, Cu2+ and Cd2+ were found to be reversible non-competitive inhibitors of the enzyme. The pH-dependence of Ki for Ag+-inhibition is sigmoidal with a pKa near 6. The Kilim values, calculated for the pH-independent region of the metal-enzyme inhibition, are close to the corresponding dissociation constants of metal-imidazole complexes, thus implying that the inhibitory effect of metal ions on enzyme activity is due to complex formation with the imidazole group of the active site histidine. The method of the two-component inhibition showed that Cu2+ and Ag+ bind to the same ligand of the enzyme molecule. The addition of Cu2+ decreases the rate of deacylation of the hydrolysis of p-nitrophenyl valerate, catalyzed by alkaline mesentericopeptidase in contrast to alpha-chymotrypsin where the acylation step is affected.

Hydrophobic interactions in the urokinase active centre. Inhibitory action of alkyl ammonium and amidinium ions: comparison with trypsin.

In order to obtain information concerning the binding site of urokinase (EC 3.4.99.26) the inhibitory action of aliphatic ammonium and amidinium ions has been studied. The comparison between the corresponding K1 values for urokinase and trypsin shows that although the two enzymes possess a hydrophobic binding pocket of presumably equal length, they differ mainly in their behaviour towards n-alkylammonium ions with an alkyl chain longer than four methylene groups and towards benzylammonium and amidinium ions. This is an indication of a less rigid binding site in urokinase than in trypsin.

A study of the alkaline mesentericopeptidase active site by means of peptide chloromethyl ketones.

The kinetics of inactivation of alkaline mesentericopeptidase was studied using chloromethyl ketone derivatives of amino acids and peptides. It was shown that Tos-LysCH2Cl and Tos-PheCH2Cl did not influence the enzyme activity, while the inhibitory effect of Cbz-Ala-Gly-PheCH2Cl was 35 times that of Cbz-Ala-PheCH2Cl. The dependence of the pseudo-first order rate constant of the enzyme inactivation by Cbz-Ala-Gly-PheCH2Cl on pH and temperature indicated that a group with a pK of 6.59 and deltaHi of 7.7 kcal/mol was the site of the inhibitor's attack. Amino acid analysis of the modified totally inactive enzyme revealed a definite loss of histidine and after performic acid oxidation a recovery of 3-carboxymethyl histidine. The whole set of experimental data is convincing evidence on behalf of a selective alkylation of the N-3 of the active site histidine after treatment with Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl. Alkaline mesenteriocopeptidase possesses an extended active site and only a peptide chloromethyl ketone, covering a determined sequence of theenzyme molecule (S3, S2, S1, S'1, S'2, S'3 ...) is able to provide effective inhibition. The values of the inactivation constant (kinact) for Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl are close to the corresponding values reported for subtilisin Amylosacchariticus.

Kinetic studies of the alkaline mesentericopeptidase. Study on the active centre topography of alkaline mesentericopeptidase by means of bifunctional reversible inhibition.

The inhibitory effect of alkylboronic acids H(CH2)nB(OH)2(n=2-8) and Ph(CH2)n-B(OH)2, (n=0-4), on the alkaline mesentericopeptidase-catalysed hydrolysis of synthetic substrates was studied. It was shown that alkylboronic acids act as bifunctional reversible inhibitors. The borate group interacts with an ionogenic group of the enzyme with a pKa of about 6.9-7.0. The latter is probably the catalytically active imidazole of the active centre. The hydrocarbon part of the molecule also takes part in the formation of the enzyme-inhibitor complex. The dependence of the degree of the enzyme-inhibitor complex formation upon the length of the side-chain of the inhibitor indicates the presence of two binding sites on the enzyme molecule.