Remarkable stabilization of neutrophil NADPH oxidase using RacQ61L and a p67phox-p47phox fusion protein

Activation of the phagocyte NADPH oxidase occurs via assembly of cytosolic p47(phox), p67(phox), and Rac with the membrane-bound flavocytochrome b(558). Recently, we have found that p67(phox)-(1-210) (p67N) fused with p47(phox)-(1-286) (p47N) or with Rac efficiently stabilizes the oxidase in a cell-free reconstitution system. In an attempt to further stabilize the oxidase, we herein used a constitutively active Rac, RacQ61L, and examined its effect on the oxidase stability. The half-life (t(1/2)) of the activity reconstituted with wild-type Rac was 12 min at 37 degrees C, which was extended 6-fold by RacQ61L. Also, the stability of the oxidase without p47(phox) increased 8-fold using RacQ61L. RacQ61L had a higher affinity for the complex than wild-type Rac and increased the affinity of p67N for the complex. Far-western blotting showed an enhanced binding between RacQ61L and p67N. The oxidase was stabilized by nanomolar FAD, and RacQ61L lowered the FAD concentration required. The combination of RacQ61L and a fusion protein consisting of p67N and p47N produced an extremely stable enzyme (t(1/2) = 184 min at 37 degrees C). The effectiveness of RacQ61L and fusion proteins on stabilization was in the following order: p67N-Rac < p67N + RacQ61L < or = p67N-RacQ61L < p67N-p47N + RacQ61L. These results indicate that a tightly bound ternary complex of p67(phox), Rac, and p47(phox) is very effective in maintaining the oxidase and confirm that the longevity of the activated state requires continuous association of these components. This simple and efficient method of stabilization may provide a useful tool to elucidate the nature of the activated oxidase.     

Distribution of rat C5a anaphylatoxin receptor

The anaphylatoxin, complement 5a (C5a), plays a key role in mediating various inflammatory reactions following complement activation. Several investigators have reported that C5a receptor (C5aR) is expressed in non-myeloid cells under certain conditions or in different cell lines. In our study, the abundance of C5aR-positive myeloid cells in rats depended on the organs examined. C5aR was usually expressed at the site of exposure to pathogens, such as in salivary gland or lung, and was up-regulated in liver in the inflammatory state induced by lipopolysaccharide (LPS) administration. Furthermore, the increased expression of C5aR antigen was not accompanied by an increase in C5aR mRNA in Kupffer cells following LPS challenge.     

Molecular characteristics of tissue-bound semicarbazide-sensitive amine oxidase (SSAO) in guinea pig tissues

Various mammalian tissues contain a tissue-bound amine oxidizing enzyme distinct from mitochondrial outer membrane enzyme, monoamine oxidase (MAO, EC 1.4.3.4), termed semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6). An increase in SSAO activity was found in patients suffering from vascular disorders such as diabetes and diabetic complications. It has previously been shown that 2-bromoethylamine (2-BEA) is a potent, and selective suicidal inhibitor of tissue-bound SSAO. The aim of this study was to investigate the interaction of this suicidal SSAO inhibitor with the tissue-bound enzyme in guinea pig lung, kidney, stomach, and heart homogenates. The conditions necessary for this inhibitor to titrate the concentrations of this enzyme were also determined. 2-BEA appears to interact with SSAO, as reported previously for this enzyme from different sources, in a manner consistent with an irreversible, "suicide" reaction. Because of this property, 2-BEA could be used to titrate the concentrations of SSAO active centers in these tissues under the appropriate conditions employed. Although some possible non-specific binding of the inhibitor to sites other than the active center of the enzyme, metabolism of this inhibitor and/or presence of enzyme subtypes was hypothesized, the molecular characteristics of SSAO in these tissues (Km, Vmax values, enzyme efficiencies, approximate enzyme concentrations, and molecular turnover numbers) towards the substrate kynuramine (0.1 mM) at pH 7.4 and 37 degrees C have been estimated.     

Agonist-promoted heteromeric oligomerization between adenosine A(1) and P2Y(1) receptors in living cells

We have explored the process of oligomerization of G protein-coupled purinergic receptors, adenosine A(1) receptor (A(1)R) and P2Y(1) receptor (P2Y(1)R), in intact HEK293T cells by means of modified bioluminescence resonance energy transfer technology (BRET(2)) that offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared to traditional BRET. This approach identified both constitutive and agonist-promoted heteromeric oligomerization between Myc-tagged P2Y(1)R fused to a donor, Renilla luciferase (Myc-P2Y(1)R-Rluc) and HA-tagged A(1)R fused to an acceptor, a different form of green fluorescent protein (HA-A(1)R-GFP(2)). The BRET(2) signal increased in a time-dependent manner in the cells expressing HA-A(1)R-GFP(2)/Myc-P2Y(1)R-Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y(1)R antagonist MRS2179. A high degree of HA-A(1)R-GFP(2) and Myc-P2Y(1)R-Rluc co-localization in the co-transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A(1)R and P2Y(1)R can form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous activation of both receptors.     

Typings of Campylobacter jejuni isolated from patients of 2 outbreak cases by genotypic and phenotypic methods

We compared Campylobacter jejuni strains isolated from the patient stools associated with two food-borne diarrheal outbreak cases by the serotypic methods (Lior and Penner systems) and the genotypic methods (restriction fragment length polymorphism (RFLP) of flaA gene and pulsed-field gel electrophoresis (PFGE)). Fla-RFLP was based on the digestion of 410 bp DNA fragment by MboI restriction enzyme amplified from a 5' portion of C. jejuni flaA gene. Six distinctive fla-RFLP patterns were identified by examining 29 serotype reference strains and 58 strains isolated from the patients infected with C. jejuni independently. In the first outbreak case, 4 isolates were shown to be the same patterns each other by the fla-RFLP and PFGE, and by the Lior serotyping, except the Penner system that serotyped into 2 distinct types. On the other hand, in the second case, out of 10 isolates, 5 isolates were identical by the both genotypic and the both serotypic methods, and 4 isolates were not differentiated by the fla-RFLP and Penner system, but were separated into 4 types by PFGE in a little difference. The rest isolate was completely different from the other isolates by the all of methods used now. The findings suggest that the second case occurred by the infection of at least 3 different strains of C. jejuni.     

Preferential detection of pro-carboxypeptidase R by enzyme-linked immunosorbent assay

We generated two monoclonal antibodies (mAbs), 2A16 and 10G1, against pro-carboxypeptidase R (proCPR), also known as thrombin activatable fibrinolysis inhibitor (TAFI). By use of these mAbs, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) system to detect proCPR. Since the amount of the antigen detectable by the ELISA was essentially the same in fresh plasma and serum incubated at 37 C for 1 hr, we concluded that the ELISA system detected not only proCPR, but also inactivated CPR generated from proCPR. However, an appreciable amount of proCPR remained unactivated in serum. For extensive activation of proCPR in plasma, thrombin and thrombomodulin complexes (TTM) can be used together with CaCl2. Following extensive conversion of proCPR to CPR by T-TM and CaCl2, converting plasma to serum (T-TM serum), antigenicity became undetectable by ELISA. Further analysis revealed that 2A16 reacts only with proCPR although 10G1 reacts with proCPR, active CPR and inactivated CPR. Therefore, we concluded that the ELISA system preferentially detects proCPR and not CPR. Our sandwich ELISA system utilizing 2A16 and 10G1 provides a suitable method for detecting proCPR and can be used to determine levels of proCPR in plasma samples from patients.     

Induction of Neurite Outgrowth in PC12 Cells Treated with Temperature-Controlled Repeated Thermal Stimulation

To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine.     

Characterization of a Novel Plasmid,pMAH135, from Mycobacterium avium Subsp. hominissuis

Mycobacterium avium complex (MAC) causes mainly two types of disease. The first is disseminated disease in immunocompromised hosts, such as individuals infected by human immunodeficiency virus (HIV). The second is pulmonary disease in individuals without systemic immunosuppression, and the incidence of this type is increasing worldwide. M. avium subsp. hominissuis, a component of MAC, causes infection in pigs as well as in humans. Many aspects of the different modes of M. avium infection and its host specificity remain unclear. Here, we report the characteristics and complete sequence of a novel plasmid, designated pMAH135, derived from M. avium strain TH135 in an HIV-negative patient with pulmonary MAC disease. The pMAH135 plasmid consists of 194,711 nucleotides with an average G + C content of 66.5% and encodes 164 coding sequences (CDSs). This plasmid was unique in terms of its homology to other mycobacterial plasmids. Interestingly, it contains CDSs with sequence homology to mycobactin biosynthesis proteins and type VII secretion system-related proteins, which are involved in the pathogenicity of mycobacteria. It also contains putative conserved domains of the multidrug efflux transporter. Screening of isolates from humans and pigs for genes located on pMAH135 revealed that the detection rate of these genes was higher in clinical isolates from pulmonary MAC disease patients than in those from HIV-positive patients, whereas the genes were almost entirely absent in isolates from pigs. Moreover, variable number tandem repeats typing analysis showed that isolates carrying pMAH135 genes are grouped in a specific cluster. Collectively, the pMAH135 plasmid contains genes associated with M. avium’s pathogenicity and resistance to antimicrobial agents. The results of this study suggest that pMAH135 influence not only the pathological manifestations of MAC disease, but also the host specificity of MAC infection.