Oral toxicity to high level sodium fluoride causes impairment of autophagy.

Autophagy is a highly conserved intracellular digestion process that degrades damaged proteins and organelles but the biological roles of autophagy in pathological aspects of oral tissues remain largely unknown. We sought to elucidate the function of autophagy, especially its interplay with apoptosis and oxidative stress, in the oral toxicity induced by exposure to 5 mM sodium fluoride (NaF). Human cementoblasts (HCEM-2) in culture were exposed to 5 mM NaF for 5 min, after which cell viability and cell apoptosis were assessed using the MTS assay and an Annexin V-FITC/PI apoptosis detection kit, respectively. Quantitative RT-PCR and Western blotting were performed to characterize the expression levels of markers for autophagy, apoptosis, and oxidative stress. Senescence-resistant (SAMR1) mice were exposed to 5 mM NaF in their drinking water from 12 to 58 weeks. Micro-computed tomography was used to measure changes in their alveolar bone while immunohistochemistry and immunofluorescence staining was used to evaluate protein expression levels. HCEM-2 cells exposed to 5 mM NaF had decreased levels of autophagy, as shown by reduced expression levels of ATG5, Beclin-1 and LC3-II, elicited apoptosis, which in turn induced oxidative stress and inflammation, as manifested by elevated levels of Bax, cleaved caspase-3, SOD1 and phospho NF-κB. Treatment of mice with 5 mM NaF resulted in histological abnormalities in periodontal tissues, induced excessive oxidative stress and apoptosis, and reduced autophagy. Micro-computed tomography analysis demonstrated that 5 mM NaF caused a decrease in bone areas of mice compared with controls. Exposure to 5 mM NaF induced RANKL (receptor activator of nuclear factor κB ligand) and cathepsin K expression in periodontal tissues, while ATG5 and Beclin-1 expression was abrogated by 5 mM NaF. Taken together, our findings suggest that 5 mM NaF elicits oral toxicity that contributes to excessive apoptosis, oxidative stress, and defective autophagy, which aggravates periodontal tissue damage.

Phenytoin-induced gingival overgrowth caused by death receptor pathway malfunction.

OBJECTIVE: In this study, we investigated the role of phenytoin (PHT) in death receptor-induced apoptosis of gingival fibroblasts to clarify the mechanism of PHT-induced gingival overgrowth. METHODS: Human gingival fibroblasts were cultured to semiconfluence and treated with PHT (0.025, 0.1, 0.25, and 1.0 µM) for 48 h, and then, the apoptotic cell numbers were relatively determined by absorptiometry. After 24 h of 0.25 µM PHT treatment, caspase activity was measured by absorptiometry, apoptotic and cell cycle phase distribution was analyzed by flow cytometry, expression levels of apoptotic genes were quantified by real-time qPCR, and expression of apoptotic proteins was detected by Western blot analysis. After 48 h of 0.25 µM PHT treatment, appearance of apoptotic cells was detected by TUNEL assay. RESULTS: PHT treatment decreased the proportion of apoptotic cells in gingival fibroblasts compared to a serum-free control culture in response to the protein changes as follows: PHT upregulated c-FLIP and, in turn, downregulated FADD, caspase-8, and caspase-3; PHT upregulated c-IAP2 and downregulated TRAF2; PHT downregulated caspase-9 and caspase-3 via decreased RIPK1 activity and increased Bcl-2 activity. CONCLUSION: PHT-induced gingival overgrowth may result from the above-mentioned mechanisms involving apoptosis inhibition in gingival fibroblasts.

Photoreceptor spectral tuning by colorful, multilayered facet lenses in long-legged fly eyes (Dolichopodidae).

The facet lenses of the compound eyes of long-legged flies (Dolichopodidae) feature a striking, interlaced coloration pattern, existing of alternating rows of green-yellow and orange-red reflecting facets, due to dielectric multilayers located distally in the facet lenses (Bernard and Miller. Invest Ophthalmol 7:416-434 (1968). We investigated this phenomenon in the dolichopodid Dolichopus nitidus by applying microspectrophotometry, electron microscopy and optical modeling. The measured narrow-band reflectance spectra, peaking at ~540 and ~590 nm with bandwidth ~105 nm, are well explained by a refractive index oscillating sinusoidally in six periods around a mean value of about 1.44 with amplitude 0.6. The facet lens reflectance spectra are associated with a spectrally restricted, reduced transmittance, which causes modified spectral sensitivities of the underlying photoreceptors. Based on the modeling and electroretinography of the dolichopodid Condylostylus japonicus we conjecture that the green and orange facets narrow the spectral bandwidths of blue and green central photoreceptors, respectively, thus possibly improving color and/or polarization vision.

Possible pharmacotherapy for nifedipine-induced gingival overgrowth: 18α-glycyrrhetinic acid inhibits human gingival fibroblast growth.

BACKGROUND AND PURPOSE: This investigation aimed to establish the basis of a pharmacotherapy for nifedipine-induced gingival overgrowth. Gingival overgrowth has been attributed to the enhanced growth of gingival fibroblasts. In this study, we investigated the effects of 18-α-glycyrrhetinic acid (18α-GA) on growth, the cell cycle, and apoptosis and on the regulators of these processes in gingival fibroblasts isolated from patients who presented with nifedipine-induced gingival overgrowth. EXPERIMENTAL APPROACH: Gingival fibroblasts were cultured in medium containing 1% FBS with/without 10 µM 18α-GA for 24 or 48 h, and the cell number, cell cycle phase distribution, relative DNA content, apoptotic cell number and morphological characteristics of the cells undergoing apoptosis were measured together with the levels of proteins that regulate these processes and the level of caspase activity. KEY RESULTS: 18α-GA significantly decreased cell numbers and significantly increased the percentage of cells in the sub-G1 and G0 /G1 phases of the cell cycle and the number of apoptotic cells. Nuclear condensation and fragmentation of cells into small apoptotic bodies appeared in the fibroblasts treated with 18α-GA. In addition, 18α-GA significantly decreased the protein levels of cyclins A and D1, CDKs 2 and 6, phosphorylated Rb (ser(780) and ser(807/811)), Bcl-xL and Bcl-2 and increased the protein levels of p27, cytosolic cytochrome c, pro-caspase-3, and cleaved caspase-3 and the activities of caspases 3 and 9. CONCLUSIONS AND IMPLICATIONS: 18α-GA inhibited gingival fibroblast growth by suppressing the G1 /S phase transition and inducing apoptosis. In conclusion, 18α-GA may be used as a pharmacotherapy for nifedipine-induced gingival overgrowth.

Cholesterol homeostasis in the ovaries of neonatally diethylstilbestrol-treated mice.

In the ovary of neonatally DES-treated mice, lipid droplets accumulation was observed in the hypertrophied interstitial tissues. Our previous results demonstrated that the impaired steroidogenesis in the ovary of neonatally DES-treated mice was caused by altered gonadotropins levels, and resulted in the hypertrophy of ovarian interstitial cells. We speculated that lipid droplets in the ovary mainly consisted of cholesterol. This study was aimed to examine the effects of neonatal DES on cholesterol homeostasis in the ovary. The serum and ovarian total cholesterol concentrations in 3-month-old neonatally DES-treated mice were significantly higher than those in the neonatally oil-treated mice, but triglyceride concentrations were not altered. In the ovary of neonatally DES-treated mice, expression of Hmgcr, a rate-limiting enzyme in de novo cholesterol biosynthesis, was reduced but expression of Ldlr and Scarb1, involved in cholesterol uptake, was not changed. These results suggest that cholesterol uptake is not altered in the ovary of 3-month-old neonatally DES-treated mice. However, the expression of Acat1, the microsomal acyl coenzyme A cholesterol acyltransferase which is involved in cholesterol esterification and storing was increased compared with that in the ovary of neonatally oil-treated mice. Since ovarian steroidogenesis in neonatally DES-treated mice was impaired, synthesized and/or obtained cholesterol from the blood may not be used sufficiently. Thus, in the ovary of neonatally DES-treated mice, cholesterol is esterified by ACAT1 and stored in the interstitial cells.

Biochemical characterisation of urease from urease-positive thermophilic campylobacter (UPTC).

This study aims to characterise biochemically urease from an atypical Campylobacter lari, namely urease-positive thermophilic Campylobacter (UPTC). Urease was purified from cells of a Japanese UPTC isolate (CF89-12) using phenyl-Sepharose chromatography. Two protein components (estimates molecular masses 24 kDa and 61 kDa) were obtained that appeared to be structural proteins of urease (subunits A and B), and these were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE). The native molecular weight for the final purified UPTC urease was estimated to be approximately 186,000 Da which is close to the calculated molecular weight (182,738 Da) based on all six open reading frames of UPTC CF89-12 urease genes (ureA, B, E, F, G and H), as described previously. Moreover, an active band was observed on phenol red staining after a nondenaturing native PAGE of the crude extract from the UPTC cells. In addition, the purified urease of UPTC CF8912 showed enzyme activity over a broad pH range (pH 6-10), with maximal activity at pH 8.0. The urease was also stable against heat treatment, with almost no loss of enzyme activity seen following 60-min incubation at temperatures of 20-60 degrees C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.

Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli.

AIMS: The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC. METHODS AND RESULTS: Primers and TaqMan probes were designed to amplify and quantify one gene (eae, stx1, stx2, elt, est, virB, aggR, astA, and afaB) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0.99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7.1 x 10(2) to 1.1 x 10(4) CFU ml(-1), depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g(-1)) were found to be positive by the method. CONCLUSIONS: The present system allows for the efficient and simultaneous determination of various DEC pathotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.

Light on the moth-eye corneal nipple array of butterflies.

The outer surface of the facet lenses in the compound eyes of moths consists of an array of excessive cuticular protuberances, termed corneal nipples. We have investigated the moth-eye corneal nipple array of the facet lenses of 19 diurnal butterfly species by scanning electron microscopy, transmission electron microscopy and atomic force microscope, as well as by optical modelling. The nipples appeared to be arranged in domains with almost crystalline, hexagonal packing. The nipple distances were found to vary only slightly, ranging from about 180 to 240 nm, but the nipple heights varied between 0 (papilionids) and 230 nm (a nymphalid), in good agreement with previous work. The nipples create an interface with a gradient refractive index between that of air and the facet lens material, because their distance is distinctly smaller than the wavelength of light. The gradient in the refractive index was deduced from effective medium theory. By dividing the height of the nipple layer into 100 thin slices, an optical multilayer model could be applied to calculate the reflectance of the facet lenses as a function of height, polarization and angle of incidence. The reflectance progressively diminished with increased nipple height. Nipples with a paraboloid shape and height 250 nm, touching each other at the base, virtually completely reduced the reflectance for normally incident light. The calculated dependence of the reflectance on polarization and angle of incidence agreed well with experimental data, underscoring the validity of the modelling. The corneal nipples presumably mainly function to reduce the eye glare of moths that are inactive during the day, so to make them less visible for predators. Moths are probably ancestral to the diurnal butterflies, suggesting that the reduced size of the nipples of most butterfly species indicates a vanishing trait. This effect is extreme in papilionids, which have virtually absent nipples, in line with their highly developed status. A similar evolutionary development can be noticed for the tapetum of the ommatidia of lepidopteran eyes. It is most elaborate in moth-eyes, but strongly reduced in most diurnal butterflies and absent in papilionids.

Butterfly wing colours: scale beads make white pierid wings brighter.

The wing-scale morphologies of the pierid butterflies Pieris rapae (small white) and Delias nigrina (common jezabel), and the heliconine Heliconius melpomene are compared and related to the wing-reflectance spectra. Light scattering at the wing scales determines the wing reflectance, but when the scales contain an absorbing pigment, reflectance is suppressed in the absorption wavelength range of the pigment. The reflectance of the white wing areas of P. rapae, where the scales are studded with beads, is considerably higher than that of the white wing areas of H. melpomene, which has scales lacking beads. The beads presumably cause the distinct matt-white colour of the wings of pierids and function to increase the reflectance amplitude. This will improve the visual discrimination between conspecific males and females.

Action spectrum of foraging behavior of the Japanese yellow swallowtail butterfly, Papilio xuthus.

This paper describes the action spectrum of foraging behavior of a butterfly, Papilio xuthus. We first established an experimental protocol to evaluate learning and discrimination of monochromatic light by the butterflies. We trained butterflies to feed on sucrose solution at the window illuminated with certain monochromatic light produced through a monochromator. After confirming that they learned the monochromatic light, after 10 days of training, we tested the butterflies one by one. We presented training wavelengths for each individual at different intensities, and recorded whether they perform foraging behavior under freely-flying as well as tethered conditions. Freely-flying butterflies responded to light by visiting the window and searching for nectar around it, whereas tethered butterflies responded by extending their proboscides towards the window. The light intensity required to elicit 50% response for each tested monochromatic light was plotted. The resulting action spectrum for the visit was rather flat with the maximum sensitivity a 420 nm, whereas the spectrum for the proboscis extension had prominent peaks at 380, 500 and 600 nm. The difference in action spectra indicates that the visit and the proboscis extension are controlled by two independent mechanisms at least in part.

Spectral organization of the eye of a butterfly, Papilio.

This review outlines our recent studies on the spectral organization of butterfly compound eyes, with emphasis on the Japanese yellow swallowtail butterfly, Papilio xuthus, which is the most extensively studied species. Papilio has color vision when searching for nectar among flowers, and their compound eyes are furnished with six distinct classes of spectral receptors (UV, violet, blue, green, red, broadband). The compound eyes consist of many ommatidia, each containing nine photoreceptor cells. How are the six classes of spectral receptors arranged in the ommatidia? By studying their electrophysiology, histology, and molecular biology, it was found that the Papilio ommatidia can be divided into three types according to the combination of spectral receptors they contain. Different types of ommatidia are distributed randomly over the retina. Histologically, the heterogeneity appeared to be related to red or yellow pigmentation around the rhabdom. A subset of red-pigmented ommatidia contains 3-hydroxyretinol in the distal portion, fluorescing under UV epi-illumination. The red, yellow and fluorescing pigments all play crucial roles in determining the spectral sensitivities of receptors. Spectral heterogeneity and random array of ommatidia have also been found in other lepidopteran species. Similarities and differences between species are also discussed.

The photoreceptor localization confirms the spectral heterogeneity of ommatidia in the male small white butterfly, Pieris rapae crucivora.

The compound eye of Pieris rapae crucivora contains ventrally three types of histologically distinct ommatidia. An ommatidium contains nine photoreceptors, four of which (R1-4) construct the distal tier of the rhabdom. We determined the sensitivity spectra of the R1-4 distal photoreceptors in each type of ommatidia by intracellular electrophysiology and identified UV, blue, double-peaked blue, green, and a green receptor with depressed sensitivity in the violet. We localized these receptors in each type of ommatidia by injecting dye after the recording. In type I ommatidia the R1 and R2 cells are UV and blue receptors. When R1 is UV sensitive, R2 is always blue sensitive, or vice versa. R3 and R4 in type I are both green receptors. In type II, R1 and R2 are both double-peaked blue receptors and R3 and R4 are both green receptors with depressed sensitivity in the violet. In type III, R1 and R2 are both UV, and R3 and R4 are green receptors. The double-peaked blue, and green receptors with depressed sensitivity in the violet in type II ommatidia have depressed sensitivity at 420 nm, which is probably due to the filtering effect of a fluorescing material present in the type II ommatidia. Spectral heterogeneity of ommatidia seems to be a common design of insect compound eyes.

An outbreak of gastroenteritis in Osaka, Japan due to Escherichia coli serogroup O166:H15 that had a coding gene for enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1).

In an outbreak of gastroenteritis on 23 July 1996, in Osaka, Japan, 54 of 91 persons who had attended a meeting the previous day became ill. Escherichia coli O166:H15 was isolated from stool specimens of patients (29/33, 88%). Laboratory tests for other bacterial pathogens and viruses were negative. The E. coli 0166 organisms did not adhere to HEp-2 cells in a localized, diffuse, or enteroaggregative manner. The organisms did not express known enterotoxigenic E. coli (ETEC) colonization factors. In polymerase chain reaction tests, the bacteria did not have coding genes for shigatoxin of enterohemorrhagic E. coli (EHEC), heat-labile, or heat-stable enterotoxin of ETEC, attachment and effacement (eaeA) of EPEC, or invasion (invE) of enteroinvasive E. coli (EIEC). Consequently, they could not be assigned to any of the recognized diarrhoeagenic groups of E. coli: EPEC, ETEC, EHEC, EIEC, enteroaggregative E. coli (EAggEC), or diffusely adhering E. coli. However, the organisms possessed the EAggEC heat-stable enterotoxin (EAST1) gene. To our knowledge, this is the first report of an outbreak caused by E. coli that did not have well-characterized virulence genes other than EAST1. The isolates showed the same DNA banding pattern in pulsed-field gel electrophoresis after digestion with the restriction enzymes XbaI or NotI. Three O166:H15 strains isolated from two sporadic cases and another outbreak during 1997-8 were distinct, indicating that multiple clones have spread already. We propose that diarrhoeal specimens should be examined for E. coli possessing the EAST1 gene.

Accuracy of portable infusers under hyperbaric oxygenation conditions.

Three types of portable infusers with different infusion mechanisms were evaluated with regard to their accuracy during a hyperbaric oxygenation protocoL The power driving the pump is provided by either a balloon, a spring or a vacuum mechanism. Performance during hyperbaric oxygenation (HBO) varied between the devices, probably due to the difference in driving mechanism. Flow delivery by the vacuum type infuser is substantially affected by HBO. Doubling the ambient pressure approximately doubled flow delivery from the vacuum type device. We suggest that other devices are more suitable for use in this clinical situation. We conclude that it is desirable to check the performance of any infuser intended for use during hyperbaric oxygenation and to be mindful of potential differences among such devices.

Polarisation-dependent colour vision in Papilio butterflies.

Butterflies of the genus Papilio have polarisation-sensitive photoreceptors in all regions of the eye, and different spectral types of receptor are sensitive to different e-vector orientations. We have studied the consequences of this eye design for colour vision in behavioural tests and find that Papilio spp. see false colours due to the polarisation of light. They discriminate between vertically and horizontally polarised light of the same colour in the contexts of oviposition and feeding. The discrimination depends on the spectral composition of the stimuli. In the blue and probably in the green range, discrimination does not depend on intensity. However, colour discrimination is influenced by polarisation. Thus, colour and polarisation processing are not separated in the visual system of Papilio spp. From these results, we propose hypotheses about which photoreceptors contribute to colour vision in Papilio spp. and what adaptational value such a system might have for the butterflies. Finally, we give examples for other eyes that have a similar structure.

[Extracranial metastasis of glioblastoma to the lung and heart with a histological resemblance to small cell carcinoma of the lung: an autopsy case].

An autopsy case of small cell glioblastoma, showing multiple extracranial metastases, is reported with special reference to histopathological differentiation from metastatic small cell carcinoma. Widely spread lesions in the bilateral lungs were developed after an operation and chemo-radiotherapy for glioblastoma, and the lung lesions led to fatal respiratory failure. Postmortem examination revealed multiple tumors in the lung, lymph nodes, and the heart, as well as local invasion of the primary tumor to the dura, skull, and the scalp. The mechanism of extracranial metastasis of brain tumor is discussed.

[Clinical significance of eosinophilic cationic protein in serum and bronchoalveolar lavage fluid of adult patients with mycoplasmal pneumonia].

Pathogenic mechanisms of mycoplasmal pneumonia is not fully understood at present though some kind of cell-mediated hypersensitivity is closely related to its mechanisms. Though eosinophilia in peripheral blood are sometimes revealed in patient with mycoplasmal pneumonia, it is not unclear whether eosinophils related to its pathogenesis, or not. We evaluated the clinical significance of ECP in serum and BAL fluid in patients with mycoplasmal pneumonia. The diagnosis of mycoplasmal pneumonia was confirmed both by serological diagnosis from paired serum and by the polymerase chain reaction (PCR) methods using specific primers of the Mycoplasma pneumoniae for detecting specific DNA from bronchial washing fluids. ECP level in serum were measured in 27 patients (11 male, 16 female, average age 31.7 yo) with mycoplasmal pneumonia by ELISA methods. ECP level in BALF were also measured in ten of all patients. The level of ECP in serum was high in 17 cases (63%) of the total cases. In addition the level of ECP in BALF was also high in all tested patients (10 cases). There was a correlation between serum ECP level and days from onset. There was also a correlation between serum ECP level and WBC counts, the degree of PaO2. These results suggested that ECP derived from activated eosinophils in the lung might in part play a role in the pathogenesis of mycoplasmal pneumonia.

Retinal regionalization and heterogeneity of butterfly eyes.

The regional characteristics of the eyes of butterflies from different families have been surveyed using epi-illumination microscopy, utilizing the eyeshine visible due to the tapetum situated proximally to the rhabdom. All butterflies studied have a high spatial acuity in the frontal region. The facet diameter varies slightly across the eye, and the interommatidial angle and the eye parameter p are especially large dorsally. Whereas the ommatidial lattice is generally highly regular, the eyeshine colours distinctly depend on the species. Sometimes the eyeshine is locally uniform, but often it is heterogeneous. It is hypothesized that the regional characteristics as well as the local heterogeneity are adaptations that optimize spectral discrimination.

Colour constancy in the swallowtail butterfly Papilio xuthus.

We have recently shown that the Japanese yellow swallowtail butterfly Papilio xuthus uses colour vision when searching for food. In the field, these butterflies feed on nectar provided by flowers of various colours not only in direct sunlight but also in shaded places and on cloudy days, suggesting that they have colour constancy. Here, we tested this hypothesis. We trained newly emerged Papilio xuthus to feed on sucrose solution on a paper patch of a certain colour under white illumination. The butterflies were then tested under both white and coloured illumination. Under white illumination, yellow- and red-trained butterflies selected the correctly coloured patch from a four-colour pattern and from a colour Mondrian collage. Under four different colours of illumination, we obtained results that were fundamentally similar to those under white illumination. Moreover, we performed critical tests using sets of two similar colours, which were also correctly discriminated by trained butterflies under coloured illumination. Taken together, we conclude that the butterfly Papilio xuthus exhibits some degree of colour constancy when searching for food.

Ultraviolet and violet receptors express identical mRNA encoding an ultraviolet-absorbing opsin: identification and histological localization of two mRNAs encoding short-wavelength-absorbing opsins in the retina of the butterfly Papilio xuthus.

This paper describes the primary structures of two opsins of short-wavelength-absorbing visual pigments deduced from the mRNA sequences in the retina of the Japanese yellow swallowtail butterfly Papilio xuthus. A phylogenetic analysis of the amino acid sequences indicates that one of these visual pigments is of the ultraviolet-absorbing type and that the other is of the blue-absorbing type. We identified the photoreceptor cells that express these mRNAs by histological in situ hybridization. The mRNA of the ultraviolet type is expressed in two distinct photoreceptor types previously identified as ultraviolet and violet receptors, providing the first molecular biological evidence that different types of spectral receptor probably express a visual pigment with an identical amino acid sequence. The mRNA of the blue type is expressed exclusively in cells classified as blue receptors.