Purification, characterisation and reconstitution of glutaconyl-CoA decarboxylase, a biotin-dependent sodium pump from anaerobic bacteria

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X-100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate-binding site are located on the same side of the membrane. The decarboxylase was solubilized with Triton X-100 and purified by affinity chromatography on monomeric avidin-Sepharose. The enzyme is composed of three types of polypeptides: the group of alpha chains (Mr 120000-140000) containing the biotin, the beta chain (60000) and an apparently hydrophobic gamma chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na+ channel. The beta and gamma chains but not the alpha chain could be labelled by N-ethyl-[14C]maleimide. Similar decarboxylases but with much smaller biotin peptides (Mr 15000-20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum. The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na+ uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K+ indicating an electrogenic Na+ pump. The coupling between Na+ transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2. The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl-CoA decarboxylase are able to ferment glutamate and require 10 mM Na+ for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.     

Phosphorylation of the Pseudomonas aeruginosa response regulator AlgR is essential for type IV fimbria-mediated twitching motility

The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P. aeruginosa.     

Preparative Syntheses of Guanylate-Rich Oligonucleotides Using the Phosphotriester Method in Solution

Abstract

The guanylate-rich fragments: 150 mg d(G₄T₄), 180 mg d(T₄G₄), 350 mg d(T₄G₄T₄), 30 mg d(G₄T₄G₄), 85 mg d(T₄G₄T₄G₄)were synthesized using the triester method. By enzymatic ligation of aliquots the 36mer d(G₄T₄G₄T₄G₄T₄G₄T₄G₄) is obtained.     

Invasion by activated macrophages requires delivery of nascent membrane‐type‐1 matrix metalloproteinase through late endosomes/lysosomes to the cell surface

Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane‐type‐1 matrix metalloproteinase (MT1‐MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1‐MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1‐MMP. Upon lipopolysaccharide (LPS) activation, MT1‐MMP synthesis dramatically increases 10‐fold at the surface by 15 hours. MT1‐MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R‐SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q‐SNARE complex Stx4/SNAP23 to regulate MT1‐MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1‐MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1‐MMP transport through this pathway could reduce macrophage migration and the resulting inflammation.     

Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell-cycle phase-dependent degradation in yeast

Using co-immunoprecipitation combined with MS analysis, we identified the alpha' subunit of casein kinase 2 (CK2) as an interaction partner of the atypical Rio1 protein kinase in yeast. Co-purification of Rio1p with CK2 from Deltacka1 or Deltacka2 mutant extracts shows that Rio1p preferentially interacts with Cka2p in vitro. The C-terminal domain of Rio1p is essential and sufficient for this interaction. Six C-terminally located clustered serines were identified as the only CK2 sites present in Rio1p. Replacement of all six serine residues by aspartate, mimicking constitutive phosphorylation, stimulates Rio1p kinase activity about twofold in vitro compared with wild-type or the corresponding (S > A)(6) mutant proteins. Both mutant alleles (S > A)(6) or (S > D)(6) complement in vivo, however, growth of the RIO1 (S > A)(6) mutant is greatly retarded and shows a cell-cycle phenotype, whereas the behaviour of the RIO1 (S > D)(6) mutant is indistinguishable from wild-type. This suggests that phosphorylation by protein kinase CK2 leads to moderate activation of Rio1p in vivo and promotes cell proliferation. Physiological studies indicate that phosphorylation by CK2 renders the Rio1 protein kinase susceptible to proteolytic degradation at the G(1)/S transition in the cell-division cycle, whereas the non-phosphorylated version is resistant.     

Adult index patient with Currarino syndrome due to a novel HLXB9 mutation, c.336dupG (p.P113fsX224), presenting with Hirschsprung's disease, cephalgia, and lumbodynia

BACKGROUND: The symptom triad of autosomal dominant Currarino syndrome (CS; MIM #176450) consists of anorectal malformation, a sacral bone defect, and presacral masses. Mutations in the homeoboxHLXB9 gene have already been described in a subset of sacrococcygeal anomalies characterized by partial sacral agenesis. CASE: We report a 28-year-old male patient with Currarino syndrome due to a heterozygous novel frame-shift mutation c.336dupG (p.P113fsX224) in the homeoboxHLXB9 gene. CONCLUSIONS: Molecular diagnostics may be helpful in cases of Hirschsprung's disease accompanied by other symptoms suggestive for Currarino syndrome, since it can lead to major complications such as perianal sepsis, meningitis, and malignant transformation.     

Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine particles

In ambient aerosols, ultrafine particles (UFP) and their agglomerates are considered to be major factors contributing to adverse health effects. Reactivity of agglomerated UFP of elemental carbon (EC), Printex 90, Printex G, and diesel exhaust particles (DEP) was evaluated by the capacity of particles to oxidize methionine in a cell-free in vitro system for determination of their innate oxidative potential and by alveolar macrophages (AMs) to determine production of arachidonic acid (AA), including formation of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), reactive oxygen species (ROS), and oxidative stress marker 8-isoprostane. EC exhibiting high oxidative potential induced generation of AA, PGE2, LTB4, and 8-isoprostane in canine and human AMs. Printex 90, Printex G, and DEP, showing low oxidative capacity, still induced formation of AA and PGE2, but not that of LTB4 or 8-isoprostane. Aging of EC lowered oxidative potential while still inducing production of AA and PGE2 but not that of LTB4 and 8-isoprostane. Cellular ROS production was stimulated by all particles independent of oxidative potential. Particle-induced formation of AA metabolites and ROS was dependent on mitogen-activated protein kinase kinase 1 activation of cytosolic phospholipase A2 (cPLA2) as shown by inhibitor studies. In conclusion, cPLA2, PGE2, and ROS formation was activated by all particle types, whereas LTB4 production and 8-isoprostane were strongly dependent on particles' oxidative potential. Physical and chemical parameters of particle surface correlated with oxidative potential and stimulation of AM PGE2 and 8-isoprostane production.     

Synthesis, characterization, and biological properties of cyanine-labeled somatostatin analogues as receptor-targeted fluorescent probes

We present the synthesis and characterization of the somatostatin receptor-specific peptide H(2)N-(D-Phe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, which is labeled with a carboxylated indodicarbo- and an indotricarbocyanine dye at the N-terminal amino group. The preparation was performed by automated solid-phase synthesis, with subsequent attachment of the cyanine dye and cleavage of the entire conjugate from the resin. The compounds display high molar absorbance and fluorescence quantum yields typical for cyanine dyes and are thus suitable receptor-targeted contrast agents for molecular optical imaging. The ability of these agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, in which the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. In contrast, the corresponding linearized derivative of the sequence H(2)N-(D-Phe)-Met-Phe-(D-Trp)-Lys-Thr-Met-Thr-OH produced only minimal cell fluorescence, hence confirming the specificity of the cyclic somatostatin analogue. Intracellular localization could be visualized by near-infrared (NIR) fluorescence microscopy. In conclusion, receptor-specific peptides are promising tools for designing site-directed optical contrast agents for use in molecular optical imaging.