S-nitrosylation-dependent inactivation of Akt/protein kinase B in insulin resistance

Inducible nitric-oxide synthase (iNOS) has been implicated in many human diseases including insulin resistance. However, how iNOS causes or exacerbates insulin resistance remains largely unknown. Protein S-nitrosylation is now recognized as a prototype of a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe the mechanism of inactivation of Akt/protein kinase B (PKB) in NO donor-treated cells and diabetic (db/db) mice. NO donors induced S-nitrosylation and inactivation of Akt/PKB in vitro and in intact cells. The inhibitory effects of NO donor were independent of phosphatidylinositol 3-kinase and cGMP. In contrast, the concomitant presence of oxidative stress accelerated S-nitrosylation and inactivation of Akt/PKB. In vitro denitrosylation with reducing agent reactivated recombinant and cellular Akt/PKB from NO donor-treated cells. Mutated Akt1/PKBalpha (C224S), in which cysteine 224 was substituted by serine, was resistant to NO donor-induced S-nitrosylation and inactivation, indicating that cysteine 224 is a major S-nitrosylation acceptor site. In addition, S-nitrosylation of Akt/PKB was increased in skeletal muscle of diabetic (db/db) mice compared with wild-type mice. These data suggest that S-nitrosylation-mediated inactivation may contribute to the pathogenesis of iNOS- and/or oxidative stress-involved insulin resistance.     

Intracellular cAMP controls a physical association of V-1 with CapZ in cultured mammalian endocrine cells

V-1, an ankyrin repeat protein with the activity to control tyrosine hydroxylase (TH) gene expression and transmitter release in PC12D cells, associates with CapZ, an actin capping protein, and thereby regulates actin polymerization in vitro. In this study, immunoprecipitation and Western blot analysis showed that V-1 was physically associated with CapZ-beta in PC12D transfectants overexpressing V-1. These proteins were co-localized in the soma of Purkinje cells of rat cerebellum as assayed by immunohistochemistry. Furthermore, in the V-1 transfectants, the amount of CapZ which physically associated with V-1 was steeply reduced at 2h after treatment with forskolin, but was thereafter increased to reach its initial level at 12h after forskolin-treatment. These results suggest that the association of V-1 with CapZ is controlled by a cAMP-dependent signalling pathway probably to play a functional role in the regulatory mechanism of actin dynamics in the endocrine system and the central nervous system.     

Measurement of the carbohydrate-binding specificity of lectins by a multiplexed bead-based flow cytometric assay

Carbohydrate binding underlies many cell recognition events. Here, we describe a multiplexed glyco-bead array method for determining the carbohydrate-binding specificities of plant lectins using a bead-based flow cytometric analysis. N-glycans including high mannose, hybrid, and complex types and O-glycans from glycoproteins were immobilized on multiplexed beads, and the specificities of 13 kinds of sugar chains were monitored within 2 h in a single reaction. This strategy is easy, rapid, reproducible, and suitable for small samples and allows the reliable and simultaneous elucidation of sugar-binding properties under identical conditions.     

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.     

Mammalian Mitochondrial DNA Replication Intermediates Are Essentially Duplex but Contain Extensive Tracts of RNA/DNA Hybrid

We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.     

Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2-and TLR4-independent signaling

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.     

Parkin potentiates ATP-induced currents due to activation of P2X receptors in PC12 cells

Loss-of-function mutations of the parkin gene causes an autosomal recessive juvenile-onset form of Parkinson's disease (AR-JP). Parkin was shown to function as a RING-type E3 ubiquitin protein ligase. However, the function of parkin in neuronal cells remains elusive. Here, we show that expression of parkin-potentiated adenosine triphosphate (ATP)-induced currents that result from activation of the P2X receptors which are widely distributed in the brain and involved in neurotransmission. ATP-induced inward currents were measured in mock-, wild-type or mutant (T415N)-parkin-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in wild-type parkin-transfected cells. However, the immunocytochemical study showed no apparent increase in the number of P2X receptors or in ubiquitin levels. The increased currents were attenuated by inhibition of cAMP-dependent protein kinase (PKA) but not protein kinase C (PKC) or Ca2+ and calmodulin-dependent protein kinase (CaMKII). ATP-induced currents were also regulated by phosphatases and cyclin-dependent protein kinase 5 (CDK5) via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32), though the phosphorylation at Thr-34 and Thr-75 were unchanged or rather attenuated. We also tried to investigate the effect of alpha-synuclein, a substrate of parkin and also forming Lysine 63-linked multiubiquitin chains. Expression of alpha-synuclein did not affect the amplitude of ATP-induced currents. Our finding provides the evidence for a relationship between parkin and a neurotransmitter receptor, suggesting that parkin may play an important role in synaptic activity.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

Isolation and characterization of novel denitrifying alkalithermophiles, AT-1 and AT-2

Two novel denitrifying alkalithermophilic bacteria, AT-1 and AT-2, were isolated from manure-amended soil. The isolates grew at 35–65°C with an optimum temperature at 50–60°C, and pH 6.5–10.0 with an optimum pH at 9.5. Both isolates were Gram-positive, facultative anaerobic, non-motile rod-shaped bacteria. A phylogenetic analysis based on 16S rRNA sequence data indicated that both AT-1 and AT-2 are members of the genus Anoxybacillus. DNA-DNA hybridization revealed moderate relatedness between AT-1 and AT-2 and one phylogenetically related strain, A. pushchinensis K1 (69.5 and 69.1%, respectively). Comparative analysis of morphology and biochemical characteristics of the two isolates also showed similarity to A. pushchinensis K1. Based on these results, we identified AT-1 and AT-2 as A. pushchinensis. To our knowledge, this is the first report of denitrifying bacterium isolated from alkalithermophilic Anoxybacillus spp.     

Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin

Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.