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.

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.

Preoperative administration of rebamipide significantly lowers body temperature and circulating interleukin-6 in gastric cancer patients after gastrectomy.

BACKGROUND/AIMS: It has recently been reported that rebamipide (OPC-12759) inhibits inflammatory cytokines and activation of neutrophils. The aim of the present study was to investigate the effects of preoperative administration of rebamipide on parameters of systemic inflammatory response syndrome (SIRS) and serum levels of inflammatory cytokines in gastric cancer patients after gastrectomy. PATIENTS AND METHODS: We measured the parameters of SIRS, circulating cytokines and acute phase reactants in patients after (i). distal gastrectomy with D2 lymph node dissection (group 1, n = 10); (ii). distal gastrectomy with D2 lymph node dissection following administration of rebamipide (group 2, n = 10), and (iii). laparoscopy-assisted distal gastrectomy (LADG) with D1 lymph node dissection (group 3, n = 10). Group 2 was administered 100 mg of rebamipide 3 times/day after meals for 7 days before surgery. RESULTS: Among the parameters of SIRS, a difference was observed in body temperature on day 3. The mean body temperature of group 2 was significantly lower than group 1 (p = 0.006), and was similar to group 3. In proinflammatory cytokines, a significant difference was observed in the serum levels of interleukin (IL)-6. On day 1 the IL-6 levels of group 2 were significantly lower than those of group 1 (p < 0.001). The changes in the IL-6 level of group 2 were similar to group 3, except in the very early postoperative phase. However, other proinflammatory cytokines, such as IL-8 and IL-10, were not detected, and there was no difference in C-reactive protein between the 3 groups. CONCLUSION: Preoperative administration of rebamipide significantly decreased postoperative body temperatures and circulating IL-6 in gastric cancer patients after gastrectomy to levels similar to those of patients with LADG.

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.

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.

Suppressive effect of norzoanthamine hydrochloride on experimental osteoporosis in ovariectomized mice.

Norzoanthamine is an alkaloid isolated from a colonial zoanthid. We examined the effects of norzoanthamine hydrochloride on bone weight, strength and morphology in ovariectomized mice, a postmenopausal osteoporosis model. Norzoanthamine hydrochloride significantly suppressed the decrease in femoral weight and bone biomechanical parameters caused by ovariectomy without an increase in uterine weight. This means that norzoanthamine hydrochloride does not have an estrogen-like effect on reproductive organs. Morphological observations of longitudinally ground sections of the humeri showed that norzoanthamine hydrochloride administration (2 mg/kg/d, p.o.) completely suppressed the loss of trabecular bone. Furthermore, norzoanthamine hydrochloride thickened the cortical bone. Based on these results, norzoanthamine hydrochloride may act as both a suppressor of bone resorption and an enhancer of bone formation in vivo.

Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-dihydroxyvitamin D(3).

Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyte-inducing agent 1alpha,25-dihydroxyvitamin D(3). We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50-55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophage-induced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation.

Nd:YAG laser influence on sound dentin bond strength.

OBJECTIVE: In the present study, the authors evaluated tensile bond strength of composite resin to dentin treated with Nd:YAG laser before and after bonding procedures. SUMMARY BACKGROUND DATA: Lasers have been widely used in dentistry and have contributed to the development of new technologies. Adhesive systems have been indicated for most dental procedures and have produced good results. Studies concerned with the combined use of this adhesive system was the subject of this study. METHODS: Thirty noncarious human anterior teeth, freshly extracted, for periodontal reasons, were used. After grinding the buccal dentinal surface until its exposition, specimens were separated into three groups and received the following treatments: (group 1) control--acid etching plus primer plus bond; (group 2) laser plus acid etching plus primer plus bond; (group 3) acid etching plus primer plus bond plus laser. Specimens of composite resin (Z100, 3M, Saint Paul, MN) were constructed on an inverted truncated 3-mm diameter cone mold. Tensile bond strength was performed using Inströn Universal machine, at 0.5 mm/min speed. RESULTS: Analysis of variance (ANOVA) (p < 0.05) determined that the type of dentinal treatments used had an influence on tensile bond strength. Tukey's test, however, showed that group 1 (15.46) and 3 (15.67) had similar results both of which were higher than group 2 (4.57). CONCLUSIONS: Based on the results obtained, one can conclude that group 1 (without laser) and group 3 (laser after bonding) had similar results, both higher than those observed for group 2 (laser before bonding). These results indicate that more research is needed about how a hybrid layer is formed when laser radiation is used.

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.

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.

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.

[Clinical evaluation of Azasetron Hydrochloride: a new selective 5-HT3 receptor antagonist--antiemetic profile and plasma concentration in transcatheter arterial chemoembolization using CDDP for unresectable hepatocellular carcinoma].

We performed a clinical evaluation on the antiemetic profile and the plasma concentration of Azasetron Hydrochloride (a new selective 5-HT3 receptor antagonist), in transcatheter arterial chemoembolization using CDDP for unresectable hepatocellular carcinoma. Antiemetic effects were examined in 32 patients in the serotone group (administration of serotone 10 mg + methylprednisolone 125 mg) and in 77 patients of the control group (administration of metoclopramide 20-30 mg + methylprednisolone 500 mg). The response rate and the CR ratio in serotone group was 97% and 66%, respectively. These results were statistically higher than in the control group. Although all patients had chronic liver diseases, no side effects and complications related to administration of serotone were observed. The average area under the concentration (AUC) curve of plasma serotone in five patients with liver cirrhosis was 531 ng.h/ml, which was greater than that of a healthy volunteer. In conclusion, serotone is a new, safe and useful antiemetic drug in TACE therapy for hepatocellular carcinoma.

Positions of disulfide bonds in yam (Dioscorea japonica) acidic class IL (class IV) chitinase.

Yam acidic class I chitinase belongs to a low molecular weight subclass of class I (class IL; corresponds to class IV) chitinase. The positions of disulfide bonds in this chitinase were examined. Chitinase protein was digested with acid protease and thermolysin, and the resulting disulfide bond containing peptides were separated by reversed-phase HPLC and detected using the SBD-F (7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid ammonium salt) method. Four intradisulfide bonds containing peptides were purified and three disulfide bonds in the catalytic domain were identified as Cys-66 and Cys-115, Cys-128 and Cys-136, and Cys-218 and Cys-250. Location of disulfide bonds in the catalytic domain was identical to that of barley class II chitinase but different from rye class II chitinase at the C-terminal. Conservation of S-S bonds at the N-terminal half of the catalytic domain between class I and class II chitinases strongly suggests that this region is important for formation of the active site.

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.

[Pulmonary lymphangiomyomatosis with tuberous sclerosis and endolymphatic stromal myosis of the uterus].

Pulmonary lymphangiomyomatosis is a rare disorder that affects only women of reproductive age. The lung shows numerous cystic changes and striking proliferation of smooth muscle around the airways, blood vessels, and lymphatic channels. We encountered a patient with pulmonary lymphangiomyomatosis who had repeated pneumothorax and bloody sputum over 10 years. The patient also had other lesions, including intracranial calcification, renal angiomyolipoma and endolymphatic stromal myosis of the uterus. The first two findings are known as tuberous sclerosis, and lymphangiomyomatosis is considered to be a form fruste of tuberous sclerosis. However, we know of no previous reports of uterine lesions in this disease. Lymphangiomyomatosis may be a systemic disorder of smooth muscle.

Resident gynecologists and total hysterectomy.

We examined the specific number of surgeries necessary for a three-year obstetrics and gynecology resident to acquire proficiency in two types of hysterectomies. Improvement in the technical skills of the residents was assessed using surgical time and blood loss, and resected tumor weight was chosen as the factor representing the difficulty of the surgery. Regarding abdominal total hysterectomy (ATH), early residents (less than 25 ATH experience) performed relatively easier surgeries, and improvement in technical skill was manifested as reduced blood loss by mid residents (25 to 49 ATH) and as shortened surgical time by later residents (75 or more ATH). Regarding vaginal total hysterectomy (VTH), blood loss for earlier residents (less than 15 VTH) was greater than that for the staff, and there was a significant difference between staff surgical time and that for each resident group. These data suggest that performing more than 75 ATH during the residency period of three years is adequate to establish proficiency in this type of surgery, but that the execution of 25 VTH is insufficient and that residents require more training to learn VTH.

[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.

Tissue-type plasminogen activator predicts endocrine responsiveness of human pancreatic carcinoma cells.

BACKGROUND: Estrogen receptor (ER) has been found in human pancreatic carcinoma, but the potential benefit of endocrine therapy never has been assessed adequately. The aim of this study was to determine whether the presence of ER can be used as an indicator of hormone responsiveness, and whether modulation of tissue-type plasminogen activator (t-PA) by ER can identify hormone-responsive pancreatic carcinomas. METHODS: The authors investigated ER status and hormonal regulation of t-PA in nine human pancreatic carcinoma cell lines, AsPC-1, BxPC-3, Capan-1, Capan-2, Hs-700T, Hs-766T, MiaPaCa-2, PANC-1, and SUIT-2. Furthermore, to examine whether estrogen dependency of t-PA production in pancreatic carcinoma cells correlated with responsiveness to endocrine therapy, the in vivo effects of various endocrine agents on the growth of the nine pancreatic cell lines transplanted into nude mice were examined. RESULTS: In a 17 beta-estradiol (E2)-binding assay, three of the nine pancreatic carcinoma cell lines (BxPC-3, Capan-2, and MiaPaCa-2) contained measurable levels of estradiol binding sites in vitro and in vivo using tumors transplanted into nude mice. Although t-PA was present in the culture medium in eight of the nine pancreatic carcinoma cell lines (not in Hs-700T), t-PA production was regulated by estrogen via an ER system in vitro only in the Capan-2 cell line. E2 injection into tumor-bearing mice showed acceleration of tumor growth only in Capan-2 tumors. Administration of a competitive ER antagonist, toremifene, and a luteinizing hormone-releasing hormone analogue, leuprorelin acetate (LEU), to Capan-2-bearing mice significantly reduced the rate of tumor growth, although there was no actual shrinkage of tumor mass. These agents failed to exert any antitumor effect on the other eight pancreatic cell lines. Although aromatase inhibitors, CGS 20267 and vorozole did not modify the in vivo growth of the nine pancreatic carcinoma cell lines significantly, the combined use of aromatase inhibitors with LEU exhibited a synergistic antitumor effect on Capan-2-bearing mice. Medroxyprogesterone acetate treatment significantly reduced the tumor volume of Capan-2 and also caused delayed growth in two other cell lines, AsPC-1 and MiaPaCa-2. CONCLUSIONS: The estrogen dependency of t-PA production may clarify the functional state of ER in human pancreatic carcinoma cells. This finding may aid in planning endocrine therapy for patients with this lethal cancer.

The amino acid sequence of copper pheasant lysozyme.

The amino acid sequence of copper pheasant lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 20, 77, and 113 for Lady Amherst's pheasant lysozyme and seven amino acid substitutions at positions 3, 15, 20, 41, 113, 121, and 124 for hen lysozyme. Phenylalanine at position 20 was newly detected in avian lysozyme.