NADPH oxidase 2 (NOX2) enzyme activation in patients with chronic inflammatory demyelinating polyneuropathy.

BACKGROUND AND PURPOSE: Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired immunomediated condition affecting the peripheral nervous system where probably macrophages are the primary effector cells for demyelination. Reactive oxygen species (ROS), catalyzed by the NOX family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, can induce peroxidation and are potentially injurious to myelin. Our aim was to assess the activity of NOX2, an isoform of NOX, in a series of CIDP patients and to analyze the effect of intravenous immunoglobulin (IVIg) on NOX2. METHODS: Thirty CIDP patients treated with IVIg and 30 control subjects were enrolled. To evaluate NOX2 activity, neutrophil and monocyte oxidative burst was measured directly in fresh whole blood using the Phagoburst™ assay, a fluorescence-activated cell sorting method. The mean fluorescence intensity, emitted in response to different stimuli, leads to the production of ROS and corresponds to the percentage of oxidizing cells and their enzymatic activity. RESULTS: Mean fluorescence intensity values for granulocyte and monocyte burst in patients (mean 633.3, SD 191; mean 111.8, SD 28.5) were different from those measured in healthy controls (granulocytes, mean 436.6, SD 137.0, P = 0.0003; monocytes, mean 78.2, SD 17.3, P = 0.000001). Moreover, IVIg administration increased both granulocyte (P = 0.005) and monocyte (P = 0.0009) burst. CONCLUSION: Our findings demonstrate that oxidative burst is significantly increased in CIDP patients and that treatment with IVIg enhances oxidative values, thus representing a possible IVIg therapeutic effect linked to a regulatory effect of ROS. Based on this, the development of treatments targeting the specific activation of NOX may be beneficial in autoimmune disorders.

Injured cardiomyocytes promote dental pulp mesenchymal stem cell homing.

BACKGROUND: The heart is unable to regenerate its tissues after severe injuries. Stem cell therapy appears to be one of the most promising approaches, though preclinical results are hitherto contradictory and clinical trials scanty and/or limited to phase-I. The limited knowledge about stem cell early homing in infarcted cardiac tissues can concur to this scenario. METHODS: The stem cell migration was assessed in in-vitro and ex-vivo models of heart ischemia, employing a rat dental pulp stem cell line (MUR-1) that shares the same ontogenic progenitors with portions of the heart, expresses markers typical of cardiac/vascular-like progenitors and is able to differentiate into cardiomyocytes in-vitro. RESULTS: Here, we demonstrated that the MUR-1 can reach the injured cells/tissue and make contacts with the damaged cardiomyocytes, likely through Connexin 43, N-cadherin and von Willebrand Factor mediated cell-cell interactions, both in in-vitro and ex-vivo models. Furthermore, we found that SDF-1, FGF-2 and HGF, but not VEGF are involved as chemotactic factors in MUR-1 migration, notifying a similarity with neural crest cell behavior during the organogenesis of both the splanchnocranium and the heart. CONCLUSIONS: Herein we found a similarity between what happens during the heart organogenesis and the early migration and homing of MUR-1 cells in ischemic models. GENERAL SIGNIFICANCE: The comprehension of molecular aspects underlying the early phases of stem cell migration and interaction with damaged organ contributes to the future achievement of the coveted stem cell-mediated organ regeneration and function preservation in-vivo.

Biodiversity of Geodermatophilaceae isolated from altered stones and monuments in the Mediterranean basin.

An investigation was made into the occurrence and biodiversity of Geodermatophilaceae on 78 samples of altered stone surfaces from 24 monuments and natural stones in the Mediterranean basin; it was found that the total microbial counts ranged between 0 and 10(7) cfu g(-1) dry weight. Members of the Geodermatophilaceae family were isolated from 22 of the 78 samples examined, with the incidence of Geodermatophilaceae colonies in the cultivable population ranging from 1% to 100%. The highest percentage was found in six samples of markedly deteriorated stone. Sixty-five strains randomly isolated from the plates were clustered in six different groups by amplified 16S rDNA restriction analysis (ARDRA) using five different restriction enzymes. Twenty-five strains, representing all the ARDRA haplotypes, were characterized further by partial sequencing (350-550 bp) of the 16S rDNA and by analysing 76 morphological, metabolic and physiological properties. The strains were associated with three well-separated clusters of the genera Geodermatophilus, Blastococcus and Modestobacter. On the basis of 16S rDNA sequence and ARDRA analysis, only two strains were found to be related to the two reference strains of Geodermatophilus. All the others could be grouped with Blastococcus aggregatus (19 strains) or the Antarctic species Modestobacter multiseptatus (44 strains), suggesting that it is these two groups, rather than Geodermatophilus, that tend to colonize the stone surfaces, and that Modestobacter-like strains are also found in temperate/Mediterranean climates. From the BOX-polymerase chain reaction (PCR) data, it can be seen that the Modestobacter-like strains, belonging to the most represented ARDRA haplotype (haplotype B, 34 strains), are very polymorphic and that, over a stone surface, there is a wide genetic diversity at the microsite level.

Analysis of allosteric effector binding sites of potato ADP-glucose pyrophosphorylase through reverse genetics.

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme of bacterial glycogen and plant starch synthesis as it controls carbon flux via its allosteric regulatory behavior. Unlike the bacterial enzyme that is composed of a single subunit type, the plant AGPase is a heterotetrameric enzyme (alpha2beta2) with distinct roles for each subunit type. The large subunit (LS) is involved mainly in allosteric regulation through its interaction with the catalytic small subunit (SS). The LS modulates the catalytic activity of the SS by increasing the allosteric regulatory response of the hetero-oligomeric enzyme. To identify regions of the LS involved in binding of effector molecules, a reverse genetics approach was employed. A potato (Solanum tuberosum L.) AGPase LS down-regulatory mutant (E38A) was subjected to random mutagenesis using error-prone polymerase chain reaction and screened for the capacity to form an enzyme capable of restoring glycogen production in glgC(-) Escherichia coli. Dominant mutations were identified by their capacity to restore glycogen production when the LS containing only the second site mutations was co-expressed with the wild-type SS. Sequence analysis showed that most of the mutations were decidedly nonrandom and were clustered at conserved N- and C-terminal regions. Kinetic analysis of the dominant mutant enzymes indicated that the K(m) values for cofactor and substrates were comparable with the wild-type AGPase, whereas the affinities for activator and inhibitor were altered appreciably. These AGPase variants displayed increased resistance to P(i) inhibition and/or greater sensitivity toward 3-phosphoglyceric acid activation. Further studies of Lys-197, Pro-261, and Lys-420, residues conserved in AGPase sequences, by site-directed mutagenesis suggested that the effectors 3-phosphoglyceric acid and P(i) interact at two closely located binding sites.

Investigation of subunit function in ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase (AGPase), a key regulatory enzyme in higher plant starch biosynthesis, is composed of a pair of large and small subunits (alpha(2)beta(2)). Current evidence suggests that the large subunit has primarily a regulatory function, while the small subunit has both regulatory and catalytic roles. To define the structure-function relationship of the large subunit (LS), the LS of potato AGPase was subjected to chemical mutagenesis and coexpressed with the wild-type (WT) small subunit (SS) cDNA in an AGPase defective Escherichia coli strain. An LS mutant (M143) was isolated, which accumulated very low levels of glycogen compared to the WT recombinant AGPase, but maintained normal catalytic activity when assayed under saturating conditions. Sequence analysis revealed that M143 has a single amino acid change, V463I, which lies adjacent to the C-terminus. This single mutation had no effect on the Km for ATP and Mg(2+), which were similar to the WT enzyme. The K(m) for glucose 1-P, however, was sixfold higher than the WT enzyme. These results suggest that the LS plays a role in binding glucose 1-P through its interaction with the SS.

Isolation and characterization of a higher plant ADP-glucose pyrophosphorylase small subunit homotetramer.

ADP-glucose pyrophosphorylase (AGPase) is the allosterically regulated gateway for carbon entry into transient and storage starch in plants as well as glycogen in bacteria. This enzyme plays a key role in the modulation of photosynthetic efficiency in source tissues and directly determines the level of storage starch in sink tissues, thus influencing overall crop yield potential. AGPase is a tetrameric enzyme; in higher plants it consists of two regulatory large subunits (LS) and two catalytic small subunits (SS), while in cyanobacteria and prokaryotes the enzyme is homotetrameric. The potato SS gene in pML10 was mutated by hydroxylamine and mutants were screened for elevated homotetrameric activity by iodine vapor staining. This search strategy led to the isolation of SS mutants (SUP-1, TG-15) that had pyrophosphorylase activity in the absence of the LS. TG-15 has a leucine to phenylalanine change at position 48 (L(48)F) that corresponds to a phenylalanine residue at the analogous position in the Escherichia coli homotetrameric AGPase as well as a valine to isoleucine change at position 59 (V(59)I). TG-15 was partially purified and kinetic analysis revealed substrate and effector affinities equal to wild type heterotetrameric enzyme with the exception of ATP binding.

Marmoricola aurantiacus gen. nov., sp. nov., a coccoid member of the family Nocardioidaceae isolated from a marble statue.

A Gram-positive, aerobic bacterium with coccoid cells occurring singly, in pairs and in clusters was isolated from the surface of a marble statue. The peptidoglycan contain LL-diaminopimelic acid as diagnostic diamino acid and a single glycine residue as interpeptide bridge (type A3 gamma). The major menaquinone is MK-8(H4). The cellular fatty acid pattern consists of straight chain saturated and monounsaturated components and 10-methyl octadecanoic (tuberculostearic) acid as the only branched chain fatty acid. Phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol occur as characteristic polar lipids. The DNA G + C composition is 72 mol%. According to its phylogenetic position and 16S rDNA signature nucleotides, the organism is a member of the family Nocardioidaceae. The combination of chemotaxonomic characteristics is unique within this family and supports the description of a new genus and new species, Marmoricola aurantiacus. The type strain is strain BC 361T (= DSM 12652T).

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens.

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50,900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42 degrees C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 micrograms/ml) and not inhibited by antipain dihydrochloride (120 micrograms/ml), aprotinin (4 micrograms/ml), bestatin (80 micrograms/ml), chymostatin (50 micrograms/ml), E-64 (20 micrograms/ml), leupeptin (4 micrograms/ml), Pefabloc SC (2000 micrograms/ml), pepstatin (4 micrograms/ml), phosphoramidon (660 micrograms/ml), or phenylmethylsulfonyl fluoride (400 micrograms/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.