Production and characterization of recombinant P1 adhesin essential for adhesion,gliding, and antigenic variation in the human pathogenic bacterium,Mycoplasma pneumoniae

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.     

Neuronal nitric oxide synthase (NNOS) catalyzes one-electron reduction of 2,4,6-trinitrotoluene, resulting in decreased nitric oxide production and increased nNOS gene expression: implication for oxidative stress

To determine the mechanism of 2,4,6-trinitrotoluene (TNT)-induced oxidative stress involving neuronal nitric oxide synthase (nNOS), we examined alterations in enzyme activity and gene expression of nNOS by TNT, with an enzyme preparation and rat cerebellum primary neuronal cells. TNT inhibited nitric oxide formation (IC(50) = 12.4 microM) as evaluated by citrulline formation in a 20,000 g cerebellar supernatant preparation. A kinetic study revealed that TNT was a competitive inhibitor with respect to NADPH and a noncompetitive inhibitor with respect to L-arginine. It was found that purified nNOS was capable of reducing TNT, with a specific activity of 3900 nmol of NADPH oxidized/mg/min, but this reaction required CaCl(2)/calmodulin (CaM). An electron spin resonance (ESR) study indicated that superoxide (O(2)(.-)) was generated during reduction of TNT by nNOS. Exposure of rat cerebellum primary neuronal cells to TNT (25 microM) caused an intracellular generation of H(2)O(2), accompanied by a significant increase in nNOS mRNA levels. These results indicate that CaM-dependent one-electron reduction of TNT is catalyzed by nNOS, leading to a reduction in NO formation and generation of H(2)O(2) derived from O(2)(.-). Thus, it is suggested that upregulation of nNOS may represent an acute adaptation to an increase in oxidative stress during exposure to TNT.     

Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.     

Sulfite induces adherence of polymorphonuclear neutrophils to immobilized fibrinogen through activation of Mac-1 beta2-integrin (CD11b/CD18)

Sulfite is a major air pollutant which can cause respiratory tract inflammation characterized by an influx of polymorphonuclear neutrophils (PMN). We have previously shown that human PMN can produce sulfite either spontaneously or in response to stimulation with lipopolysaccharide. We now demonstrate that sulfite activates PMN to adhere to immobilized fibrinogen via the beta2-integrin Mac-1 (CD11b/CD18). Mac-1 expression is not altered by treatment with this agent. Although unaffected by pertussis toxin, sulfite-triggered PMN adhesion was abrogated by pretreating cells with the membrane-impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a modifier of thiol groups on the cell surface. These results suggest that sulfite-induced PMN adhesion is dependent on a modification of thiols at the cell surface. Given its potent antioxidant and antimicrobial activities, sulfite may act as an endogenous mediator in host defense and/or inflammation.     

Activation of Pyk2/RAFTK induces tyrosine phosphorylation of alpha-synuclein via Src-family kinases

The hyperthermophilic archaeon Methanococcus jannaschii uses several non-canonical enzymes to catalyze conserved reactions in glycolysis and gluconeogenesis. A highly diverged gene from that organism has been proposed to function as a phosphoglycerate mutase. Like the canonical cofactor-independent phosphoglycerate mutase and other members of the binuclear metalloenzyme superfamily, this M. jannaschii protein has conserved nucleophilic serine and metal-binding residues. Yet the substrate-binding residues are not conserved. We show that the genes at M. jannaschii loci MJ0010 and MJ1612 encode thermostable enzymes with phosphoglycerate mutase activity. Phylogenetic analyses suggest that this gene family arose before the divergence of the archaeal lineage.     

Diethylstilbestrol attenuates antioxidant activities in testis from male mice

It has been reported that acute exposure to diethylstilbestrol (DES) induces apoptosis in the testis, and antioxidants play a role in preventing DES-induced tissue damage. In this study, the effect of chronic exposure to DES on the antioxidants was examined in the testis and liver. Eight-week old male ICR mice were treated subcutaneously with various doses of DES for 20 days. Morphologically apparent apoptotic changes, 4-hydroxy-2-nonenal-positive cells and TUNEL-positive DNA-fragmentation, were demonstrated in the testis, but were minimal in the liver. Activities of antioxidants such as glutathione (GSH) peroxidase and GSH S -transferase decreased in both the liver and testis. The activity of Mn-superoxide dismutase (SOD) decreased in the liver but increased in the testis. The activity of Cu, Zn-SOD decreased in the liver but was unchanged in the testis. On Western and Northern blots, gamma-glutamylcysteine synthetase ( γ-GCS), a rate limiting enzyme of GSH synthesis, was increased in the liver dependent on the dose of DES. However, the expression of γ-GCS was reduced in the testis. Since quinones, metabolites of DES, generate reactive oxygen species, which damage DNA, antioxidants are important to prevent the damage. The data suggest that antioxidant activities are impaired by DES, and the levels of GSH are related to DES-induced apoptosis in the testis.     

Compound-specific deltaD-delta13C analyses of n-alkanes extracted from terrestrial and aquatic plants

Stable hydrogen and carbon isotopic compositions of individual n-alkanes were determined for various terrestrial plants (33 samples including 27 species) and aquatic plants (six species) in natural environments from Japan and Thailand. In C3 plants, n-alkanes extracted from angiosperms have a deltaD value of -152+/-26 per thousand (relative to Standard Mean Ocean Water [SMOW]) and delta13C value of -36.1+/-2.7 per thousand (relative to Peedde Belemnite [PDB]), and those from gymnosperms have a deltaD value of -149+/-16 per thousand and delta13C value of -31.6+/-1.7 per thousand. Angiosperms have n-alkanes depleted in 13C relative to gymnosperms. n-Alkanes from C4 plants have a deltaD value of -171+/-12 per thousand and delta13C value of -20.5+/-2.1 per thousand, being a little depleted in D and much enriched in 13C compared to C3 plants. n-Alkanes of CAM plants are a little depleted in D and vary widely in delta13C relative to those of C3 and C4 plants. In aquatic plants, n-alkanes from freshwater plants have a deltaD value of -187+/-16 per thousand and delta13C value of -25.3+/-1.9 per thousand, and those from seaweeds have a deltaD value of -155+/-34 per thousand and delta13C value of -22.8+/-1.0 per thousand. All n-alkanes from various plant classes are more depleted in D and 13C relative to environmental water and bulk tissue, respectively. In addition, the hydrogen and carbon isotopic fractionations during n-alkane synthesis are distinctive for these various plant classes. While C3 plants have smaller isotopic fractionations in both D and 13C, seaweed has larger isotopic fractionations.     

A potential mechanism for the impairment of nitric oxide formation caused by prolonged oral exposure to arsenate in rabbits

We have recently found evidence for impairment of nitric oxide (NO) formation and induction of oxidative stress in residents of an endemic area of chronic arsenic poisoning in Inner Mongolia, China. To investigate the underlying mechanisms responsible for these phenomena, a subchronic animal experiment was conducted using male New Zealand White rabbits. After 18 weeks of continuous exposure of rabbits to 5 mg/l of arsenate in drinking water, a significant decrease in systemic NO production occurred, as shown by significantly reduced plasma NO metabolites levels (76% of control) and a tendency towards decreased serum cGMP levels (81.4% of control). On the other hand, increased oxidative stress, as shown by significantly increased urinary hydrogen peroxide (H(2)O(2)) (120% of control), was observed in arsenate-exposed rabbits. In additional experiments measuring aortic tension, the addition of either the calcium ionophore A23187 or acethylcholine (ACh) induced a transient vasoconstriction of aortic rings prepared from arsenate-exposed rabbits, but not in those prepared from control animals. This calcium-dependent contractility action observed in aorta rings from arsenate-exposed rabbits was markedly attenuated by the superoxide (O2(.-)) scavenging enzyme Cu, Zn-SOD, as well as diphenyleneiodonium (DPI) or N(G)-nitro-L-arginine methyl ester (L-NAME), which are inhibitors for nitric oxide synthase (NOS). However, the cyclooxygenase inhibitor indomethacin or the xanthine oxidase blocker allopurinol had no effect on this vasoconstriction. These results suggest that arsenate-mediated reduction of systemic NO may be associated with the enzymatic uncoupling reaction of NOS with a subsequent enhancement of reactive oxygen species such as O2(.-), an endothelium-derived vasoconstricting factor. Furthermore, hepatic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)), a cofactor for NOS, were markedly reduced in arsenate-exposed rabbits to 62% of control, while no significant change occurred in cardiac L-arginine levels. These results suggest that prolonged exposure of rabbits to oral arsenate may impair the bioavailability of BH(4) in endothelial cells and, as a consequence, disrupt the balance between NO and O2(.-) produced from endothelial NOS, such that enhanced free radicals are produced at the expense of NO.