Tat pathway-mediated translocation of the Sec pathway substrate OprM, an outer membrane subunit of the resistance nodulation division xenobiotic extrusion pumps, in Pseudomonas Aeruginosa.

BACKGROUND: Pseudomonas aeruginosa produces the Sec and Tat protein secretion machineries. The latter appears to be involved in the secretion of virulence factors, including phospholipase C (PlcH), and hence is a potential target of chemotherapeutic agents. METHODS: The signal sequence of OprM, the outer membrane subunit of the xenobiotic extrusion pumps, was substituted with that of PlcH. The antibiotic susceptibility of oprM-deficient cells expressing the hybrid protein PlcH-OprM was evaluated using the agar dilution method. RESULTS: The PlcH-OprM-expressing cells showed resistance to various MexAB-OprM substrate antibiotics. To evaluate the translocation route of PlcH-OprM, tatC encoding an indispensable component of the Tat machinery was knocked out in oprM-deficient cells. The tatC-oprM double mutant expressing PlcH-OprM exhibited antibiotic hypersusceptibility like the oprM-deficient cells, indicating that PlcH-OprM was translocated across the inner membrane exclusively through the Tat system. CONCLUSIONS: This system can be used for the screening of Tat system inhibitors and will be an excellent model for the study of secretion and biogenesis of the ß-barrel outer membrane proteins.

Effect of Ethiopian multiflora honey on fluconazole-resistant Candida species isolated from the oral cavity of AIDS patients.

This study aimed to determine the antifungal effect of Ethiopian multiflora honey against Candida species isolated from the oral cavity of AIDS patients. Oral rinses were obtained from 13 AIDS patients and cultured on CHROMagar plates at 37°C for 48 hours. Candida species were identified by microbiological and molecular techniques. The antifungal effect of the honey sample on Candida was investigated by an agar dilution technique. Susceptibility of the Candida species to fluconazole was tested following a semi-modified microdilution method. Growth of both fluconazole-susceptible and -resistant Candida species was inhibited with a minimum fungicidal concentration (MFC) of 35-40% (v/v) honey. The MFC of different Candida species was not significantly different (P > 0.05). From the total of 25 Candida isolates tested for susceptibility, 11 (44%), eight (32%) and six (24%) of the isolates were sensitive (minimum inhibitory concentrations [MICs] < 8 µg/mL), susceptible (dose-dependent: MICs 16-32 µg/mL) and resistant (MICs > 64 µg/mL) to fluconazole, respectively. Ethiopian multiflora honey has antifungal activity against fluconazole-resistant Candida species isolated from the oral cavity of AIDS patients. This supports the existing folkloric practice of using honey to treat oral lesions. Nevertheless, identification of the bioactive agents in honey, their clinical evaluation and pharmacological standardization are crucial.

Ceragenin CSA-13 exhibits antimicrobial activity against cariogenic and periodontopathic bacteria.

INTRODUCTION: Ceragenin CSA-13 is a bile-acid-based mimic of endogenous antimicrobial peptides and shares a mechanism of action with many of these antimicrobial agents. Because CSA-13 is not peptide based, it is not a substrate for the proteases that are found in the oral cavity, which are capable of degrading antimicrobial peptides. Furthermore, the simplicity of the ceragenins makes them easier to prepare and purify than antimicrobial peptides. In this study, we examined the antimicrobial activities of CSA-13 against oral pathogens and found that this compound was bactericidal against all of the strains tested. METHODS: The strains used were isolates of Streptococcus mutans and Porphyromonas species. Minimum inhibitory concentrations (MIC) were determined using agar dilution methods. In susceptibility testing, viable counts were determined after incubation with CSA-13. RESULTS: CSA-13 was potent against all 23 strains tested with MICs of 1-8 microg/ml for S. mutans and 1-16 microg/ml for 24 strains of the genus Porphyromonas. The MIC(50) was 2 and the MIC(90) was 8 mug/ml for S. mutans. MIC ranges for protease-positive P. gingivalis and P. cangingivalis were 2-16 microg/ml, and 1-2 microg/ml for protease-negative P. circumdentaria. CSA-13 interacted with lipopolysaccharide-sensitized erythrocytes at a concentration of 5.0-20.0 microg/ml. CONCLUSION: CSA-13 displays broad-spectrum activity against cariogenic and periodontopathic bacteria. CSA-13 was effective against protease-positive Porphyromonas. It was shown to bind to erythrocytes coated with lipopolysaccharide and lipoteichoic acid from diverse bacterial strains. These results suggest that CSA-13 may be useful for the prevention and treatment of oral microbial diseases.

Novel risk stratification of patients with neuroblastoma by genomic signature, which is independent of molecular signature.

Human neuroblastoma remains enigmatic because it often shows spontaneous regression and aggressive growth. The prognosis of advanced stage of sporadic neuroblastomas is still poor. Here, we investigated whether genomic and molecular signatures could categorize new therapeutic risk groups in primary neuroblastomas. We conducted microarray-based comparative genomic hybridization (array-CGH) with a DNA chip carrying 2464 BAC clones to examine genomic aberrations of 236 neuroblastomas and used in-house cDNA microarrays for gene-expression profiling. Array-CGH demonstrated three major genomic groups of chromosomal aberrations: silent (GGS), partial gains and/or losses (GGP) and whole gains and/or losses (GGW), which well corresponded with the patterns of chromosome 17 abnormalities. They were further classified into subgroups with different outcomes. In 112 sporadic neuroblastomas, MYCN amplification was frequent in GGS (22%) and GGP (53%) and caused serious outcomes in patients. Sporadic tumors with a single copy of MYCN showed the 5-year cumulative survival rates of 89% in GGS, 53% in GGP and 85% in GGW. Molecular signatures also segregated patients into the favorable and unfavorable prognosis groups (P=0.001). Both univariate and multivariate analyses revealed that genomic and molecular signatures were mutually independent, powerful prognostic indicators. Thus, combined genomic and molecular signatures may categorize novel risk groups and confer new clues for allowing tailored or even individualized medicine to patients with neuroblastoma.

Bcl-2 is a key regulator for the retinoic acid-induced apoptotic cell death in neuroblastoma.

Retinoic acid (RA) has been shown to induce neuronal differentiation and/or apoptosis, and is widely used as a chemotherapeutic agent for treating the patients with neuroblastoma. However, the therapeutic effect of RA is still limited. To unveil the molecular mechanism(s) inducing differentiation and apoptosis in neuroblastoma cells, we compared CHP134 and NB-39-nu cell lines, in which all-trans-RA (ATRA) induces apoptosis, with LA-N-5 and RTBM1 cell lines, in which it induces neuronal differentiation. Here, we found that Bcl-2 was strongly downregulated in CHP134 and NB-39-nu cells, whereas it was abundantly expressed in LA-N-5 and RTBM1 cells. ATRA-mediated apoptosis in CHP134 and NB-39-nu cells was associated with a significant activation of caspase-9 and caspase-3 as well as cytoplasmic release of cytochrome c from mitochondria in a p53-independent manner. Enforced expression of Bcl-2 significantly inhibited ATRA-mediated apoptosis in CHP134 cells. In addition, treatment of RTBM1 cells with a Bcl-2 inhibitor, HA14-1, enhanced apoptotic response induced by ATRA. Of note, two out of 10 sporadic neuroblastomas expressed bcl-2 at undetectable levels and underwent cell death in response to ATRA in primary cultures. Thus, our present results suggest that overexpression of Bcl-2 is one of the key mechanisms to give neuroblastoma cells the resistance against ATRA-mediated apoptosis. This may provide a new therapeutic strategy against the ATRA-resistant and aggressive neuroblastomas by combining treatment with ATRA and a Bcl-2 inhibitor.

Increased expression of proapoptotic BMCC1, a novel gene with the BNIP2 and Cdc42GAP homology (BCH) domain, is associated with favorable prognosis in human neuroblastomas.

Differential screening of the genes obtained from cDNA libraries of primary neuroblastomas (NBLs) between the favorable and unfavorable subsets has identified a novel gene BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1). Its 350 kDa protein product possessed a Bcl2-/adenovirus E1B nineteen kDa-interacting protein 2 (BNIP2) and Cdc42GAP homology domain in the COOH-terminus in addition to P-loop and a coiled-coil region near the NH2-terminus. High levels of BMCC1 expression were detected in the human nervous system as well as spinal cord, brain and dorsal root ganglion in mouse embryo. The immunohistochemical study revealed that BMCC1 was positively stained in the cytoplasm of favorable NBL cells but not in unfavorable ones with MYCN amplification. The quantitative real-time reverse transcription-PCR using 98 primary NBLs showed that high expression of BMCC1 was a significant indicator of favorable NBL. In primary culture of newborn mice superior cervical ganglion (SCG) neurons, mBMCC1 expression was downregulated after nerve growth factor (NGF)-induced differentiation, and upregulated during the NGF-depletion-induced apoptosis. Furthermore, the proapoptotic function of BMCC1 was also suggested by increased expression in CHP134 NBL cells undergoing apoptosis after treatment with retinoic acid, and by an enhanced apoptosis after depletion of NGF in the SCG neurons obtained from newborn mice transgenic with BMCC1 in primary culture. Thus, BMCC1 is a new member of prognostic factors for NBL and may play an important role in regulating differentiation, survival and aggressiveness of the tumor cells.

Reduced inflammatory pain in mice deficient in the differential screening-selected gene aberrative in neuroblastoma.

Differential screening-selected gene aberrative in neuroblastoma (Dan) protein is produced in small neurons of dorsal root ganglia. Thermal and mechanical allodynia and Fos expression in the spinal dorsal horn evoked by inflammation and neuropathic pain were investigated using Dan-deficient mice. Mice showed pain reactions induced by the introduction of complete Freund's adjuvant (CFA) into their hind paw (inflammatory pain model) and after sciatic nerve ligation (neuropathic pain model). In the inflammatory pain model, thermal and mechanical pain thresholds in Dan-deficient mice were significantly higher than those of wild-type mice. The number of Fos-immunoreactive cells in the dorsal horn during the inflammatory period was significantly less in Dan-deficient mice. However, in the neuropathic pain model, no differences in thermal hypersensitivity, mechanical allodynia, or the number of Fos-immunoreactive cells in the dorsal horn were observed between the mice. These data suggest that Dan may be a neuromodulator in inflammatory pain.

Immunology and functional genomics of Behçet's disease.

Behçet's disease (BD) is a multisystemic inflammatory disorder. Although the cause and pathogenesis of BD are still unclear, there is evidence for genetic, immunologic and infectious factors at the onset or in the course of BD. This review focuses on the functional genomics and immunology of BD. HLA-B51 is the major disease susceptibility gene locus in BD. An increased number of gammadelta T cells in the peripheral blood and in the involved tissues have been reported. However, the T cells at the sites of inflammation appear to be a phenotypically distinct subset. There is also a significant gammadelta T cell proliferative response to mycobacterial 65-kDa heat shock protein peptides. Homologous peptides derived from the human 60-kDa heat shock protein were observed in BD patients. There is evidence that natural killer T cells may also play a role in BD.

Sensitivity of genera Porphyromonas and Prevotella to the bactericidal action of C-terminal domain of human CAP18 and its analogues.

This paper reports the effect of the synthesized 27-amino acid sequence in the C-terminal domain of human CAP18 (hCAP18), a human cationic antibacterial protein or cathelicidin, on certain strains belonging to the genera Porophyromonas and Prevotella. The domain binds lipopolysaccharides (LPS) from Porophyromonas gingivalis and Porophyromonas circumdentaria as well as enterobacterial LPS. Two analogues of hCAP18, designated LL/CAP18 and FF/CAP18, were also tested to determine whether additional activity was obtained. The analogue peptides replaced with hydrophobic and cationic amino acid residues showed more potent bactericidal and LPS-binding activities than the original one.

Induction of intestinal IgA and IgG antibodies preventing adhesion of verotoxin-producing Escherichia coli to Caco-2 cells by oral immunization with liposomes.

AIMS: To examine the efficacy of liposome oral administration to induce systemic and mucosal immune responses against verotoxin-producing Escherichia coli (VTEC) and the effect of the induced antibodies on the binding of the bacteria to Caco-2 cells. METHODS AND RESULTS: Mice were immunized orally with VTEC antigen and monophosphoryl lipid A (MPL)-containing liposomes composed of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylserine and cholesterol (1 : 1 : 2, molar ratio) (PS-liposome). After immunization, significant IgA and IgG responses to VTEC were induced in both serum and the intestinal lavage fluid in all mice tested. Furthermore, anti-VTEC IgA and IgG antibodies in the lavage fluid effectively inhibited the adhesion of VTEC to Caco-2 cells. CONCLUSIONS: Oral immunization with liposome-associated E. coli O157:H7 antigen can induce significant systemic and mucosal antibody responses against the bacterial antigen and antibodies produced in the intestinal tract, thus functioning as inhibitors for preventing VTEC infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Oral PS-liposome vaccines containing MPL have the potential usefulness for the induction of a protective mucosal immune response against intestinal diseases.

Expression of the Dan gene during chicken embryonic development.

The Dan gene was first identified as the putative rat tumor suppressor gene and encodes a protein structurally related to Cerberus and Gremlin in vertebrates. Xenopus DAN, as with Cerberus and Gremlin, was demonstrated to block bone morphogenetic protein (BMP) signaling by binding BMPs, and to be capable of inducing additional anterior structures by ectopic overexpression in Xenopus embryos. DAN, thus, is suggested to play pivotal roles in early patterning and subsequent organ development, as in the case of other BMP antagonists. In this report, we isolated the chicken counterpart of Dan. Chicken Dan is mainly expressed in the cephalic and somitic mesoderm and several placodes during organ development.

[A novel neurotransmitter, DAN, mediates pain sensation in the spinal dorsal horn].

Differential screening-selected gene aberrative in neuroblastoma(DAN) belongs to a novel gene family(DAN family) that includes the head-inducing factor, Cerberus, and dorsaling factor, Gremlin. It has been suggested that DAN family members control diverse processes in growth, development and the cell cycle. Here, we demonstrate that DAN is produced in the small neurons of the dorsal root ganglion(DRG) and transported to the nerve terminals in the spinal dorsal horn in adult rats. Furthermore, intrathecal injection of an antibody to DAN suppressed pain sensations induced by the application of complete Freund's adjuvant and carageenan into the rat hindpaw, and the amount of DAN mRNA in the DRG neurons and of DAN in the spinal dorsal horn were increased in the inflammatory models. These data suggest that DAN in a novel neurotransmitter and/or modulator in the primary sensory nerve fibers for pain sensation.

Therapeutic effect of anti-TNF-alpha antibody and levofloxacin (LVFX) in a mouse model of enterohemorrhagic Escherichia coli O157 infection.

The ability of an anti-TNF-alpha antibody to confer protection against enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated in germfree IQI mice. The use of an antibiotic levofloxacin (LVFX) alone or with the antibody was also studied. Protection included an increase in survival rate. Treatment with the anti-TNF-alpha antibody inhibited the histological signs associated with EHEC infection but did not prevent the colonization of EHEC or production of Shiga toxin (Stx). No clinical signs were observed and EHEC was completely eliminated in the mouse model receiving both anti-TNF-alpha antibody and LVFX. Anti-TNF-alpha antibody suppressed inflammatory cytokine response in the mouse kidney and brain by EHEC infection.

Retinoic acid-induced apoptosis of the CHP134 neuroblastoma cell line is associated with nuclear accumulation of p53 and is rescued by the GDNF/Ret signal.

BACKGROUND: Neuroblastoma (NBL) is one of the most common solid malignancies in childhood and is derived from the sympathetic precursor cells. Although p53, a tumor suppressor, has been reported to be rarely mutated in NBLs, it is sequestered abnormally in the cytoplasm of the NBL cell. The mechanism and functional role of the abnormal intracellular localization of p53 remain unclear. PROCEDURE: Here, we established an in vitro system of apoptosis model using a NBL cell line CHP134 which also showed a cytoplasmic sequestration of p53. The treatment of the cells with 1 or 5 microM all-trans retinoic acid (RA) induced moderate neurite outgrowth followed by massive death of CHP134 cells by days 5 to 6. RESULTS: TUNEL staining showed that the cell death was due to apoptosis. Immunofluorescent stain demonstrated that p53 was strongly positive in the nucleus on day 5, which was accompanied with induction of p21WAF1. In addition, expression of caspase-3 was also increased during the cell death. Intriguingly, the RA treatment induced expression of Ret tyrosine kinase receptor in CHP134 cells. CONCLUSIONS: The addition of ligands, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN), inhibited apoptosis as well as nuclear accumulation of p53 in the cell. The present results suggest that the RA-induced apoptosis of NBL cells is associated with activation of both the caspase cascade and the p53-mediated pathway with its nuclear translocation. The neurotrophic signal through the GDNF-Ret system may prevent the neuronal cell death.

Comparison of maximum bite force and dentate status between healthy and frail elderly persons.

The purpose of the present study was to (1) determine the standard value of maximum bite force and to (2) compare the maximum bite force of the elderly between healthy and frail subjects. Subjects included 349 healthy elderly individuals (149 males, 200 females) and 24 frail elderly individuals (seven males, 17 females) ranging from 65 to 74 years of age. Maximum bite force was evaluated using a Dental Prescale system. The maximum bite force of the healthy subjects was significantly higher than that of the frail subjects in both males (P=0.020) and females (P=0.015). However, no significant difference was observed in the number of present teeth between the healthy and frail subjects. Median of maximum bite force in healthy males was 408.0 N, and that of the healthy females was 243.5 N. These results suggest that the frail elderly have latent bite force problems.

Structural characterization of the O-antigenic polysaccharide chain of Porphyromonas circumdentaria NCTC 12469.

Structural studies were carried out on an O-antigenic polysaccharide moiety derived from Porphyromonas circumdentaria NCTC 12469, a reference strain of Porphyromonas species. The polysaccharide chain was composed of D-glucose, D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-galactosamine in a molar ratio of 1:2:1:1. On the basis of results from 1H- and 13C-NMR spectroscopic analyses including COSY, TOCSY, and HMQC experiments together with results of Smith degradation, methylation analysis, and partial acid hydrolysis, it is concluded that the polysaccharide chain has a pentasaccharide repeating unit of -->6)-beta-D-Glcp-(1-->6)-beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)-beta-D-GalpNAc-(1-->. The immunoreaction between P. circumdentaria LPS and the corresponding antiserum was strongly inhibited by the pentasaccharide fragment (Glc-Gal-Gal-GlcNAc-GalNAc) isolated from partial acid hydrolysis of the above polysaccharide, suggestive of O-antigen specific antibodies in the used antiserum.

Inhibition of Vero cell cytotoxic activity in Escherichia coli O157:H7 lysates by globotriaosylceramide, Gb3, from bovine milk.

In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2. Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC). N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively. N-4 and N-5 GSLs migrated below globoside on the TLC plate. N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody. Furthermore, the effect of bovine milk Gb3 on VT2-induced cytotoxicity was investigated. We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2.

In vivo synergy between green tea extract and levofloxacin against enterohemorrhagic Escherichia coli O157 infection.

We studied the synergistic effects of Japanese green tea extract (JGTE) and levofloxacin (LVFX) against enterohemorrhagic Escherichia coli (EHEC) infection in a gnotobiotic mouse model. Mice fed on JGTE conferred a significant degree of protection against an oral challenge with EHEC. Complete elimination of the bacteria from the mice, was however, difficult. The combination of JGTE and LVFX increased the survival rate and reduced damage to target organs. Thus, dietary supplementation with JGTE improved the therapeutic effects of antibiotic treatment.

[A patient with neuroborreliosis presenting gadolinium-enhanced MRI lesions in bilateral facial nerves].

We report a 33-year-old man with bilateral facial paralysis due to neuroborreliosis. About three weeks after rhinorrhea and fever lasting four days, he noticed fatigue in the legs and paresthesia in all four extremities. Another week later, he developed paresthesia in his tongue and bilateral facial muscle weakness, and was admitted to our hospital. On admission, neurological examination revealed moderate bilateral facial muscle weakness, mild paresthesia in the tongue and four extremities, and decreased Achilles tendon reflex bilaterally. Mild pleocytosis and increased protein were found in the cerebrospinal fluid (CSF). IgM antibodies that reacted with the antigens of Borrelia garinii and Borrelia afzelii were found in his serum. Clinically and serologically, he was thus diagnosed as having neuroborreliosis. Brain MRI revealed gadolinium-enhanced lesions of the bilateral facial nerves in the facial nerve canal portion. After three weeks of treatment with 100 mg/day doxycycline and 2 g/day ceftriaxone sodium, his symptoms and CSF abnormalities were rapidly improved. Although facial nerve paralysis is a major symptom of neuroborreliosis, the present report is the first to detect the inflammatory lesions of the facial nerves in the facial nerve canal portion by MRI.