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Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Background: Although clinical restenosis within 1 year after percutaneous coronary intervention has been remarkably reduced with the advent of drug-eluting stents (DES), the late catch-up (LCU) phenomenon remains an issue despite medical advances. The aim of this study was to investigate the incidence and predictive factors of the LCU phenomenon in an unselected population treated with first-generation DES., Methods: A total of 923 patients treated with DES between June 2004 and August 2008 were analyzed. The LCU phenomenon was defined as secondary revascularization 1 year after index stenting. Retreatment for very late stent thrombosis was considered as part of the LCU phenomenon., Results: Incidence of the LCU phenomenon was seen in 33 patients (3.6%). Very late stent thrombosis was observed in 5 patients (0.6%) and very late in-stent restenosis was observed in 28 patients (3.0%). At the 12-month landmark analysis, the cumulative rate of cardiac death was significantly higher in patients with the LCU phenomenon than in those without any target lesion revascularization (9.0% vs. 0.9%, p<0.001). In the multivariate analysis, hemodialysis [odds ratio (OR) 6.07, p=0.003], number of stents (OR 1.58, p=0.02), and coronary bifurcation lesions (OR 2.06, p=0.048) were identified as independent predictors of the LCU phenomenon., Conclusion: The LCU phenomenon is associated with serious consequences and adverse events and remains an important issue in modern practice, despite medical advances. DES should be deployed with a minimum number of stents, and special consideration must be given to patients on hemodialysis and those with coronary bifurcation lesions., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Aims: Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression-secretion system., Methods and Results: The esp gene was fused with the N-terminal Sec-dependent signal sequence of the B. choshinensis cell wall protein and a C-terminal hexa-histidine-tag gene. The recombinant Esp was expressed and secreted into the optimized medium as an immature form and subsequently activated by thermolysin. The mature Esp was easily purified by a single purification step using nickel affinity chromatography and showed proteolytic activity as well as Staph. aureus biofilm destruction activity., Conclusions: The purification yield of the developed extracellular production system was 5 mg recombinant mature Esp per 20-ml culture, which was much higher than that of an intracellular production system in Escherichia coli (3 mg recombinant Esp per 1-l culture)., Significance and Impact of the Study: Our findings will be a powerful tool for the production and purification of recombinant Esp and also applicable to a large variety of recombinant proteins used for basic researches and biotechnological applications., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Fibronectin-binding protein A (FnBPA) and FnBPB are important adhesins for Staphylococcus aureus infection. We constructed fnbA and/or fnbB mutant strains from S. aureus SH1000, which possesses intact rsbU, and studied the role of these adhesins in in vitro and in vivo infections. In intravenous infection, all fnb mutants caused a remarkable reduction in the colonization rate in kidneys and the mortality rate of mice. fnbB mutant caused a more severe decrease in body weight than that caused by fnbA mutant. Serum levels of interleukin-6 and nuclear factor κB (NF-κB) activation in spleen cells were remarkably reduced in fnbA or fnbA fnbB mutant infections; however, there was no significant reduction in fnbB mutant infections. In in vitro cellular infection, FnBPA was shown to be indispensable for adhesion to and internalization by nonprofessional phagocytic cells upon ingestion by inflammatory macrophages and NF-κB activation. However, both FnBPs were required for efficient cellular responses. The results showed that FnBPA is more important for in vitro and in vivo infections; however, cooperation between FnBPA and FnBPB is indispensable for the induction of severe infection resulting in septic death.

Commensal bacteria are known to inhibit pathogen colonization; however, complex host-microbe and microbe-microbe interactions have made it difficult to gain a detailed understanding of the mechanisms involved in the inhibition of colonization. Here we show that the serine protease Esp secreted by a subset of Staphylococcus epidermidis, a commensal bacterium, inhibits biofilm formation and nasal colonization by Staphylococcus aureus, a human pathogen. Epidemiological studies have demonstrated that the presence of Esp-secreting S. epidermidis in the nasal cavities of human volunteers correlates with the absence of S. aureus. Purified Esp inhibits biofilm formation and destroys pre-existing S. aureus biofilms. Furthermore, Esp enhances the susceptibility of S. aureus in biofilms to immune system components. In vivo studies have shown that Esp-secreting S. epidermidis eliminates S. aureus nasal colonization. These findings indicate that Esp hinders S. aureus colonization in vivo through a novel mechanism of bacterial interference, which could lead to the development of novel therapeutics to prevent S. aureus colonization and infection.

In a recent report, ATP, which was possibly secreted by some intestinal bacteria, was shown to cause colitis in mice via Th17 cell differentiation. However, the ATP-secreting bacteria have not been isolated and identified. In the present study, we report that Enterococcus gallinarum, which is a vancomycin-resistant Gram-positive coccus isolated from mice and humans, secretes ATP.

Endovascular treatment (stenting) has evolved as an effective and safe treatment modality for symptomatic subclavian and innominate artery disease. Most of these patients have comorbid conditions associated with atherosclerotic vascular disease, which is responsible for the access site and increased difficulty of procedure. We report a case of symptomatic innominate artery stenosis with concomitant atherosclerotic disease of the abdominal aorta successfully treated with using coronary devices and the pull-through technique via the bilateral brachial approach.

BRAK/CXCL14 is a chemokine that is expressed in many normal cells and tissues but is absent from or expressed at very low levels in transformed cells and cancerous tissues including head and neck squamous cell carcinoma (HNSCC). We reported previously that the forced expression of BRAK/CXCL14 in HNSCC cells decreased the rate of tumor formation and size of tumor xenografts in athymic nude mice and SCID mice, suggesting that expression level of the gene is important for tumor suppression. In order to study the regulatory mechanisms governing the expression of this gene, we determined the transcriptional start site and promoter motifs of the gene. The major transcriptional start site determined by 5'rapid amplification of cDNA end method was located 283 bp downstream of the first proposed site of the gene. Determination of luciferase activities of reporter gene constructs with various deletions or mutations showed that an atypical TATA-like sequence, TATTAA was essential for the transcription of the gene and that the AP-1 binding sequence and tandem GC box were necessary for stimulating the expression of the gene in human squamous epithelial cells. The human DNA region was highly homologous (95% base identity) to the mouse gene. In addition, okadaic acid, an inhibitor of serine/threonine phosphatases 1, 2A and 2B, stimulated TATTAA sequence and AP-1 binding-sequence dependent promoter activity as well as increased the level of BRAK/CXCL14 mRNA, indicating that these sequences are essential for the regulation of BRAK/CXCL14 gene expression in the cells.

Objectives: To investigate stent deformation by torsional stress after implantation at the ostium of a model coronary artery., Background: Little is known about coronary stent deformation, especially the association between stent design and deformation at the coronary ostium. Recent reports have suggested that mechanical factors are important for stent restenosis., Methods: A coronary ostium model was constructed and three different stents (Express(2), Cypher, and Tsunami, n=5 each) were implanted at the aorto-ostial junction. Differences of stent deformation were assessed after exposure to torsional stress. Intravascular ultrasound was used to measure the luminal area along each stent. Then the extent and pattern of plastic deformation were compared between the three stent types., Results: The Express(2) stents and Cypher stents both showed significant deformation (P<.0001 and P=.045, respectively) adjacent to the ostium, whereas only a minimal decrease of luminal area was observed with the Tsunami stent. In the central and distal parts of each stent, the decrease of luminal area was minimal and no differences were noted among the three types. Sudden fracture of a Cypher stent strut occurred during the experiment., Conclusion: Differences of structural characteristics influence permanent plastic deformation at sites where continuous stress occurs, such as the coronary ostium. A more elastic design may show better resistance to such stress.

A synthetic route is described for collagen-like polypeptides constructed from O-phospho-L-hydroxyproline [Hyp(PO(3)H(2))] residues. Using the synthetic polypeptides and a natural protein, gelatin, fine fibers and their network structures (ESNWs) were prepared via electrospinning. The composite ESNWs can induce the mineralization of calcium phosphate. The phosphoryl groups of the Hyp(PO(3)H(2)) residues affect both the crystalline phase and amount of the calcium phosphate, depending on the chemical structure in the repeating sequence. The composite ESNWs can be developed as a biocompatible replacement of the extracellular matrix of hard tissues, and thus can be applied as dental materials for restoration of dental cavities or as a sealant for pits and fissures.

Neutrophils play a crucial role in the host response to infection with Staphylococcus aureus, which is a major human pathogen capable of causing life-threatening disease. Interleukin-8 (IL-8) is a potent chemoattractant and activator of neutrophils. We previously reported that S. aureus secretes a factor that suppresses IL-8 production by human endothelial cells. Here we isolated an inhibitor of IL-8 production from the supernatant and identified it as staphylococcal beta-hemolysin. Beta-hemolysin reduced IL-8 production without cytotoxicity to endothelial cells. Pretreatment with beta-hemolysin decreased the expression of both IL-8 mRNA and protein induced by tumor necrosis factor alpha (TNF-alpha). Migration of neutrophils across TNF-alpha-activated endothelium was also inhibited by beta-hemolysin. In contrast, beta-hemolysin had no effect on intercellular adhesive molecule 1 expression in activated endothelial cells. These results showed that beta-hemolysin produced by S. aureus interferes with inflammatory signaling in endothelial cells and may help S. aureus evade the host immune response.

We examined the effect of alpha(1)-adrenoceptor subtype-specific stimulation on L-type Ca2+ current (I(Ca)) and elucidated the subtype-specific intracellular mechanisms for the regulation of L-type Ca2+ channels in isolated rat ventricular myocytes. We confirmed the protein expression of alpha(1A)- and alpha(1B)-adrenoceptor subtypes at the transverse tubules (T-tubules) and found that simultaneous stimulation of these 2 receptor subtypes by nonsubtype selective agonist, phenylephrine, showed 2 opposite effects on I(Ca) (transient decrease followed by sustained increase). However, selective alpha(1A)-adrenoceptor stimulation (> or =0.1 micromol/L A61603) only potentiated I(Ca), and selective alpha(1B)-adrenoceptor stimulation (10 mumol/L phenylephrine with 2 micromol/L WB4101) only decreased I(Ca). The positive effect by alpha(1A)-adrenoceptor stimulation was blocked by the inhibition of phospholipase C (PLC), protein kinase C (PKC), or Ca2+/calmodulin-dependent protein kinase II (CaMKII). The negative effect by alpha(1B)-adrenoceptor stimulation disappeared after the treatment of pertussis toxin or by the prepulse depolarization, but was not attributable to the inhibition of cAMP-dependent pathway. The translocation of PKCdelta and epsilon to the T-tubules was observed only after alpha(1A)-adrenoceptor stimulation, but not after alpha(1B)-adrenoceptor stimulation. Immunoprecipitation analysis revealed that alpha(1A)-adrenoceptor was associated with G(q/11), but alpha(1B)-adrenoceptor interacted with one of the pertussis toxin-sensitive G proteins, G(o). These findings demonstrated that the interactions of alpha(1)-adrenoceptor subtypes with different G proteins elicit the formation of separate signaling cascades, which produce the opposite effects on I(Ca). The coupling of alpha(1A)-adrenoceptor with G(q/11)-PLC-PKC-CaMKII pathway potentiates I(Ca). In contrast, alpha(1B)-adrenoceptor interacts with G(o), of which the betagamma-complex might directly inhibit the channel activity at T-tubules.

A species-specific quantitative detection method involving 5' nuclease real-time polymerase chain reaction using a minor groove binder probe that was designed from the sodA gene was developed for Staphylococcus epidermidis. This method distinguished S. epidermidis from other staphylococci and specifically quantified the bacterium. This study shows that the method is useful for the identification and quantitative detection of S. epidermidis.

Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci. Each species has different characteristics, and a difference in pathology is also seen in compromised hosts. Therefore, the development of a species-specific simple detection method for the identification of these staphylococci is important. Here, a species-specific real-time PCR assay is reported that targets the superoxide dismutase A-encoding gene of these bacteria. Primers were designed with a base that was non-complementary with regard to the other bacteria. This base was at the 3' end of the primer (3' mismatch primer) and conferred high specificity. These primers were then evaluated using real-time PCR. They reacted only with the target bacterium. In addition, stable quantitative reactions were observed when experiments were performed using genomic DNA extracted from varying numbers of staphylococci cells (10(1)-10(7) cells). These results indicate that this method is useful for the identification and quantitative analysis of S. capitis, S. haemolyticus and S. warneri.

Objective: A comparison of aspiration catheters that have been approved for real-world use was carried out in vitro., Background: Myocardial damage occurs during therapeutic aspiration of thrombus. The relative efficiency of aspiration may be important in this regard., Methods: Using saline and human clot, nine aspiration catheters were compared Thrombuster III(R)N (6 Fr and 7 Fr), ZEEK (6 Fr), Rebirth (7 Fr), Eliminate (6 Fr and 7 Fr), Pronto (6 Fr), and Export(R) (6 Fr and 7 Fr). Tracking was assessed from the resistance required to pass the catheter through a vessel model. Pushability was determined from the difference between the load at the hand piece and tip of the catheter during advancement through the vessel model., Results: The Thrombuster III(R)N (6 Fr and 7 Fr) showed significantly better aspiration performance, although the ranking order of the catheters was not the same for saline and clot. The Thrombuster III(R)N also showed the best tracking with low resistance and was the easiest catheter to advance, as evaluated based on pushability., Conclusions: In the present in vitro evaluation system, the Thrombuster III(R)N performed better than other catheters.

Recent reports have shown that Staphylococcus aureus infection increases the expression of cytokines and cell adhesion molecules in endothelial cells and enhances leucocyte migration, thereby resulting in bacterial elimination. In this study, we analysed the production of the chemokine interleukin (IL)-8 in human umbilical vein endothelial cells (HUVEC) infected with several S. aureus strains by using reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found that the avirulent strains (00-51 and 00-62) increased IL-8 production but the virulent strains (A17 and A151) decreased it at both the mRNA and protein levels. We considered that the inhibition of IL-8 production depended on certain inhibitory factor(s) secreted by bacteria. This was because S. aureus also abolished IL-8 expression in HUVEC treated with cytochalasin D, and the addition of culture supernatants of strains A17 and A151 decreased IL-8 production in HUVEC. This factor(s) in the bacterial culture supernatant inhibited both basal and tumour necrosis factor (TNF)-alpha-induced IL-8 production. In contrast, no inhibitory effect was observed on monocyte chemotactic protein-1 (MCP-1) production. These results indicate that S. aureus can down-regulate IL-8 release in endothelial cells through the secretion of inhibitory factor(s), and this may result in decreased neutrophil recruitment, thus interfering with the host immune response to bacterial infection.

Many pathogens colonize host tissues by binding to the extracellular matrix via their cell surface adhesion molecules, which are called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). Staphylococcus aureus expresses several of these adhesion molecules, some of which bind to fibronectin. Of these adhesion molecules, fibronectin-binding proteins play a role in the pathogenicity of S. aureus, although it is not yet clear whether they enhance its virulence. We have previously shown that fibronectin-bound S. aureus is efficiently phagocytosed by thioglycolate-induced mouse peritoneal macrophages. Bacterial ingestion is mediated by Very Late Antigen-5 (VLA-5; alpha5beta1 integrin) and is accompanied by the formation of adhesion complexes. Here we show that the expression of VLA-5 is restricted to thioglycolate-induced inflammatory macrophages and is not found in the resident macrophages. When cells were in suspension, alpha5 integrin was not expressed on the surface of either resident or inflammatory macrophages, whereas in adherent cells, this integrin was distributed on the surface of inflammatory but not resident macrophages. A high level of this integrin was present in the cytoplasmic region only in inflammatory macrophages. In agreement with this, fibronectin-mediated phagocytosis of S. aureus was observed only in the inflammatory macrophages. In inflammatory macrophages ingesting fibronectin-bound S. aureus, alpha5 integrin was concentrated close to the phagocytosed bacteria. This change in distribution was not found in macrophages ingesting untreated bacteria. Together with our previous work, these results indicate that, upon ingestion of fibronectin-bound S. aureus, VLA-5 accumulates in the area of phagocytosis in inflammatory macrophages, where it forms adhesion complexes.

Gallbladder cancer is an asymptomatic disease in the early stage and no therapeutic measure is available except surgical intervention. The prognosis for patients with advanced,i.e., unresectable or metastatic disease is dismal, with median survival usually being less than 6 months if not treated with chemotherapy. To date, chemotherapy for gallbladder cancer has been limited by the absence of agents with effective cytotoxic activity. Thalidomide has been shown to have antiangiogenic and immunomodulatory effects, including the inhibition of vascular endothelial growth factor, basic fibroblast growth factor and tumor necrosis factor alpha. Celecoxib is a potent selective COX-2 inhibitor. The reported biological consequences of COX-2 up-regulation include inhibition of apoptosis, increased metastatic potential and promotion of angiogenesis. These events may contribute to cell transformation and tumor progression. Antiangiogenesis represents a significant new strategy for cancer treatment. Therefore,it is important to accept a wide range of different inhibitors such as thalidomide and selective COX-2 inhibitors with conventional cytotoxic agents. Here we show a case of unresectable gallbladder cancer with remarkable improvement in CA19-9 and prolongation of life.

Previous studies have shown that transient no-reflow during coronary intervention but with Thrombosis in Myocardial Infarction (TIMI) grade 3 flow at the completion of the procedure is associated with increased in-hospital and 6-month mortality. We hypothesized that the use of intravascular ultrasound before intervention could identify morphologic features that were predictive of transient no-reflow in patients who had acute coronary syndrome (ACS). We analyzed 220 patients with ACS who had suitable intravascular ultrasound images that were acquired before intervention. We defined "transient no-reflow" as TIMI grade 0, 1, or 2 flow during the procedure and TIMI grade 3 flow at the completion of the procedure. We defined "reflow" as good coronary flow (TIMI grade 3 flow) during and after the procedure. Patients were categorized to a transient no-reflow group (n = 20) or a reflow group (n = 200). In the transient no-reflow group, vessel area and amount of plaque burden in the culprit lesion were significantly greater than in the reflow group (vessel 20.8 +/- 5.4 vs 16.4 +/- 6.2 mm(2), p < 0.01; plaque burden 0.90 +/- 0.03 vs 0.83 +/- 0.08, p < 0.001). The presence of ruptured plaque, lipid pool-like images, and thrombus formation were significantly higher in the transient no-reflow group than in the reflow group. Multivariate analysis identified the presence of thrombus formation (odds ratio 4.53, 95% confidence interval 1.03 to 20.0, p = 0.04) and larger plaque burden (odds ratio 1.79, 95% confidence interval 1.01 to 3.23, p = 0.05) as independent predictors of transient no-reflow. In conclusion, lesion morphologies are different for transient no-reflow and reflow. These findings suggest that the presence of thrombus formation and large plaque burden increase the risk for developing transient no-reflow during coronary intervention for ACS.

Objectives: The toxicity of microglass fibers (MG), one of the man-made mineral fibers, has not been sufficiently evaluated. The aim of the current study was to evaluate the cytotoxicity of MGin vitro., Methods: Alveolar macrophages were obtained from the bronchoalveolar lavage of male F344/N rats. The macrophages were exposed to MG at concentrations of 0, 40, 80, 160 and 320 μg/ml. The effects of MG on the macrophages were examined by cell magnetometry, LDH assay and morphological observation., Results: In the cell magnetometry experiment, a significant delay of relaxation (the reduction of remanent magnetic field strength) was observed in the cells treated with 160 and 320 μg/ml of MG in a dose-dependent manner. A significant increase in LDH release was also observed in the cells with 160 and 320 μg/ml in a dose-dependent manner. Changes in the cytoskeleton were observed after exposure to MG by immunofluorescent microscopy using an α-tubulin antibody., Conclusions: The cytotoxicity of MG on alveolar macrophages was demonstrated with cell magnetometry. The mechanism of the toxic effects of MG was related to cytoskeleton damage.

Rock wool (RW), a type of man-made mineral fiber (MMMF), is a building material used as an asbestos substitute for heat insulation, fire resistance, and reinforcement. RW is included in group 3 of the IARC classification. In the present study, the cytotoxicity of RW was investigated by cell magnetometry, enzyme assay, DNA ladder detection, and electron microscopic morphological evaluation in comparison with chrysotile fibers (CF). Specimens were prepared by 18-h incubation of Fischer rat alveolar macrophages in the presence of RW fibers as the study material, CF as positive control, and phosphate-buffered saline (PBS) as negative control, together with a relaxation indicator, Fe3O4, except for morphological evaluation, followed by additional procedures of external magnetization and subsequent 20-min remanent magnetic field measurement for magnetometric evaluation, and macrophage DNA extraction for evaluating possible apoptosis by DNA ladder detection. In magnetometry, relaxation, a marker of cytotoxicity, was rapid in both the RW- and PBS-treated groups, while it was delayed in both the long and short CF-treated groups. Differences in percent lactate dehydrogenase (LDH) release between the RW-treated group and PBS-treated group were not significant, but those between the RW-treated group and short CF-treated group were statistically significant. A DNA ladder was not detected in any of the study groups. Electron micrographs showed that RW did not cause any change, but CF caused changes in macrophages. Thus, magnetometric measurements suggested no cytotoxicity of RW. We plan, in the future, to evaluate the safety of RW by magnetometric measurement and morphological observation of the lungs in in vivo inhalation experiments.

Since alveolar macrophages play an important role in the clearance of inhaled dust from air-ways, these cells have been used as a target for various toxic chemicals. Alveolar macrophages obtained from bronchoalveolar lavage of Syrian golden hamsters were concurrently exposed in vitro to Fe(3)O(4), as an indicator for magnetometry, and various concentrations of cadmium oxide (CdO) in this study. A rapid decrease of the remnant magnetic field, called relaxation, was observed after the cessation of an external magnetic field stimulus in macrophages concurrently exposed to phosphate-buffered saline or CdO at 0.1 microg/ml, while relaxation was delayed in those concurrently exposed to 1, 25, or 50 microg/ml CdO. Therefore, the concentration of CdO affecting relaxation in vitro was estimated at between 0.1 and 1 microg/ml. Release of LDH activity from CdO-exposed macrophages into the medium significantly increased at levels of 25 and 50 microg/ml CdO. Apoptosis was not detected in macrophages exposed to CdO by the DNA ladder detection method or morphological observations. Electron-microscopic examination revealed severe membrane damage and vacuolar changes in macrophages exposed to CdO. Since delayed relaxation is thought to occur by (1). disrupted cytoskeleton-driven random rotation of phagosomes containing iron oxide particles, (2). significant lactate dehydrogenase (LDH) activity release, and (3). detachment of cell membranes, CdO is considered to affect macrophage functions.

Staphylococcus aureus Cowan I and a clinically isolated coagulase-negative Staphylococcus strain, S. saprophyticus 10312, were found to have two fibronectin binding proteins, FnBPA and FnBPB. While both staphylococci bound to serum fibronectin to a similar extent, fibronectin binding significantly increased the phagocytic activity of macrophages against S. aureus (by ca. 150%) but not against S. saprophyticus. This enhancing effect of fibronectin was inhibited by an RGD sequence-containing peptide and also by anti-very late antigen 5 antibody. This suggests that the effect is mediated by very late antigen 5 expressed on macrophages. In macrophages ingesting fibronectin-bound Cowan I, alpha(5) and beta(1) chains were associated with the cytoskeleton. Cytosolic signaling factors such as paxillin, c-Src, and c-Csk were also associated with the cytoskeleton. On the contrary, beta(3) integrin transiently disappeared from the cytoskeleton when macrophages ingested the fibronectin-treated S. aureus Cowan I. Furthermore, the Src kinase family tyrosine kinase Lyn dissociated from the cytoskeleton. These cellular components did not respond in a fibronectin-dependent manner when macrophages phagocytosed S. saprophyticus. This means that only fibronectin-treated S. aureus Cowan I induces the accumulation of very late antigen 5, which in turn induces the association of paxillin and tyrosine kinases. It is thought that the phagocytic activity of macrophages against fibronectin-treated S. aureus was increased by signaling via the activation of very late antigen 5.

The present study was conducted to clarify the toxicity of Indium arsenide (InAs) particles to alveolar macrophages of hamsters by cytomagnetometry, enzyme release assays and morphological examinations. One million alveolar macrophages obtained from hamsters were exposed to 60 microg of ferrosoferric oxide and 2, 4, 10 and 20 microg of InAs particles. Relaxation, which is the rapid decline of strength of the remanent magnetic fields radiating from the alveolar macrophages, was insignificantly delayed and decay constants were not changed due to exposure to such doses of InAs. Because the relaxation is thought to be associated with the cytoskeleton, the exposure to InAs may not have impaired their motor function. An LDH release assay and morphological findings indicate slight damage to macrophages. DNA electrophoresis and the TUNEL method revealed neither necrotic changes nor apoptotic changes. Thus, InAs particles at such doses hardly cause cytostructural changes and cell death.

The concept of superantigens is well-known and widely accepted. In this brief communication, we analyze the behaviour of antigen-presenting cells after T-cell activation by staphylococcal enterotoxin B, a representative superantigen. We tried to activate murine T cells by inflammatory mouse peritoneal macrophage in the presence of staphylococcal enterotoxin B, but no T-cell activation was observed. We, therefore, analyzed surface-specific antigens of the macrophages. They expressed insufficient amounts of MHC class II, CD80 and CD86 molecules on the surface of the cells. On the contrary, increased amounts of MHC class II and CD86 molecules on the cell surfaces were observed after incubation with interferon gamma. Interferon gamma-primed macrophages were found to be competent to activate T cells in the presence of staphylococcal enterotoxin B. To our surprise, these macrophages underwent apoptosis in parallel with T-cell activation.

Staphylococcus aureus was previously shown to be internalized by murine fibroblast. We examined the intracellular events of S. aureus ingested by BALB/3T3 cells. After uptake of strains A191 and A151, isolates from atopic lesion, and a laboratory strain, Cowan I, for 1 hr, BALB/3T3 cells were incubated with 1.25 microg/ml lysostaphin. Laddering of the DNA in multiples of approximately 180 bp occurred within 4 hr following bacterial addition in BALB/3T3 cells infected with A191 and within 18 hr in BALB/3T3 cells infected with A151: histochemical staining by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method revealed that the rate of the fragmentation of nucleic DNA in Cowan I-infected BALB/3T3 cells at 21 hr following bacterial addition was 0.52 +/- 0.25%, significantly higher than that in the control cells. Transmission electron micrographs of BALB/3T3 cells at 4 hr following A191 addition showed that the apoptotic features, including electron-dense nucleus and plasma membrane blebbing, occurred in some cells in which many staphylococci escaped the endosome and went on to cell division. At the same time, A151 organisms enclosed with endosome membrane were static in the intact BALB/3T3 cells. The significant increase of A191 was confirmed by counting intracellular live bacteria during 2- to 6-hr incubation. These results suggest that internalized S. aureus escapes the endosome, multiplies and induces apoptosis in the fibroblast cell.

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase-negative staphylococci (CNS) strains for comparison. Fibronectin-reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose-dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin-binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.

We previously showed that a relatively high dose of LPS induced the selective translocation of protein kinase C-beta (PKC-beta) in LPS-responsive mouse macrophages. This result suggested that phosphatidylinositol-specific phospholipase C (PLC) might be activated in the upstream of PKC-beta. Stimulation of C3H/HeN mouse macrophages by LPS induced the characteristic phosphatidylinositol-1,4,5-trisphosphate (IP3) response, that is, a biphasic response consisting of a rapid increase occurring within the first 1 min, and another increase beginning at around 1 min after stimulation. Only the first response was disappeared when cells were treated with a platelet-activating factor receptor antagonist. LPS-inducible TNF-alpha gene activation, however, was not suppressed by the same antagonist, but suppressed by PKC inhibitors. LPS-stimulated macrophage lysates showed tyrosine phosphorylation of some proteins, and the strongest phosphorylation was observed at molecular mass of 140 kDa. The phosphorylation of this protein started at 40 s after LPS stimulation and continued to increase. Anti-PLC-gamma2 Ab seemed to recognize the same protein as the tyrosine-phosphorylated 140-kDa protein. A low dose of LPS (1 ng/ml) could not induce the tyrosine phosphorylation of this protein. Furthermore, LPS induced only the first phase change, but not the second phase increase in LPS-hyporesponsive C3H/HeJ mouse macrophages. These results indicate that the first phase rapid IP3 change, which is also seen in HeJ macrophages, is mediated via a platelet-activating factor receptor, and is not responsible for TNF-alpha production, while the second phase change mediated by a molecule other than CD14 is responsible for PKC-beta translocation and TNF-alpha production. The results also suggest that the later IP3 change is considered to be mediated through a gamma2 type of phosphatidylinositol-specific PLC.

Translocation of protein kinase C (PKC) after PMA or LPS stimulation has been studied in thioglycolate (TGC)-elicited murine peritoneal macrophages. Among the PKC subtypes we examined (alpha, beta, gamma, delta, and epsilon) by indirect immunostaining and immunoblot analysis, conventional PKC-beta, as well as novel PKC-delta and PKC-epsilon were found to exist in TGC-elicited C3H/HeN mouse macrophages. Translocation of PKC-beta to the Triton-stable cytoskeleton could be seen in macrophages after stimulation by both PMA and LPS. On the other hand, novel PKCs redistributed only after PMA stimulation. Macrophages obtained from LPS-nonresponsive C3H/HeJ mice also exhibited PKC-beta, and the m.w., cellular distribution, and cellular content of this enzyme could not be distinguished from those of C3H/HeN macrophages. These macrophages exhibited PKC-delta and PKC-epsilon, as did the C3H/HeN macrophages. In these macrophages, however, LPS did not induce any remarkable change in the intracellular distribution of PKC-delta and PKC-epsilon or PKC-beta, whereas PMA was able to induce the translocation of PKC-beta to the cytoskeleton. These results suggest that LPS stimulation induces selective redistribution of PKC-beta in LPS-responsive macrophages, whereas a defect related to LPS unresponsiveness exists in C3H/HeJ mouse macrophages before the PKC activation. Translocation of PKC-beta can be understood to be an important event in LPS signaling in macrophages.

We reported previously that a reorganization of microfilaments can be observed when macrophages are stimulated with lipopolysaccharide (LPS). This reorganization is not detected with current methods in macrophages of an LPS-nonresponsive C3H/HeJ mouse. These results suggest that the observed microfilament response might be involved in a macrophage-activating process induced by LPS. To investigate this, we studied the effect of cytochalasin D (CD), which inhibits reorganization of microfilaments, on LPS-induced tumor necrosis factor (TNF) production. A concentration of CD incapable of affecting filamentous actin by itself was used in these experiments. When this concentration of CD was added after LPS stimulation, microfilament reorganization and TNF production were inhibited. The suppressive effect of CD on TNF production was confirmed by the observation that TNF-alpha mRNA expression was also inhibited by CD. This inhibitory effect of CD was not specific to TNF, because the production of interleukin-1 and prostaglandin E2 were also inhibited. These effects of CD were observed only when CD was added within the first 20 min after LPS stimulation. These results suggest that the CD-sensitive microfilament response is essential in the signaling pathway for the production of certain monokines in LPS-stimulated macrophages.

A protein phosphatase inhibitor, tautomycin induces blebs on the surface of human myeloid leukemia K562 cells within 10 min. In this paper we examined the cytoskeleton of tautomycin-treated cells. In the presence of tautomycin, cells with blebs turned into segmented forms with smooth surfaces after 15 min and into smooth round shapes without microprotuberance after 60 min. Observation with fluorescence microscopy showed that F-actin detached from the plasma membrane at the site of the blebs. Further treatment with tautomycin induced the accumulation of F-actin at the segmentation centers. Under electron microscopy, an electron dense ring-structure was detected at the segmentation center. Tautomycin did not induce major changes of the microtubule network although F-actin accumulated near the microtubule organizing center. The amount of F-actin increased in tautomycin-treated cells. These results indicate that the morphological changes are induced by reorganization of actinfilaments.

The effects of BY-1949, a novel dibenzoxazepine derivative, injected into the cerebral ventricle on noxious stimulus-induced responses of regional blood flow in the cortex and neuronal activity in the nucleus basalis of Meynert were studied in male rats. These induced responses were markedly enhanced by administration of BY-1949 (16.4 +/- 0.5 ng/100 g, S.E.M.). These data indicate that BY-1949 acts on the central nervous system to modulate the response to a noxious stimulus of regional cerebral blood flow and neuronal activity in the nucleus basalis of Meynert.

Lipopolysaccharide (LPS), a potent activating substance of macrophages, induced the reorganization of microfilaments in macrophages obtained from C3H/HeN mice. At 1 min after LPS addition, a slight disassembly of actin was observed. At 2 to 4 min, there was a gradual assembly; then, at 5 and 6 min, a subsequent rapid disassembly occurred. We employed two methods to observe this process. One was the RITC-phalloidin staining of actin filaments and the other was the extraction of monomeric actin and unstable actin filaments with Triton X-100 solution. The results obtained by the two methods were basically in agreement. Nevertheless, there was a discrepancy between the results from the two methods, concerning the ratio of assembly and disassembly. The RITC-phalloidin staining was more sensitive in detecting actin assembly and less sensitive in detecting the disassembly than the extraction with Triton X-100 solution was. This difference suggests that some of the unstable filaments, which were extracted with Triton X-100 solution and fixed with formalin, were formed during the LPS-induced reorganization process. This reversible actin assembly could not be observed in the LPS-nonresponder, C3H/HeJ mouse macrophages. We concluded that the observed process could be attributed to LPS-signal triggering pathways subsequent to LPS binding and that a necessary component to initiate effective LPS-signaling, which is probably deficient in C3H/HeJ mice, is involved in this reorganization process of LPS-stimulated macrophages.

The design, construction and a brief indication of the advantages of the stabilized canine retractor have already been explained (Adams, 1983). It is the purpose of the present investigation to clarify and evaluate the physical characteristics of the new form and to ascertain the precise nature and degree of the differences between it and the hitherto widely used standard form of this spring.