Endocytoscopy for in situ real-time histology of oral mucosal lesions.

This study investigated the utility of endocytoscopy, a novel emerging endoscopic system, for in situ real-time histology of oral mucosal lesions. Endocytoscopy involves the use of a contact light microscopy system with 380-fold magnification. With the development of endoscopic instruments, it has become possible to observe the abnormal microvascular and capillary patterns of tumour cells. The resolution of the endoscopic image is improved in situ, and a more detailed diagnosis is possible. In this study, endocytoscopy along with other diagnostic modalities was used in nine patients. Normal mucous membranes and oral malignant lesions were observed. Endocytoscopy enabled the pathological diagnosis of oral malignancies in situ and the observation of both structural and cytological atypia. In the future, it is expected that pathological diagnoses will be made in situ by direct viewing of living cells. This technique has the potential to allow an 'optical biopsy'.

Comparison of human mesenchymal stem cells derived from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous tooth pulp.

Populations of pluripotent stem cells were isolated from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous teeth and their multipotentiality properties compared. Osteogenic, chondrogenic, adipogenic, and neurogenic differentiation potentials were examined. Bone marrow mesenchymal stem cells (BMMSCs) and synovial fluid-derived cells (SFCs) showed the highest levels of osteogenesis as expressed by alkaline phosphatase (ALP) activity (0.54±0.094 U/mg protein and 0.57±0.039 U/mg protein, respectively; P=0.60) and by osteocalcin (BGLAP; determined by real-time RT-PCR). SFCs showed the highest levels of chondrogenesis as expressed by ALP activity (1.75±0.097 U/mg protein) and of COL2A1 and COL10A1 by real-time PCR. In terms of adipogenesis, lipid vesicles were observed in the BMMSCs and SFCs. Dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) exhibited neurogenesis potential, as shown by increases in expression of class III ß-tubulin (TUBB3) and microtubule-associated protein 2 (MAP2) on RT-PCR. Variability was found in the differentiation potential corresponding to the tendency of the original tissue to differentiate. It is suggested that the cell type should be selected depending on the regenerative treatment regimen.

Emergence and prevention measures for multidrug resistant Pseudomonas aeruginosa in catheter-associated urinary tract infection in spinal cord injury patients.

OBJECTIVE: To evaluate measures for preventing multidrug resistant Pseudomonas aeruginosa (MDRP) in catheter-associated urinary tract infection (CAUTI) in spinal cord injury patients. SETTING: Spinal Cord Injury Unit of Hyogo Prefectural Hyogo Prefectural Rehabilitation Center, Kobe, Japan. METHODS: We defined MDRP as resistance to amikacin, imipenem and levofloxacin. We had eight cases of MDRP-causing CAUTI in hospitalized neurogenic bladder patients caused by spinal cord injury in 2 months. Pulse-field gel electrophoresis (PFGE) was performed for epidemiological studies. We assessed prevention measures against MDRP emergence from the 2nd month, such as surveillance of CAUTI and infection control, and evaluated the outcomes of these measures over a total of 8 months. RESULTS: Our PFGE results showed that these eight MDRP isolates could be considered as closely related strains. We concluded that this was an MDRP outbreak that was causing CAUTI. The isolated ratio of MDRP began to decrease over 4 months of surveillance and significantly decreased in the 4th quarter (7th and 8th months) compared with the 1st quarter (1st and 2nd months) (P=0.021) even though urinary tract device usage significantly increased over the same period (P<0.001). CONCLUSION: We experienced an outbreak of emergent MDRP causing CAUTI in neurogenic bladder patients with spinal cord injury. Our preventive measures for isolating the outbreak, including surveillance, may have led to the decrease we observed in the ratio of MDRP isolated.

Smad9 is a new type of transcriptional regulator in bone morphogenetic protein signaling.

Smad1, Smad5 and Smad9 (also known as Smad8) are activated by phosphorylation by bone morphogenetic protein (BMP)-bound type I receptor kinases. We examined the role of Smad1, Smad5, and Smad9 by creating constitutively active forms (Smad(DVD)). Transcriptional activity of Smad9(DVD) was lower than that of Smad1(DVD) or Smad5(DVD), even though all three Smad(DVD)s associated with Smad4 and bound to the target DNA. The linker region of Smad9 was sufficient to reduce transcriptional activity. Smad9 expression was increased by the activation of BMP signaling, similar to that of inhibitory Smads (I-Smads), and Smad9 reduced BMP activity. In contrast to I-Smads, however, Smad9 did not inhibit the type I receptor kinase and suppressed the constitutively active Smad1(DVD). Smad9 formed complexes with Smad1 and bound to DNA but suppressed the transcription of the target gene. Taken together, our findings suggest that Smad9 is a new type of transcriptional regulator in BMP signaling.

Antimicrobial photodynamic therapy using a diode laser with a potential new photosensitizer, indocyanine green-loaded nanospheres, may be effective for the clearance of Porphyromonas gingivalis.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) is a new treatment method for the removal of infectious pathogens using a photosensitizer and light of a specific wavelength, e.g., toluidine blue with a wavelength of about 600 nm. We explored a new photosensitizer and focused on indocyanine green (ICG), which has high absorption at a wavelength of 800-805 nm. We investigated the bactericidal effect of PDT on Porphyromonas gingivalis using a new photosensitizer, ICG-loaded nanospheres with an 805 nm wavelength low-level diode laser irradiation. METHODS: We designed ICG-loaded nanospheres coated with chitosan (ICG-Nano/c) as a photosensitizer. A solution containing Porphyromonas gingivalis (10(8)  CFU/mL) with or without ICG-Nano/c (or ICG) was prepared and irradiated with a diode laser or without laser irradiation as a negative control. The irradiation settings were 0.5 W with a duty ratio of 10%, for 3-100 ms in repeated pulse (RPT) or continuous wave mode. CFU were counted after 7 d of anaerobic culture. RESULTS: We observed that ICG-Nano/c could adhere to the surface of P. gingivalis. When ICG-Nano/c was used for aPDT, irradiation with RPT 100 ms mode gave the lowest increase in temperature. Laser irradiation with ICG-Nano/c significantly reduced the number of P. gingivalis (i.e., approximately 2-log10 bacterial killing). The greatest bactericidal effect was found in the RPT 100 ms group. However, laser irradiation (RPT 100 ms) with ICG, as well as without photosensitizer, had no effect on the number of bacteria. CONCLUSIONS: Within the limits of this study, ICG-Nano/c with low-level diode laser (0.5 W; 805 nm) irradiation showed an aPDT-like effect, which might be useful for a potential photodynamic periodontal therapy.

The effects of C-type natriuretic peptide on craniofacial skeletogenesis.

C-type natriuretic peptide (CNP) is a potent stimulator of long bone and vertebral development via endochondral ossification. In the present study, we investigated the effects of CNP on craniofacial skeletogenesis, which consists of both endochondral and membranous ossification. Morphometric analyses of crania from CNP knockout and transgenic mice revealed that CNP stimulates longitudinal growth along the cranial length, but does not regulate cranial width. CNP markedly increased the length of spheno-occipital synchondrosis in fetal murine organ cultures, and the thickness of cultured murine chondrocytes from the spheno-occipital synchondrosis or nasal septum, resulting in the stimulation of longitudinal cranial growth. Mandibular growth includes endochondral and membranous ossification; although CNP stimulated endochondral bone growth of condylar cartilage in cultured fetal murine mandibles, differences in the lengths of the lower jaw between CNP knockout or transgenic mice and wild-type mice were smaller than those observed for the lengths of the upper jaw. These results indicate that CNP primarily stimulates endochondral ossification in the craniofacial region and is crucial for midfacial skeletogenesis.

Raman spectroscopic study on the solvation of p-aminobenzonitrile in supercritical water and methanol.

Raman spectra of the C[triple bond]N stretching vibration of p-aminobenzonitrile (ABN) have been investigated in water, methanol, and cyclohexane under sub- and supercritical conditions, and in acetonitrile under subcritical condition. In all solvent fluids covering the supercritical region, the vibrational frequency of the C[triple bond]N stretching mode decreased with increasing solvent density from the gaseous region to the medium density region rho(r) approximately = 2, where rho(r) is the reduced density by the critical density of the solvent. However, from the medium density region to the higher density region, the vibrational frequency turned to increase with the solvent density. The temperature-induced low frequency shift of the C[triple bond]N stretching Raman band was also ascertained by the measurement of the temperature dependence of Raman spectrum of ABN vapor above 543 K. The electronic absorption spectra in the UV region of ABN were also measured under the same experimental conditions. The absorption peak energies decreased with an increase of the solvent density, except in water above rho(r) = 2.8. The vibrational frequency shift in cyclohexane was explained by a sum of contributions of the repulsive interaction, the mean field attractive interaction, and the pure temperature effect probably due to the hot-band contribution. The residual frequency shift after the subtraction of the repulsive and temperature effects in water and methanol showed the low frequency shift with increasing solvent density from rho(r) congruent with 0 to 2.8. However, above rho(r) congruent with 2.8 in water, the residual shift showed a high frequency shift with increasing solvent density. The electronic state calculations based on the PCM model using the density functional theory (DFT) indicated that the solvent polarity change caused the low frequency shift of the C[triple bond]N stretching mode, which was also correlated with the shift of the electronic absorption spectrum. The results of the DFT calculations on the cluster of ABN with water molecules and the molecular dynamics simulations indicated that the high frequency shift of the C[triple bond]N stretching mode in water above rho(r) congruent with 2.8 could be due to the hydrogen bonding between water and ABN.

Molecular analysis of Clostridium difficile at a university teaching hospital in Japan: a shift in the predominant type over a five-year period.

Clostridium difficile isolates recovered from patients admitted to a teaching hospital in Japan over a 5-year period were analyzed. Two molecular typing systems, PCR ribotyping and pulsed-field gel electrophoresis (PFGE) analysis, were used. Twenty-six PCR ribotypes were found among the 148 isolates. The predominant type at our hospital appeared to shift during the study period, from PCR ribotype a in 2000 (15/33, 45%) to PCR ribotype f in 2004 (18/28, 64%). By using PFGE with thiourea added to both the gel and running buffer, all 148 Clostridium difficile isolates were successfully classified into 37 types and 61 subtypes. The PCR ribotype f isolates were further classified into four types and 11 subtypes by PFGE. The PFGE patterns of the 11 subtypes differed from each other by only 1 to 4 bands, suggesting that these differences might reflect genetic changes during patient-to-patient transmission over the 5-year period analyzed, and that PCR ribotype f isolates might be outbreak-related. In addition, the PCR ribotype f was identical to the PCR ribotype designated smz, which is reported to have caused multiple outbreaks in Japan. These results confirmed that PCR ribotype f (type smz) has specific virulence or survival factors that make it more likely to cause nosocomial outbreaks at Japanese hospitals. PCR ribotype 027, which has been reported to have caused recent outbreaks in North America and Europe, was recovered from one patient in this study; however, this strain was community-acquired. Our findings emphasize the importance of monitoring specific strains to control and prevent nosocomial infection.

Inhibitory effects of macrocarpals on the biological activity of Porphyromonas gingivalis and other periodontopathic bacteria.

BACKGROUND/AIMS: Macrocarpals, which are phloroglucinol derivatives contained in eucalyptus leaves, exhibit antimicrobial activity against a variety of bacteria including oral bacteria. This study examined effects of macrocarpals A, B, and C on periodontopathic bacteria, especially Porphyromonas gingivalis. METHODS: Macrocarpals A, B, and C were purified from a 60% ethanol-extract of Eucalyptus globules leaves. To investigate antibacterial activity, representative periodontopathic bacteria were cultured in media with or without various amounts of macrocarpals; subsequently, the optical density at 660 nm was measured. Macrocarpal inhibition of P. gingivalis Arg- and Lys-specific proteinases was assessed by spectrofluorophotometric assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The effect of macrocarpals on P. gingivalis binding to saliva-coated hydroxyapatite beads was examined with (3)H-labeled P. gingivalis. RESULTS: Growth of P. gingivalis was inhibited more strongly than growth of Prevotella intermedia or Prevotella nigrescens and Treponema denticola by macrocarpals, however, Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum were much more resistant. Macrocarpals inhibited P. gingivalis Arg- and Lys-specific proteinases in a dose-dependent manner. The enzyme-inhibitory effect of macrocarpals was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in which hemoglobin degradation by P. gingivalis proteinase was inhibited by macrocarpals. P. gingivalis binding to saliva-coated hydroxyapatite beads was also strongly attenuated by macrocarpals. CONCLUSIONS: Macrocarpals A, B and C demonstrated antibacterial activity against periodontopathic bacteria. Among tested bacteria, P. gingivalis displayed the greatest sensitivity to macrocarpals; additionally, its trypsin-like proteinase activity and binding to saliva-coated hydroxyapatite beads were inhibited by macrocarpals. These results indicate that eucalyptus leaf extracts may be useful as a potent preventative of periodontal disease.

Death receptor 3 (DR3) gene duplication in a chromosome region 1p36.3: gene duplication is more prevalent in rheumatoid arthritis.

The death receptor 3 (DR3) gene is a member of the apoptosis-inducing Fas gene family. In the current study, fluorescence in situ hybridization (FISH) and Fiber-FISH revealed the existence of a second DR3 gene approximately 200 kb upstream of the original DR3 gene. The existence of the duplicated DR3 gene was confirmed by sequencing the corresponding human artificial chromosome clones as well as with quantitative PCR that measured the ratio of the DR3 gene mutation (Rm), intrinsic to rheumatoid arthritis (RA) patients, by simultaneous amplification of the normal and mutated DR3 sequences. The DR3 gene duplication measured by FISH was found to be more frequent in patients with RA as compared to healthy individuals. We therefore surmise that the human DR3 gene can be duplicated and that this gene duplication is more prevalent in patients with RA.

Inhibition by the essential oils of peppermint and spearmint of the growth of pathogenic bacteria.

The effects of the, essential oils of peppermint (Mentha piperita L.), spearmint Mentha spicata L.) and Japanese mint (Mentha, arvensis L.), of four major constituents of the esssential oil of peppermint, and of three major constituents of the essential oil of spearmint, on the proliferation of Helicobacter pylori, Salmonella enteritidis, Escherichia coli O157:H7, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin sensitive Staphylococccus aureus (MSSA) were examined. The essential oils and the various constituents inhibited the proliferation of each strain in liquid culture in a dose-dependent manner. In addition, they exhibited bactericidal activity in phosphate-buffered saline. The antibacterial activities varied among the bacterial species tested but were almost the same against antibiotic-resistant and antibiotic-sensitive strains of Helicobacter pylori and S. aureus. Thus, the essential oils and their constituents may be useful as potential antibacterial agents for inhibition of the growth of pathogens.

Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS).

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

Identification of cariostatic substances in the cacao bean husk: their anti-glucosyltransferase and antibacterial activities.

The cacao bean husk has been shown to possess two types of cariostatic substances, one showing anti-glucosyltransferase (GTF) activity and the other antibacterial activity, and to inhibit experimental dental caries in rats infected with mutans streptococci. In the present study, chromatographic purification revealed high-molecular-weight polyphenolic compounds and unsaturated fatty acids as active components. The former, which showed strong anti-GTF activity, were polymeric epicatechins with C-4beta and C-8 intermolecular bonds estimated to be 4636 in molecular weight in an acetylated form. The latter, which showed bactericidal activity against Streptococcus mutans, were determined to be oleic and linoleic acids, and demonstrated a high level of activity at a concentration of 30 microgram/mL. The cariostatic activity of the cacao bean husk is likely caused by these biologically active constituents.

Caries inhibitory activity of cacao bean husk extract in in-vitro and animal experiments.

Cacao bean husk extract (CBH) was examined for inhibitory effects on the caries-inducing properties of mutans streptococci in vitro and on caries development in specific pathogen-free Sprague-Dawley rats infected with mutans streptococci. CBH reduced the growth rate of almost all oral streptococci examined, which resulted in the reduction of acid production. Furthermore, insoluble glucan synthesis by the glucosyltransferases from Streptococcus mutans MT8148R and Streptococcus sobrinus 6715 was significantly inhibited by CBH. Hence, the sucrose-dependent cell adherence of mutans streptococci was also depressed by CBH. The administration of CBH in drinking water resulted in significant reductions of caries development and dental plaque accumulation in rats infected with either Strep. sobrinus 6715 or Strep. mutans MT8148R, and the minimum cariostatic concentration was 1.0 mg/ml. These results indicate that CBH possesses powerful anticariogenic potential.

Cariostatic activity of cacao mass extract.

Chocolate is suspected to contain some caries-inhibitory substances. The cariostatic activity of cacao mass extract (CM), the main component of chocolate, was examined in vitro and in experimental animals. CM showed no detectable effects on the cellular growth and acid production of mutans streptococci. On the other hand, the cell-surface hydrophobicity of mutans streptococci was significantly reduced by the presence of CM. Furthermore, insoluble glucan synthesis by the glucosyltransferases from either Streptococcus mutans MT8148R or Strep. sobrinus 6715 was inhibited by CM, but not significantly. Hence, the sucrose-dependent cell adherence of mutans streptococci was also depressed by CM. Finally, CM in both a 40% sucrose diet and drinking water resulted in reductions of caries development and plaque accumulation in rats infected with Strep. sobrinus 6715, but not significantly. These results indicate that cacao mass extract possesses some anticariogenic potential, but its anticaries activity is not strong enough to suppress significantly the cariogenic activity of sucrose.

Structural study of anhydrous tendon chitosan obtained via chitosan/acetic acid complex.

The molecular structure and packing arrangement of anhydrous tendon chitosan was determined by the X-ray fibre diffraction method together with the linked-atom least-squares refinement technique. The specimen was prepared from chitosan/acetic acid complex which was obtained by exposing tendon chitosan to acetic acid vapour at room temperature for several days. There is high degree of orientation and crystallinity compared with the specimen obtained by the annealing method. Two chitosan chains are present in an orthorhombic unit cell of dimensions a = 8.26(2), b = 8.50(1), c (fibre axis) = 10.43(2) A and space group P2(1)2(1)2(1). The 2-fold helical chain is stabilised by O3 triple bond O5 hydrogen bond with the gt orientation of O6. There are direct hydrogen bonds (N2 triple bond O6) between adjacent chains along the a-axis, which makes a sheet structure parallel to the ac-plane. On the other hand, no hydrogen bond is found between the sheets.

Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos.

Retinoic acid (RA) is a physiological agent that has a wide range of biological activity and appears to regulate developmental programs of vertebrates. However, little is known about the molecular basis of its metabolism. Here we have identified a novel cytochrome P450 (P450RA) that specifically metabolizes RA. In vitro, P450RA converts all-trans RA into 5,8-epoxy all-trans RA. P450RA metabolizes other biologically active RAs such as 9-cis RA and 13-cis RA, but fails to metabolize their precursors, retinol and retinal. Overexpression of P450RA in cell culture renders the cells hyposensitive to all-trans RA. These functional tests in vitro and in vivo indicate that P450RA inactivates RA. The P450RA gene is not expressed uniformly but in a stage- and region-specific fashion during mouse development. The major expression domains in developing embryos include the posterior neural plate and neural crest cells for cranial ganglia. The expression of P450RA, however, is not necessarily inducible by excess RA. These results suggest that P450RA regulates the intracellular level of RA and may be involved in setting up the uneven distribution of active RA in mammalian embryos.

Macrocarpals H, I, and J from the Leaves of Eucalyptus globulus.

A 50% EtOH extract of Eucalyptus globulus leaves yielded eight phloroglucinol--sesquiterpene-coupled constituents, including three novel compounds named macrocarpals, H, I, and J. Some of these compounds possessed antibacterial activity against oral pathogenic microorganisms with MIC values ranging from 0.20 micrograms/mL to 6.25 micrograms/mL. Inhibition of glucosyltransferase activity by these compounds was also noted.