Growth, respiration and nutrient acquisition by the arbuscular mycorrhizal fungus Glomus mosseae and its host plant Plantago lanceolata in cooled soil.

Although plant phosphate uptake is reduced by low soil temperature, arbuscular mycorrhizal (AM) fungi are responsible for P uptake in many plants. We investigated growth and carbon allocation of the AM fungus Glomus mosseae and a host plant (Plantago lanceolata) under reduced soil temperature. Plants were grown in compartmented microcosm units to determine the impact on both fungus and roots of a constant 2.7 °C reduction in soil temperature for 16 d. C allocation was measured using two (13)CO(2) pulse labels. Although root growth was reduced by cooling, AM colonization, growth and respiration of the extraradical mycelium (ERM) and allocation of assimilated (13)C to the ERM were all unaffected; the frequency of arbuscules increased. In contrast, root respiration and (13)C content and plant P and Zn content were all reduced by cooling. Cooling had less effect on N and K, and none on Ca and Mg content. The AM fungus G. mosseae was more able to sustain activity in cooled soil than were the roots of P. lanceolata, and so enhanced plant P content under a realistic degree of soil cooling that reduced plant growth. AM fungi may therefore be an effective means to promote plant nutrition under low soil temperatures.

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity.

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin-agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63.

Relationship between developmental synaptic modulation and conditioning-induced synaptic change in Lymnaea.

Though adult Lymnaea are bimodal breathers, young animals breathe mainly through the skin and adults through the lung. Operant conditioning changes adult breathing behavior from aerial to cutaneous. We hypothesized that this behavioral change is caused by alterations in the neuronal circuit during both development and conditioning. We focused our study on whether the synaptic connection between RPeD1 and RPA6 neurons is modulated during development and conditioning. Our findings indicated that the RPeD1 has an excitatory monosynaptic contact with the RPA6 in young naive and operantly-conditioned adult animals. The relationship of this contact was well correlated with their respiratory behavior.

Ephrin-B1 localizes at the slit diaphragm of the glomerular podocyte.

Ephs and ephrins are a family of membrane-bound proteins that function as receptor-ligand pairs. Members of the Eph-ephrin-B family have recently been reported to regulate the paracellular permeability of epithelial cells. In this study, we analyzed the expression and the function of ephrin-B1 in glomeruli. Using immunofluorescence (IF), we found that ephrin-B1 was expressed along the glomerular capillary loop. Immunoelectron microscopy revealed that ephrin-B1 expression was restricted at the slit diaphragm. Dual labeled IF showed ephrin-B1 colocalized with the slit diaphragm proteins nephrin and CD2-associated protein. Ephrin-B1 colocalized with nephrin at the late capillary loop stage of kidney development. Additionally, injection of rats with a nephritogenic anti-nephrin antibody (ANA) reduced ephrin-B1 expression. When podocytes were cultured in vitro, they extruded processes that co-stained for ephrin-B1 and for CD2-associated protein. When these podocytes were treated in culture with small interfering RNA for ephrin-B1, CD2-associated protein was reduced in the processes, with a remaining faint perinuclear staining. We suggest that ephrin-B1 has a role in maintaining barrier function at the slit diaphragm.

Antimicrobial susceptibility of 800 anaerobic isolates from patients with dentoalveolar infection to 13 oral antibiotics.

INTRODUCTION: The aim of this study was to determine the current antimicrobial susceptibility of the principle anaerobic pathogens involved in dentoalveolar infection, to 13 oral antibiotics, and to assess the value of each antibiotic in the management of the infection. METHODS: A total of 800 isolates from patients with dentoalveolar infection (Prevotella species, Fusobacterium species, Porphyromonas species and Peptostreptococcus micros) were tested for their susceptibility to amoxicillin, amoxicillin/clavulanate, cefaclor, cefuroxime, cefcapene, cefdinir, erythromycin, azithromycin, telithromycin, minocycline, levofloxacin, clindamycin, and metronidazole using an agar dilution method. RESULTS: Although the majority of Fusobacterium strains were resistant to erythromycin, azithromycin, and telithromycin, the remaining antibiotics demonstrated a high level of antimicrobial activity. P. micros and Porphyromonas species exhibited high susceptibility to all antibiotics tested in this study. In the case of Prevotella species, resistance to amoxicillin occurred in 34% of isolates and all of these resistant strains were found to produce beta-lactamase. Susceptibility of Prevotella strains to cefaclor, cefuroxime, cefcapene, cefdinir, erythromycin, azithromycin, and minocycline was found to correlate with amoxicillin susceptibility. Amoxicillin/clavulanate, telithromycin, clindamycin, and metronidazole exhibited high antimicrobial activity even against amoxicillin-resistant strains of Prevotella species. CONCLUSION: Amoxicillin would still be advocated therefore as being a suitable first-line agent, while reduced susceptibility of Prevotella strains remains a matter of concern with penicillins. Amoxicillin/clavulanate, clindamycin, and metronidazole are useful alternatives in combating the anaerobic bacteria involved in dentoalveolar infection.

The microbial synergy of Peptostreptococcus micros and Prevotella intermedia in a murine abscess model.

This study characterized the microbial interaction of Peptostreptococcus micros and Prevotella intermedia, the major pathogens of dentoalveolar infection, using a murine model. Subcutaneous injection of P. micros cells in the dorsum of the mouse together with living cells of P. intermedia resulted in a significantly larger abscess when compared with single injection of the organisms (P < 0.02). The abscess size was also significantly increased (P < 0.05) when the plate-cultured cell suspension of P. micros was injected into mouse with the culture filtrate of P. intermedia. The heat-treated culture filtrate of P. intermedia also enhanced the virulence of P. micros. P. micros culture filtrate did not affect the virulence of P. intermedia. Interestingly, the virulence of P. micros appeared to be enhanced even when the culture filtrate of P. intermedia was injected at separate sites in the mouse. These results suggest that a heat-stable product or products of P. intermedia increase the virulence of P. micros indirectly by altering the host condition, whereas living cells of P. micros can directly enhance virulence of P. intermedia.

Antimicrobial susceptibility of major pathogens of orofacial odontogenic infections to 11 beta-lactam antibiotics.

In this study, we evaluated the current effectiveness of 11 beta-lactam antibiotics for treatment of orofacial odontogenic infections by determining the antimicrobial susceptibility of the major pathogens. The antimicrobial susceptibilities of viridans streptococci (n = 47), Peptostreptococcus (n = 67), Porphyromonas (n = 18), Fusobacterium (n = 57), black-pigmented Prevotella (n = 59) and non-pigmented Prevotella (n = 47) isolated from pus specimens of 93 orofacial odontogenic infections to penicillin G, cefmetazole, flomoxef, cefoperazone, cefoperazone/sulbactam, ceftazidime, cefpirome, cefepime, cefoselis, imipenem and faropenem were determined using the agar dilution method. Penicillin G, most cephalosporins, imipenem and faropenem worked well against viridans streptococci, Peptostreptococcus, Porphyromonas and Fusobacterium. Penicillin G and most cephalosporins, including fourth-generation agents, were not effective against beta-lactamase-positive Prevotella, though they were effective against beta-lactamase-negative strains. Cefmetazole, cefoperazone/sulbactam, imipenem and faropenem expressed powerful antimicrobial activity against beta-lactamase-positive Prevotella. In conclusion, penicillins have the potential to be first-line agents in the treatment of orofacial odontogenic infections. Most of the other beta-lactam antibiotics, including fourth-generation cephalosporins, were not found to have greater effectiveness than penicillins. In contrast, cefmetazole, cefoperazone/sulbactam, imipenem and faropenem were found to have greater effectiveness than penicillins.

Bacteriology and antimicrobial susceptibility of gram-positive cocci isolated from pus specimens of orofacial odontogenic infections.

We recently reported the beta-lactamase production and antimicrobial susceptibility of anaerobic gram-negative rods isolated from pus specimens of 93 orofacial odontogenic infections. In this report, we determine the bacteriology and antimicrobial susceptibility of bacteria other than anaerobic gram-negative rods, mainly gram-positive cocci, isolated from the same specimens. Streptococcus constellatus and Peptostreptococcus micros were frequent isolates from all types of infection examined. Peptostreptococcus prevotii, Corynebacterium species, and Eubacterium species were recovered only from dentoalveolar infections, while Gemella morbillorum was found more frequently in periodontitis than in the other infections. beta-Lactamase-positive strains were detected only in staphylococci. Ampicillin, ampicillin/sulbactam, cefazolin, cefotaxime, imipenem, erythromycin, clindamycin and levofloxacin showed high susceptibility rates (> or = 77%) against viridans streptococci, Peptostreptococcus and Gemella. Minocycline showed a high MIC90 value against viridans streptococci (32 microg/ml), and metronidazole was effective against Peptostreptococcus and Gemella. These results provide useful information for the treatment of orofacial odontogenic infections.

Effects of the antiplatelet agent TA-993 and its metabolite MB3 on the hemorheological properties of rat and human erythrocytes.

In the present study, we investigated the effects of the antiplatelet agent TA-993 and its metabolite MB3 on the hemorheological properties of rat and human erythrocytes in comparison with ticlopidine and aspirin. TA-993 and MB3 concentration-dependently lowered the viscosity of rat erythrocyte suspensions. TA-993 and MB3 inhibited both the hypotonic hemolysis of human erythrocytes and the mechanical hemolysis of rat erythrocytes induced by turbulent flow. Treatment of rats with TA-993 (10 mg/kg/day po) for 10 days significantly increased blood filterability, but ticlopidine and aspirin did not show this effect. TA-993 and MB3 enhanced the interaction of 1-anilino-8-naphthalene sulfonate (ANS), a hydrophobic probe, with human erythrocyte ghosts and reduced the fluorescence polarization in 1,6-diphenyl 1,3,5-hexatriene (DPH, a fluidity probe)-labeled human erythrocyte ghosts. TA-993 and MB3 induced aggregation of liposome suspensions prepared from acidic phospholipids. These findings suggest that TA-993 and MB3 may affect the erythrocyte membrane by interacting with acidic phospholipids and thus improve the hemorheological properties.

Colonisation and transmission of Clostridium difficile in healthy individuals examined by PCR ribotyping and pulsed-field gel electrophoresis.

Healthy adults who had not been exposed to antimicrobial agents for the preceding 4 weeks were examined for intestinal carriage of Clostridium difficile. The 1234 individuals examined were composed of seven groups: three classes of university students, hospital workers at two hospitals, employees of a company and self-defence force personnel at a local station. Overall, 94 (7.6%) individuals were positive for C. difficile by faecal culture but carriage rates among the study groups ranged from 4.2% to 15.3%. Typing by PCR ribotyping and pulsed-field gel electrophoresis demonstrated clusters of carriers colonised by a single type in each of three groups, indicating that cross-transmission of C. difficile can occur in community settings. Follow-up culture was performed on 38 C. difficile-positive individuals and C. difficile was isolated again from 12 (32%) of them 5-7 months after the initial culture; six (50%) of these 12 individuals had a new strain on repeat culture. Two or more family members were C. difficile-positive in five of 22 families examined. C. difficile with an identical type was isolated from persons within a family in only one family. These results suggest that intestinal carriage by healthy adults may play a role as a reservoir for community-acquired C. difficile-associated diarrhoea, but that cross-transmission of C. difficile does not occur frequently among family members at home.

Specific immunoglobulin E responses in ZAP-70-deficient patients are mediated by Syk-dependent T-cell receptor signalling.

ZAP-70 deficiency is a rare primary immunodeficiency characterized by the absence of peripheral CD8+ T cells and defects in T-cell receptor (TCR) signalling. T cells in ZAP-70-deficient patients are assumed to have no helper functions for B-cell immunoglobulin synthesis, whereas the patients rarely have antigen-specific antibodies. We experienced a ZAP-70-deficient patient, who had immunoglobulin E (IgE) antibodies specific to food allergens, and we investigated the mechanisms of switching to IgE in the patient. Peripheral blood mononuclear cells from the patient did not proliferate upon stimulation with the antigens but produced distinct levels of interleukin-4 (IL-4). Cell sorting analysis indicated that the cells that produced IL-4 in response to the antigens were enriched in CD4+ T cells. Purified CD4+ T cells from the patient produced IL-4 and expressed CD40L upon stimulation with anti-CD3. Moreover, CD4+ T cells pretreated with anti-CD3 induced mature epsilon transcript on naive B cells. Since the results indicated that there remained sufficient T-cell receptor (TCR)-signalling in the patient's T cells to exert antigen-specific IgE switching on B cells, we next investigated the expression of the ZAP-70-homologous kinase Syk. Syk was present in high levels in patient's CD4+ T cells and was tyrosine-phosphorylated after TCR stimulation. Inhibition of Syk by piceatannol resulted in decreased production of IL-4 and expression of CD40L on patient's CD4+ T cells. Moreover, Syk was expressed on all human T-cell leukaemia virus (HTLV-1)-transformed T-cell lines derived from peripheral blood of the patient, whereas it was low or undetectable in control lines. It was therefore concluded that specific IgE responses in the patient were most likely to be mediated by Syk-dependent TCR-signalling.

Inhibitory effects of TA-993 and its metabolite MB3 on platelet activation induced by collagen and U-46619 in human platelets.

In the present study, we investigated the effects of TA-993 and its metabolite MB3 on platelet activation in vitro. TA-993 and MB3 concentration-dependently inhibited platelet aggregation and ATP release induced by collagen in human platelets. Thromboxane (Tx) A2 formation, as determined by the production of TxB2, and the increase in intracellular Ca2+ concentration ([Ca2+]i) were also suppressed by TA-993 and MB3. TA-993 and MB3 did not inhibit TxA2 formation caused by arachidonic acid. These results suggest that the inhibition of platelet activation by TA-993 and MB3 is partly mediated by an inhibition of TxA2 formation at a step prior to cyclooxygenase. Furthermore, TA-993 and MB3 inhibited U-46619-induced platelet aggregation without blockade of the increase in [Ca2+]i, suggesting that they are likely to exert some additional effects on the intracellular events induced by Ca2+.

Incidence of beta-lactamase production and antimicrobial susceptibility of anaerobic gram-negative rods isolated from pus specimens of orofacial odontogenic infections.

The incidence of beta-lactamase production in anaerobic gram-negative rods isolated from 93 pus specimens of orofacial odontogenic infections and the antimicrobial susceptibility of these isolates against 11 antibiotics were determined. A total of 191 anaerobic gram-negative rods were isolated from the specimens. Beta-lactamase was detected in 35.6% of the black-pigmented Prevotella and 31.9% of the nonpigmented Prevotella. However, no strains among the other species isolated produced beta-lactamase. Ampicillin, cefazolin and cefotaxime showed decreased activity as regards beta-lactamase-positive Prevotella strains, whereas the activity of ampicillin/sulbactam, cefmetazole, and imipenem continued to be effective against such strains. All tested beta-lactam antibiotics were effective against Porphyromonas and Fusobacterium. Erythromycin showed decreased activity against nonpigmented Prevotella and Fusobacterium. Clindamycin, minocycline and metronidazole were powerful antibiotics against which anaerobic gram-negative rods could be tested. The present study showed that beta-lactamase-positive strains were found more frequently in the Prevotella strains than in any of the other species of anaerobic gram-negative rods. The effectiveness of adding sulbactam to ampicillin was demonstrated, as well as the difference in cephalosporin activity against beta-lactamase-positive strains.

The virulence of mixed infection with Streptococcus constellatus and Fusobacterium nucleatum in a murine orofacial infection model.

Orofacial infections are usually polymicrobial, and it is the microbial interactions of pathogenic species that cause tissue destruction. In this study, the microbial interaction between Streptococcus constellatus and Fusobacterium nucleatum was characterized using a murine orofacial infection model. A mixture of viable S. constellatus and F. nucleatum cells (both 2 x 10(8) CFU/mouse) was injected into the submandible; as a result, all of the test mice died. In contrast, none of the experimental animals monoinjected with either S. constellatus or F. nucleatum died (P<0.001), indicating that the synergism between the two resulted in the virulence. When a mixture of viable S. constellatus cells and a culture filtrate of F. nucleatum was tested, lethality and the bacterial cell count per lesion were significantly enhanced as compared with monoinjections (P<0.02). However, the virulence of F. nucleatum was not enhanced by infection of a culture filtrate of S. constellatus. The enhancement of virulence was observed even when viable S. constellatus cells and the culture filtrate of F. nucleatum were injected at separate sites. Heat treatment of the culture filtrate of F. nucleatum did not affect the enhancement. These results indicate that a heat-stable substance(s) produced by F. nucleatum contributes to the microbial synergy of S. constellatus and F. nucleatum in orofacial infections.

Spontaneous regression of hepatocellular carcinoma and review of literature.

A 68-year-old man presented with multiple hepatocellular carcinoma, which was considered to be unresectable at the first admission in January 1994. Pathological diagnosis was made by biopsy of the one lesion among them. From January 1994 to December 1997, 10 transarterial chemoembolizations and six percutaneous ethanol injection therapies were performed on the tumours in the cirrhotic liver. In February 1998 the tumour situated in the right lobe began to increase in size. The maximum tumour diameter was 6.3 cm measured by computed tomography (CT). In the beginning of May 1998 moderate ascites was present and mild hepatic encephalopathy was noticed. The patient was in the terminal stage of hepatocellular carcinoma and no further treatment was possible at that time. However, serum alpha-fetoprotein and protein induced by vitamin K absence or antagonist II dramatically decreased in June 1998. The CT scan also showed that the tumour had completely regressed without specific treatment. In February 1999 a new biopsy-proven hepatocellular carcinoma, 2 cm in diameter, developed in the lateral segment of the liver. It was well treated by percutaneous ethanol injection therapy. The patient was alive in good condition without any symptoms or tumour recurrence in June 1999. It was concluded that a rare case of spontaneous regression of hepatocellular carcinoma had occurred.

Bacteriologic features and antimicrobial susceptibility in isolates from orofacial odontogenic infections.

OBJECTIVE: The aim of this study was to obtain information for an effective antimicrobial therapy against orofacial odontogenic infections; such information was obtained from recent bacteriologic features and antimicrobial susceptibility data. STUDY DESIGN: The bacteriology and antimicrobial susceptibility of major pathogens in 163 patients with orofacial odontogenic infections to 7 antibiotics was examined. RESULTS: Mixed infection of strict anaerobes with facultative anaerobes (especially viridans streptococci) was observed most often in dentoalveolar infections, periodontitis, and pericoronitis. Penicillin (penicillin G) was effective against almost all pathogens, although it did not work well against beta-lactamase-positive Prevotella. Cefmetazole was effective against all test pathogens. Erythromycin was ineffective against viridans streptococci and most Fusobacterium. Clindamycin exerted a strong antimicrobial activity on anaerobes. Minocycline was effective against almost all the test pathogens. The antimicrobial activity of levofloxacin against viridans streptococci was not strong. CONCLUSIONS: An antibiotic that carries out antimicrobial activity against both viridans streptococci and oral anaerobes should be suitable for treatment of dentoalveolar infection, periodontitis, and pericoronitis. Penicillin remains effective as an antimicrobial against most major pathogens in orofacial odontogenic infections. Cefmetazole, clindamycin, and minocycline may be effective against most pathogens, including penicillin-unsusceptible bacteria.

Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains.

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.

Characterization of bacterial orofacial infections using a new murine model.

We devised a new murine orofacial infection model using bacteria from odontogenic infection origins and characterized the experimental infections. In this model, bacteria were injected into the submandible of mice. Streptococcus constellatus and Peptostreptococcus micros produced a single abscess at the injection site and their abscess-forming and lethal abilities were low: the median abscess-forming dose (AF(50)) of S. constellatus and P. micros were 10(8.5-10.7)and 10(10.2-10.6)cfu/mouse, and their median lethal dose (LD(50)) were >11 and 10(10.6-11)cfu/mouse, respectively. Prevotella oralis and Fusobacterium nucleatum produced multiple abscesses and their abscess-forming and lethal abilities were strong: AF(50)of P. oralis and F. nucleatum were 10(6.0-6.4)and 10(7. 0-8.7)cfu/mouse, and their LD(50)were 10(7.0-7.7)and 10(8.3-9. 9)cfu/mouse, respectively. LD(50)of P. intermedia and P. gingivalis were 10(9.4->11)and 10(8.9-9.1)cfu/mouse, respectively. Prevotella intermedia and Porphyromonas gingivalis generated a necrotizing lesion, which progressed rapidly. We conclude that this murine model could reflect human orofacial odontogenic infections and is useful to investigate the pathogenicity of causative bacteria of such infections.