Mineral trioxide aggregate suppresses pro-inflammatory cytokine expression via the calcineurin/nuclear factor of activated T cells/early growth response 2 pathway in lipopolysaccharide-stimulated macrophages.

AIM: To elucidate mechanisms by which mineral trioxide aggregate (MTA) suppresses pro-inflammatory cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. METHODOLOGY: Mineral trioxide aggregate extracts were prepared by immersing set ProRoot MTA in culture medium. RAW264.7 cells were cultured in the presence of LPS and MTA extracts. mRNA expression levels of interleukin (IL)-1α, IL-6, early growth response 2 (Egr2), suppressor of cytokine signalling 3 (Socs3) and IL-10 were quantified with reverse transcription-quantitative polymerase chain reaction. Phosphorylation of nuclear factor-kappa B (NF-κB) p65 in RAW264.7 cells was analysed by Western blotting. Intracellular calcium imaging was performed with Fluo-4 AM. The activity of nuclear factor of activated T cells (NFAT) was determined by luciferase assays. Enforced expression and silencing of Egr2 in RAW264.7 cells were carried out using an expression vector and specific RNAi, respectively. In vivo kinetics of Egr2+ cells in MTA-treated rat molar pulp tissues were examined using immunohistochemistry. Data were analysed by one-way analysis of variance, followed by the Tukey-Kramer test (P < 0.05). RESULTS: Exposure to MTA extracts resulted in reduced mRNA expression levels of IL-1α and IL-6, as well as reduced expression of phosphorylated NF-κB, in LPS-stimulated RAW264.7 cells. Exposure to MTA extracts induced Ca2+ influx, which was blocked by NPS2143, an antagonist of calcium-sensing receptor (CaSR); Ca2+ influx then triggered activation of calcineurin/NFAT signalling and enhanced mRNA expression of Egr2. Enforced expression of Egr2 in RAW264.7 cells promoted the expression of both IL-10 and Socs3. In vivo application of MTA onto rat molar pulp tissue resulted in the appearance of Egr2-expressing cells that coexpressed CD163, a typical M2 macrophage marker. CONCLUSIONS: Mineral trioxide aggregate extracts induced downregulation of IL-1α and IL-6 in LPS-stimulated RAW264.7 cells via CaSR-induced activation of calcineurin/NFAT/Egr2 signalling and subsequent upregulation of IL-10 and Socs3.

Real-time nucleic acid sequence-based amplification (NASBA) using an adenine-induced quenching probe and an intercalator dye.

AIMS: We found that an adenine base caused fluorescence quenching of a fluorescein (FL)-labelled probe in DNA:RNA hybrid sequences, and applied this finding to a nucleic acid sequence-based amplification (NASBA) method. METHODS AND RESULTS: The present NASBA method employed a probe containing an FL-modified thymine at its 3' end and ethidium bromide (EtBr) on the basis of a combination of adenine-induced quenching and fluorescence resonance energy transfer (FRET) between the FL donor and EtBr acceptor. This NASBA was used to detect Shiga toxin (STX) stx-specific mRNA in STX-producing Escherichia coli, demonstrating rapid quantification of the target gene with high sensitivity. CONCLUSION: Although the inherent quenching effect of adenine was inferior to that of guanine, FRET between the FL and EtBr moieties enhanced the adenine-induced quenching, allowing rapid and sensitive real-time NASBA detection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives a novel real-time diagnostic system based on NASBA for a sensitive mRNA (or viral RNA) detection.

Immunoreactive hepatocyte growth factor is a strong and independent predictor of recurrence and survival in human breast cancer.

Hepatocyte growth factor (HGF) is a stromally derived modulator of epithelial cell proliferation and motility. In the present study, we have measured immunoreactive (ir)-HGF concentration in tumor extracts of 258 primary human breast cancers using an enzyme-linked immunosorbent assay and have evaluated its association with disease-free and overall survival. The median value of ir-HGF concentration was 11.0 ng/100 mg protein (range, 1.4-566.7 ng/100 mg protein). Correlation analyses between ir-HGF concentration and clinicopathological factors showed that the ir-HGF level was correlated only with tumor size (P = 0.05). No significant associations were found between ir-HGF content and age, menopausal status, nodal status, histological type, histological grade, vessel involvement, estrogen receptor, progesterone receptor, type of surgery, or postoperative adjuvant therapy. Breast cancer patients with high ir-HGF concentration had a significantly shorter relapse-free (P = 0.001) and overall survival (P = 0.001) rate when compared to those with low ir-HGF concentration at the cutoff point of 21.7 ng/100 mg protein, which was determined in another group of 82 patients. In multivariate analysis, ir-HGF level was found to be the most important independent factor in predicting relapse-free and overall survival, of greater import than lymph node involvement. The putative role of HGF in breast cancer growth and metastasis is hereby strengthened.

Exposure to a dry environment enhances epidermal permeability barrier function.

Previous studies have suggested that transepidermal water movement may play an important role in epidermal homeostasis and barrier repair. Here we analyzed cutaneous barrier function, epidermal morphology, and lipid content of the stratum corneum in hairless mice maintained in a high relative humidity (RH > 80%) versus low humidity (RH < 10%) environment for 2 wk. Basal transepidermal water loss was reduced by 31% in animals maintained in a dry versus humid environment. Moreover, the number of lamellar bodies in stratum granulosum cells, the extent of lamellar body exocytosis, and the number of layers of stratum corneum increased in animals kept in a dry environment. Furthermore, the dry weight of the stratum corneum and the thickness of the epidermis also increased in a dry environment. In addition, total stratum corneum lipids increased but lipid analysis revealed no significant differences in lipid distribution. Lastly, barrier recovery following either acetone treatment or tape stripping was accelerated after prolonged prior exposure to a dry environment, while conversely, it was delayed by prior exposure to a humid environment. These studies demonstrate that environmental conditions markedly influence epidermal structure and function, and suggest mechanisms by which the environment could induce or exacerbate various cutaneous disorders.

Identification and analyses of the Xenopus TERT gene that encodes the catalytic subunit of telomerase.

The Xenopus telomerase catalytic component gene, xTERT (Xenopus telomerase reverse transcriptase), has been cloned. The production of xTERT recombinant protein together with the proposed Xenopus telomerase RNA (xTR) (Chen et al., 2000. Cell 100, 503-514) in a rabbit reticulocyte lysate system led to the reconstitution of active telomerase, indicating that both products are functional telomerase components. Both xTERT expression and telomerase activity are high from the early to the late blastula stage. However, they are decreased at the gastrula stage and thereafter, suggesting that the xTERT expression level is the primary mechanism for regulating telomerase activity in Xenopus development. This is the first report of a non-mammalian vertebrate TERT gene. Sequence comparison of xTERT with human and mouse TERTs has uncovered four regions conserved in the amino-terminal halves of vertebrate TERT proteins, the functions of which will be discussed herein.

Molecular cloning of two novel types of peptidylarginine deiminase cDNAs from retinoic acid-treated culture of a newborn rat keratinocyte cell line.

Peptidylarginine deiminases (PADs) are a group of enzymes which convert protein arginine residues to citrulline residues. Using rat muscle PAD cDNA as a probe, we obtained two novel cDNAs, PAD-R11 and PAD-R4, from immortalized rat keratinocytes treated with all-trans retinoic acid. Comparison of the deduced amino acid sequences with those of muscle and hair follicle enzymes showed high conservation in the C-terminal region. Recombinant proteins encoded by both PAD-R11 and PAD-R4 showed the enzyme activities. That of PAD-R11 showed a characteristic feature of the enzyme found in the epidermis.

1H-NMR study on the structure of lysozyme from guinea hen egg white.

The structure of lysozyme from guinea hen egg white (GEWL), which differs from hen egg white lysozyme (HEWL) by ten amino acid substitutions, was investigated by nuclear magnetic resonance (NMR) spectroscopy. GEWL and HEWL were very similar to each other in their tertiary structure as judged from the profile of 1H-NMR spectra, pH titration, and an N-acetylglucosamine trisaccharide [(GlcNAc)3 binding experiment. However, we have noticed several characteristics which distinguish GEWL from HEWL. The signal of Trp 108 indole N1H of GEWL was shifted upfield by about 0.3 ppm when compared with that of HEWL, and its hydrogen exchange was faster than that of HEWL. The pKa values of Glu 35 estimated from the pH titration curve of Trp 108 indole N1H were different between GEWL and HEWL. From a careful examination of spectral changes caused by (GlcNAc)3 binding, the changes in the chemical shift values of Trp 28 C5H and Asn 59 alpha CH of GEWL were found to be slightly larger than those of HEWL. Ile 55 of HEWL is replaced by valine in GEWL. Such a replacement may affect the neighboring hydrogen bonding between the main chain C = O of Leu 56 and Trp 108 indole N1H, resulting in a change in the microenvironment of the substrate-binding site near Trp 108.

Susceptibility of various animals and cultured cells to exfoliative toxin produced by Staphylococcus hyicus subsp. hyicus.

In piglets inoculated with partially purified exfoliative toxin (pp-shET) produced by Staphylococcus hyicus subsp. hyicus, exfoliation was observed at 12 h after injection. Chickens inoculated with the same dose of pp-shET also showed exfoliation within 30 min of injection. However, exfoliation was not demonstrated in mouse, rat, guinea pig, hamster, dog or cat inoculated with pp-shET until 24 h after injection. In cultured cell lines, especially L-929 and Hep-2, the rounding effect occurred after incubation with pp-shET for 1 h. The rounding effect was also seen in five other cultured cells (NCTC 2544, HeLa/S3, HmLu-1, CHO and BHK-21) 6-24 h after exposure to pp-shET. These round cells survived for 72 h after inoculation and formed a monolayer 24 h after changeover to a toxin-free medium. The rounding effect was observed in cells after the formation of the monolayer, but not before. It was suggested that the rounding effect was not caused by the increase in cyclic AMP in cells inoculated with pp-shET but by the cleavage of intracellular contacts.

Isolation of exfoliative toxin from Staphylococcus hyicus subsp. hyicus and its exfoliative activity in the piglet.

Exfoliative toxin was isolated from the sterile cell-free filtrate of 24 h culture of Staphylococcus hyicus subsp. hyicus strain P-1. The partial purification of exfoliative toxin produced by S. hyicus (shET) was performed by precipitation with 50-80% saturated ammonium sulfate, gel filtration on a Sephadex G-75 column and column chromatography on DEAE-cellulose. Partially purified shET (pp-shET) caused exfoliation in piglets at 8 to 12 h after intradermal or subcutaneous injection. However, heat-treated pp-shET did not cause exfoliation in piglets for up to 24 h after injection. On histopathological examination of the skin at 12 h after injection of pp-shET, an intraepidermal cleavage plane was shown between the stratum corneum and stratum granulosum and at the stratum granulosum.

Characteristics of shed snake skin permeability to indomethacin and fatty alcohols.

To investigate the utilities of a shed snake skin as a model membrane for preclinical studies of transdermal drug delivery, the flux of indomethacin was determined under various conditions by using a diffusion cell. The flux of fatty alcohols was determined and compared with that in human skin reported in references. The esterase activity of shed snake skin was also determined. It was found that the flux of indomethacin decreased with an increase of pH and the amount of ethanol in a vehicle. The flux of indomethacin increased by the addition of Azone, N-methyl-2-pyrrolidone and N,N-dimethyl-m-toluamide in the cream. The flux of fatty alcohols in shed snake skin was greater than that reported in human skin, and shed snake skin had similar esterase activity to human skin.

[Percutaneous chronic toxicity study of 10% nitroglycerin (NT-1 ointment) in rabbits].

A chronic toxicity test of 10% nitroglycerin (NT-1 ointment) was carried out in male NZW rabbits. NT-1 ointment was applied to the back skin for 26 weeks at daily doses of 15, 60 and 240 mg/kg as nitroglycerin itself, and 5-week withdrawal period was followed. Topical dermal responses to NT-1: Macroscopically, erythema, edema, scales, papules and dermal thickening were observed in response to NT-1 ointment. In the withdrawal period, however, all of them disappeared. Histopathologically, thickening of epidermis and cell infiltration were observed in response to NT-1 ointment. In addition, elongation of rete ridges and Touton giant cells were found only in 60 and 240 mg/kg groups, and hyperkeratosis only in 240 mg/kg group. At the end of withdrawal period, Touton giant cells were still found in 60 and 240 mg/kg groups, although the other dermal reactions disappeared. Systemic responses to NT-1 ointment: A slight increase in the excretion of loose or mucous feces was observed in 240 mg/kg group. The weights of right and left kidneys were increased by the administration of 240 mg/kg NT-1 ointment, and a similar trend was seen also in the weight of heart, although there found no such among-group differences at the end of withdrawal period. White blood cells, especially neutrophils, and gamma-globulin fraction were increased in response to 240 mg/kg NT-1 ointment. In conclusion, a no-toxic effect dose of NT-1 ointment as nitroglycerin itself was considered to be 15 mg/kg/day for the skin and 60 mg/kg/day for the general somatic system.

[Teratological test of 10% nitroglycerin (NT-1 ointment) in rabbits].

A teratological test of 10% nitroglycerin (NT-1 ointment) was carried out in New Zealand White rabbits. Pregnant rabbits were treated percutaneously with NT-1 ointment from day 6 to 18 of gestation at dose levels of 15, 60 and 240 mg/kg/day as nitroglycerin itself. All pregnant rabbits were killed on day 29 of gestation, and the influences of NT-1 ointment upon the performances of dams and fetuses were examined. During the treatment period, erythema was observed on the treated dorsal skin in all groups excluding the nontreatment control group. However, it disappeared soon after the cessation of NT-1 ointment administration. Influences of NT-1 ointment administration were not found at any dose levels on the food consumption and body weight changes of pregnant rabbits. In addition, influences of NT-1 ointment on the reproductive performance of dams and the development of fetuses were not observed at any dose levels. Therefore, the non-effect dose of NT-1 ointment on reproductive performance of dams and fetal development in rabbits was estimated to be 240 mg/kg/day and more as nitroglycerin itself.

[A biological study of inbred Weiser-Maples guinea pigs--urinalysis, hematological and blood-chemical values and organ weights].

Urinalysis, hematological and blood chemical examinations and measurement of organ weights were carried out for the purpose of collecting background data on Weiser-Maples (WM) which was established as an inbred strain of guinea pigs. The results were compared with those for the commercial guinea pigs, Std: Hartley (H). In addition, the measurement methods for urinary specific gravity and whether centrifugation of urine influences the results of urinalysis or not were examined. The specific gravity values calculated from the volume and weight of urine were highly correlated with those from the refraction rate. The results of urinalysis of the supernatant of centrifuged urine were similar to those of urine not centrifuged, but the results suggested that the use of the supernatant provided a more accurate measurement of urine in guinea pigs. The positive rate for urinary protein was higher in the WM strain (38.9%) than in the H strain (0%). Although the WM strain showed higher or lower values than the H strain in some items of hematological and blood chemical examinations and organ weights, we could not regard them as characteristic of the WM strain.

Flow cytometric analysis of murine splenic cells after DNFB painting on Langerhans cell deficient skin.

One week after a single painting of 50 microliters of 0.5% DNFB in acetone-olive oil on the tail skin of C3H mice, the spleens were removed from the animals. A single cell suspension was prepared from the spleens, and flow cytometric analysis was performed for L3T4 and Lyt2 positive cells, as well as for the IJk expression of the Lyt2+ cells. Results showed that the percentages and absolute numbers of Lyt2+ L3T4- cells, Lyt2- L3T4+ cells, Lyt2+ IJk+ cells, and Lyt2+ IJk- cells in the spleens of the DNFB-treated mice were not significantly different from those in the non-treated mice. However, in the treated mice, the IJk expression of Lyt2+ cells intensified. These results indicate that, following a single painting of DNFB onto Langerhans cell-deficient skin, the numbers of Lyt2+ cells do not change significantly, but do change functionally.

Atopic dermatitis-like symptoms in HR-1 hairless mice fed a diet low in magnesium and zinc.

We aimed to develop an animal model for atopic dermatitis. HR-1 hairless mice fed a diet with reduced magnesium and zinc levels were compared with mice fed a standard diet. Skin dryness and wrinkle-like changes, scratching behaviour, decreased skin water content, increased transepidermal water loss and raised blood immunoglobulin E levels were seen in the group receiving the reduced magnesium and zinc diet compared with control mice. There were no significant differences in body weight or the weight of the major organs between the two groups. Haematological examination in both groups was normal apart from increased immunoglobulin E levels in mice fed a reduced magnesium and zinc diet. These mice may be useful models of atopic dermatitis; preparation of the animals is not particularly time consuming, the reproducibility is 100%, and atopic dermatitis symptoms occur even in a specific pathogen-free environment.

Microleakage of resin-based composite restorations with ceramic inserts.

PURPOSE: To evaluate dye penetration of proximal vertical slot resin-based composite (RBC) restorations as a function of cavity size and restorative material employed (RBC, ceramic inserts with low-viscosity resin, and RBC with low-viscosity resin liner). Microleakage was measured using rank scoring and linear measurement of dye penetration and ANOVA and non-parametric tests were used to evaluate the statistical significance of the results. MATERIALS AND METHODS: Ninety erupted crack- and caries-free mandibular third molars, extracted for orthodontic reasons, were used. Proximal vertical slots were prepared on the mesial surfaces of the teeth using diamond instruments at high speed under distilled water coolant. Teeth were placed randomly into nine groups. Three sizes of diamond burs of an ultrasonic cavity preparation system were used under low energy in order to standardize cavity size and shape, each tip size being used in 30 different teeth. The gingival margins were prepared 1 mm above the cemento-enamel junction. Specimens were etched, prepared with bonding agent, restored using three different materials, and immersed in a 50% AgNO3 solution for 8 hrs and sectioned to evaluate the dye penetration. RESULTS: Statistical analysis revealed no statistically significant differences among the nine groups.

Bond strength of a resin-based composite to leucite-reinforced ceramic and dentin.

PURPOSE: To compare the in vitro tensile bond strengths of resin-based composite bonded to dentin and to a leucite-reinforced ceramic in an as-received condition (pre-silanated) or re-etched, re-silanated condition. MATERIALS AND METHODS: Caries-free human molars were ground to expose occlusal, superficial dentin. Dentin surfaces were etched with 37% phosphoric acid and rinsed, leaving a moist dentin surface. Leucite-reinforced ceramic inserts (SONICsys) also were tested as received (pre-etched, pre-silanated) or retreated with hydrofluoric acid and silane. Bonding agent (Syntac Single-Component) was applied in two layers and light cured. An all-purpose resin-based composite (Tetric Ceram) was used to prepare tensile bond strength specimens for the dentin and two ceramic groups. Fourteen specimens were prepared for each group. After 24 hours in distilled water, tensile bond strength (MPa) was measured using a testing machine. Analysis of variance was used to evaluate data. RESULTS: Bond strengths obtained were 10.2 MPa for composite bonded to dentin, 10.5 MPa for composite bonded to as-received ceramic inserts, and 9.6 MPa for composite bonded to retreated ceramic inserts, with no statistical difference between the three groups. Re-treatment of leucite-reinforced inserts did not increase or decrease bond strength to resin-based composite. Also, the bond strength of resin-based composite to dentin was similar to that of the ceramic to composite.

Effects of gentamicin on the intestinal bacterial flora of rats.

1) Normal intestinal microbial flora (NF) in feces of rats were examined before and after administration of gentamicin (GM). Although GM caused no influence on NF after 6 hours, the number of NF decreased markedly 24 hours after GM, viz., the count of coliforms, enterococci and lactobacilli was reduced to 1/10-1/100 of the control value while proteus and fusobacteria declines in number down to 1/100-1/1,000 times as low as the control value. 2) The decreased count of NF tended to recover to some degree at the end of the 1st therapeutic week and the considerably recovered count continued thereafter indicating about 1/10 times as low as the control value until a 12-week experimental period, except that the count of lactobacilli returned to almost normal. 3) Susceptibility of coliforms isolated from GM-treated rats to GM was nearly equal to that of the bacteria isolated from the untreated control rats, which revealed no development of drug resistance to GM. 4) The increasing rate of body weight was higher in GM-treated rats than in the control, and the feed intake was consequently great in the former.

Effects of gentamicin on the thyroid function of rats.

The effect of gentamicin (GM) on the thyroid function was investigated in relation with its stimulating action to the growth of rats at a very small dose level. The results may be summarized as follows: (1) Changes in 131-I-uptake of the thyroid after administration of GM were unremarkable. The change in wet weight of the thyroid was also unremarkable. Therefore, GM may not affect the thyroid itself. (2) GM-treated rats clearly showed a decrease in 131-I-triiodothyronine resine sponge uptake (trisorb test) as compared with the control animals, viz.,GM-treated rats indicated hypofunction of the thyroid. (3) Thyroxin (t4) levels in theserum, determined by 125-I-T4 resine sponge uptake (tetrasorb test), were apparentlylower in the GM-treated rats than in the control. (4) The thyroid function test for synthesis of the hormones revealed an increase in diiodotyrosine (DIT) value and decreases in 3, 5, 3'-triiodothyronine (T3) and T4 values in the GM-treated groups as compared with the values in the control. (5) In view of the above results iit was obvious that GM does not cause abnormalities on iodine-uptake, iodine oxidation, organic conversion to iodinated tyrosines, deiodination and thyroglobulin. In contrast, GM may induce abnormality on coupling reaction from DIT to T3 and T4 because the increase in DIT value and decreases in T3 and T4 values are seen following administration of GM. The whole of these facts seems to play an important role in stimulation to growth of rats after administration of the drug.

[Ultrastructural changes in the cerebellum of experimental hypothyroidism (cretinism)].

We examined the cerebellum of the rats being the experimental hypothyroidism (cretinism) on the 20th day, 35th day and 60th day by the light and electron microscope. The remarkable findings were observed to the experimental group on the 20th day. The maturational states of this group correspond to that of the 16th day of the control group. On the other hand, the remarkable pathological findings were not observed on the 35th and 60th day of the experimental group. According to the findings of the experimental rats on the 20th day, the changes of the cells were the retention of the external granule cells and the maturational changes of the internal granule cells. We recognized the retardation of the maturational timing and a disagreement of the maturational rate of the internal granule cells. The lamellar bodies being the disturbance of the mitochondrial cristae were observed. About the white matter, the myelinated nerve fibers were a small quantity in number, and the deficiency of the myelin synthesis, the maturational disturbance of oligoglia were seen. In the several axons, the lamellar bodies and the honeycomb like structures were seen. It is concluded that the maturational disturbance of the internal granule cells are due to the disturbance of the secondary protein synthesis by the hypothyroid state and the degeneration of the mitocondria. The deficiency of the myelin synthesis is related to the maturational disturbance of oligoglia.