Preparation and evaluation of orally disintegrating tablets containing taste masked microparticles of acetaminophen.

In the present work, taste masked particles of acetaminophen (AAP), a highly soluble bitter tasting drug, were developed and ODT containing the taste masked particles were prepared. Taste masked particles of AAP were prepared using different amounts of tetraglycerol polyricinoleate (TGPR) and Eudragit ®E100. Although the drug content ratio and drug recovery decreased with increasing TGPR, drug release from AAP-CR100 particles containing a large amount of TGPR was mostly suppressed for 2 min. Hence, AAP-CR100 was incorporated into ODT as taste masked particles for AAP. Three major disintegrants were used for ODT, and it was confirmed that the tensile strength of all formulations showed applicable hardness for handling. The AAP-CR100-CP(40) formulation containing crospovidone showed the shortest disintegration time and the drug release from AAP-CR100-CP(40) into pH 6.8 test solution was suppressed compared with commercial AAP tablets. Because the drug release from AAP-CR100-CP(40) into the pH 1.2 test solution was rapid, it was suggested that drug release from AAP-CR100-CP(40) is suppressed in the oral cavity, and the drug is released promptly in the stomach. Thus AAP-CR100-CP(40) may be useful as an ODT in which the dissolution of AAP in the oral cavity is suppressed.

Molecular genetic analysis of 30 families with Joubert syndrome.

Joubert syndrome (JS) is rare recessive disorders characterized by the combination of hypoplasia/aplasia of the cerebellar vermis, thickened and elongated superior cerebellar peduncles, and a deep interpeduncular fossa which is defined by neuroimaging and is termed the 'molar tooth sign'. JS is genetically highly heterogeneous, with at least 29 disease genes being involved. To further understand the genetic causes of JS, we performed whole-exome sequencing in 24 newly recruited JS families. Together with six previously reported families, we identified causative mutations in 25 out of 30 (24 + 6) families (83.3%). We identified eight mutated genes in 27 (21 + 6) Japanese families, TMEM67 (7/27, 25.9%) and CEP290 (6/27, 22.2%) were the most commonly mutated. Interestingly, 9 of 12 CEP290 disease alleles were c.6012-12T>A (75.0%), an allele that has not been reported in non-Japanese populations. Therefore c.6012-12T>A is a common allele in the Japanese population. Importantly, one Japanese and one Omani families carried compound biallelic mutations in two distinct genes (TMEM67/RPGRIP1L and TMEM138/BBS1, respectively). BBS1 is the causative gene in Bardet-Biedl syndrome. These concomitant mutations led to severe and/or complex clinical features in the patients, suggesting combined effects of different mutant genes.

Tumor necrosis factor-alpha gene promoter methylation in Japanese adults with chronic periodontitis and rheumatoid arthritis.

BACKGROUND AND OBJECTIVE: Over-expression of tumor necrosis factor-alpha (TNF-α) plays a pathological role in chronic periodontitis (CP) and rheumatoid arthritis (RA), which might be regulated by the epigenetic mechanism. The aim of the present study was to evaluate whether there is a unique methylation profile of the TNF-α gene promoter in blood cells of individuals with CP and RA. MATERIAL AND METHODS: The study participants consisted of 30 Japanese adults with RA (RA group), 30 race-matched adults with CP only (CP group) and 30 race-matched healthy controls (H group). Genomic DNA isolated from peripheral blood was modified by sodium bisulfite and analyzed, by direct sequencing, to investigate DNA methylation of the TNF-α gene promoter region. The level of TNF-α produced in mononuclear cells stimulated with Porphyromonas gingivalis lipopolysaccharide was determined using ELISA. RESULTS: Twelve cytosine-guanine dinucleotide (CpG) motifs were identified in the TNF-α promoter fragment from -343 to +57 bp. The CP group showed a significantly higher methylation rate and frequency at -72 bp than the H group (p < 0.01). The RA group exhibited significantly higher methylation rates at seven CpG motifs (-302, -163, -119, -72, -49, -38 and +10 bp), and significantly higher methylation frequencies at six CpG motifs (-163, -119, -72, -49, -38 and +10 bp), than the H group (p < 0.01 for all comparisons). The levels of TNF-α produced were significantly different between individuals with and without methylation at -163 bp (p = 0.03). CONCLUSION: These results suggest that the hypermethylated status of CpG motifs in the TNF-α gene promoter in blood cells may be unique to Japanese adults with CP and RA.

Expression of anti-Porphyromonas gingivalis peptidylarginine deiminase immunoglobulin G and peptidylarginine deiminase-4 in patients with rheumatoid arthritis and periodontitis.

BACKGROUND AND OBJECTIVE: Autoimmunity against citrullinated proteins through peptidylarginine deiminase (PAD) may be involved in the pathophysiology of rheumatoid arthritis (RA). The present study evaluated the serum levels of antibodies to citrullinated proteins and to Porphyromonas gingivalis PAD (PPAD), and the endogenous expression of PAD-4, in individuals with and without RA, as well as before and after periodontal treatment. MATERIAL AND METHODS: The study participants consisted of 52 patients with RA (RA group) and 26 age-, gender- and smoking status-matched healthy controls (non-RA group). Of the 52 patients, 26 were randomly assigned to receive oral hygiene instruction and supragingival scaling (RA subgroup). After periodontal and rheumatologic assessments, the serum levels of anti-cyclic citrullinated peptide (CCP) immunoglobulin G (IgG), anti-PPAD IgG and PAD-4 were determined using ELISA. RESULTS: The serum levels of anti-CCP IgG and anti-PPAD IgG were significantly higher in the RA group than in the non-RA group (p < 0.001 and p = 0.03). A significant, positive correlation was observed between the serum levels of anti-PPAD IgG and anti-CCP IgG (p = 0.04), but not between the serum levels of PAD-4 and anti-CCP IgG. Multiple logistic regression analyses revealed a significant association between anti-PPAD IgG responses and RA after adjustment for age, gender and smoking (p = 0.004). Supragingival scaling significantly improved the periodontal condition and disease activity of RA (p < 0.05), but failed to decrease the serum levels of anti-CCP IgG, anti-PPAD IgG and PAD-4 after 2 mo of treatment. CONCLUSION: These results might suggest an association between anti-PPAD IgG and anti-CCP IgG responses, implicating a role for PPAD in protein citrullination in patients with RA and periodontitis.

Molecular and enzymatic characterization of betaC-S lyase in Streptococcus constellatus.

BACKGROUND/AIMS: Streptococcus anginosus and Streptococcus constellatus are frequently isolated from dental abscesses and other suppurative lesions. We previously reported that betaC-S lyase from a strain of S. anginosus produced significantly more hydrogen sulfide than betaC-S lyases from other streptococci. The purpose of this study was to establish the molecular and enzymatic features of the betaC-S lyase in S. constellatus and to elucidate whether this unique capacity is common to many strains of S. constellatus and S. anginosus. METHODS: The capacity of crude extract to produce hydrogen sulfide was evaluated among 16 strains of S. constellatus, S. anginosus, and Streptococcus gordonii. The lcd gene encoding betaC-S lyase was cloned from the genomic DNA of each strain to compare the deduced amino acid sequences. The recombinant betaC-S lyases of three representative strains were purified and characterized. RESULTS: Incubation of crude extracts from all strains of S. constellatus and S. anginosus with l-cysteine resulted in the production of a large amount of hydrogen sulfide. The primary sequence of betaC-S lyase was very similar among strains of S. constellatus and S. anginosus. The kinetic properties of the betaC-S lyases purified from S. constellatus resembled those for betaC-S lyases purified from S. anginosus. In contrast, the betaC-S lyases of S. constellatus and S. gordonii differed in terms of their hydrogen sulfide production, with the former producing much more. CONCLUSION: A high level of hydrogen sulfide production, which appears to be a common feature in both S. constellatus and S. anginosus, may be associated with their abscess formation.

Anodic oxidation and hydrothermal treatment of commercially pure titanium surfaces increases expression of bone morphogenetic protein-2 in the adherent macrophage cell line J774A.1.

The surface property of commercially pure titanium (cpTi) was improved by forming a thin hydroxyapatite (HA) layer by anodic oxidation and hydrothermal treatment (HA/cpTi). We hypothesize that the adhesion of macrophages to HA/cpTi surfaces is important to the process of osseointegration. This study investigates the effect of adhesion of macrophages to HA/cpTi surfaces on the expression of bone morphogenetic protein-2 (BMP-2). The murine macrophage cell line J774A.1 was cultured on HA/cpTi and polished cpTi (S/cpTi). Macrophage cell adhesion was examined by SEM, 0-72 h following plating onto HA/cpTi and S/cpTi. BMP-2 gene expression was examined by RT-PCR analysis. The level of BMP-2 secreted into the supernatant was measured using an ELISA assay. The extent of macrophage adhesion increased with time on both the HA/cpTi and S/cpTi surfaces, with a" higher degree of spreading observed on HA/cpTi than onS/cpTi surfaces after 24 or 72 h. The ratio of BMP-2 mRNA was higher on HA/cpTi than on S/cpTi after 24 h (0.348 vs. 0, p < 0.05) and 72 h (0.584 vs. 0.189, p < 0.05). After 24 h, secretion of BMP-2 was detected in cultures grown on HA/cpTi, but not on S/cpTi. After 72 h, secretion of BMP-2 was detected in cultures grown on S/cpTi, but the levels were higher in cultures grown on HA/cpTi. These findings show that macrophages have the capacity to adhere to HA/cpTi endosseous implants and provide a source of osteoinductive cytokines that may play a key role in the process of osseointegration.

Effects of positive pressure in odontogenic keratocysts.

Intracystic fluid pressure is thought to be involved in odontogenic cyst growth. In this study, we investigated the effects of positive pressure on the expression of interleukin-1alpha (IL-1alpha), matrix metalloproteinases (MMPs), and prostaglandin E2 (PGE2) in odontogenic keratocysts to determine whether this pressure stimulates inflammatory cytokine production and signaling of osteoclastogenic events. Positive pressure enhanced the expression of IL-1alpha mRNA and protein in odontogenic keratocyst epithelial cells, and increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in a co-culture of odontogenic keratocyst fibroblasts and the epithelial cells. The pressure-induced secretions were inhibited by an interleukin-1 receptor antagonist. Recombinant human interleukin-1alpha (rhIL-1alpha) increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in the fibroblasts. Furthermore, in the fibroblasts, rhIL-1alpha enhanced the expression of macrophage colony-stimulating factor (M-CSF) mRNA, and rhIL-1alpha-induced PGE2 increased the expression of nuclear factor kappaB ligand (RANKL) mRNA. Thus, positive pressure may play a crucial role in odontogenic keratocyst growth via stimulating the expression of IL-1alpha in epithelial cells.

A drug formulation using an alginate hydrogel matrix for efficient oral delivery of the manganese porphyrin-based superoxide dismutase mimic.

In order for patients to avail of the therapeutic benefits of antioxidant drugs efficiently and conveniently, a robust oral delivery system needs to be developed. However, a common problem in oral drug delivery is ensuring that the drug remains functionally intact even after it has passed through the acidic environment of the gastrointestinal (GI) tract. To protect drugs within the GI environment, we formulated a design based on encapsulating liposomal drugs by using an alginate matrix as a carrier. The liposomal drug was composed of manganese porphyrin (Mn-por), which has been developed as a mimic of superoxide dismutase (SOD), as the therapeutic agent based on the antioxidative effect, namely superoxide (O2˙(-)) inhibitory activity. A cytochrome c assay revealed that the O2˙(-) inhibitory activity of Mn-por could be maintained even after treatment with simulated gastric and intestinal fluids. We demonstrated that oral administration of the formulated drug significantly inhibited the growth of transplanted tumors in mice. The drug formulation presented in this study would be a good candidate for orally available systems, which can effectively deliver SOD mimics.

Melanin monomers within coated vesicles and premelanosomes in melanin synthesizing cells.

We have found substantial amounts (6.6-143 and 0.5-13 micrograms/mg. protein, respectively) of 5,6-dihydroxyindole (5,6-DHI) and 5,6-dihydroxyindole-2-carboxylic acid (5,6-DHI2C), which are key intermediate monomers for the formation of the eumelanin polymer, within coated vesicle fraction of pigment cells. In addition, the amounts of these eumelanin monomers have been found to decrease along with the process of eumelanin polymer formation from coated vesicles to premelanosomes and finally to melanosomes among melanogenic subcellular compartments. Our present findings seem to indicate that coated vesicles transfer not only highly glycosylated T1-tyrosinase but also eumelanin monomers into premelanosomes.

Carbonic anhydrase in mouse salivary glands and saliva: a histochemical, immunohistochemical, and enzyme activity study.

Mouse parotid gland and saliva were studied by histochemical, immunohistochemical, and activity measurements for carbonic anhydrase. Hansson 's histochemical reaction for carbonic anhydrase revealed positive enzyme activity in the parotid acinar cell cytoplasm and little or no reaction in the secretory granules. The luminal contents in all of the glandular duct systems also reacted positively, but the duct cells themselves were only weakly positive. Ultrastructural observations confirmed the light microscope histochemical localization and, in addition, revealed luminal content activity in intercellular ducts. Purified carbonic anhydrase isolated from mouse salivary glands was used to raise antibodies in rabbits. Localization of carbonic anhydrase by direct immunolabeling with fluorescein-coupled antibody and indirect immunoperoxidase labeling revealed enzyme localization on or in the acinar cell secretory granule membrane and not in the surrounding cytoplasm. The luminal contents of the intercalated and striated ducts were also strongly positive. Stimulation of salivary secretion with phenylephrine or pilocarpine increased the amount of carbonic anhydrase in saliva. Acetatazolamide and potassium cyanate inhibited carbonic anhydrase activity. Reasons underlying the discrepancy between the histochemical and immunolabeling localization of carbonic anhydrase are discussed. It is concluded that the parotid acinar cells of mice appear to be a significant source of carbonic anhydrase in saliva but its role is enigmatic.

Surface coating of hydrophilic-hydrophobic block co-polymers on a poly(acrylonitrile) haemodialyser reduces platelet adhesion and its transmembrane stimulation.

Surface design aimed at reduced adhesion and preserved functions of platelets is of great importance for extracorporeal devices. In this study, a coating technique using hydrophilic-hydrophobic block co-polymers on a hydrophobic poly(acrylonitrile) (PAN) haemodialyser was explored. The hydrophilic block of co-polymers was composed of either poly(methoxy polyethylene glycol methacrylate) or poly(dimethyl acrylamide), and the hydrophobic block was poly(methyl methacrylate). The co-polymers were coated on the dialyser membrane by means of a solution coating method. Upon coating, the hydrophobic block of the co-polymers was anchored on a PAN membrane and the hydrophilic block oriented towards the blood-material interface. This was deduced from water wettability measurements. Significantly reduced transmembrane stimulation of platelets was observed, which was evaluated by determining the intracellular calcium ion concentration of platelets eluted through treated hollow fibres. This suppression was enhanced as the relative fraction of the hydrophilic block of the co-polymers increased. Furthermore, the number of platelets adhering to the co-polymer-coated PAN membrane was drastically reduced. Thus, coating of the hydrophilic-hydrophobic block co-polymers provided better biocompatibility on a hydrophobic PAN dialyser.

Platelet adhesion and complement activation studies on poly(N-alkyl mono and disubstituted) acrylamide derivatives.

Poly(N-alkyl mono and disubstituted) acrylamide derivatives were synthesized from poly(acryloyl chloride) by monomer analogous reaction. The polymers were characterized by FTIR-ATR and GPC. The contact angle measurements were performed to evaluate hydrophobic/hydrophilic characters of these polymers. The N-alkyl substituents changed contact angle between 55 and 75 degrees. In vitro platelet adhesion studies showed that surfaces of poly(N-alkyl substituted) acrylamides are prone to adhere platelets. Platelet spreading was more on poly(N-benzyl-N-ethyl acrylamide) surfaces in comparison to that on poly(N-benzyl-N-propionic acid ethyl ester acrylamide) and poly(N-benzyl acrylamide) surfaces. As a result of modification of amino group with N-alkyl substituents, the activations of C3a and C5a complements were suppressed 9-20% and 5-6% of native poly(acrylamides), respectively.

In vitro effect of nanoleakage expression on resin-dentin bond strengths analyzed by microtensile bond test, SEM/EDX and TEM.

This study evaluated the effect of multiple consecutive adhesive resin coatings of adhesive bonded to human dentin on nanoleakage and resin-dentin bond strength. Resin bonded dentin specimens were prepared using a total-etch adhesive (One-Step Plus) applied as multiple consecutive coating, or using two self-etch adhesive systems (iBond or Fluoro Bond). For the total-etch adhesive, resin application and air evaporation were performed 1, 2, 3, or 4 times. The self-etch adhesives were applied according to manufacturers' instructions. Resin-dentin bonded beams were prepared and immersed in water (control) or ammoniacal silver nitrate. After storage, microtensile bond strengths were measured. The fractured surfaces were examined by scanning and transmission electron microscopy (SEM and TEM), and energy-dispersive X-ray spectrometry (EDX). No significant differences in bond strength were found between water and silver nitrate storage groups. Several types of silver depositions (spotted, reticular, or water trees) were found in adhesive joints. The bond strengths of the single coated specimens of the total-etch adhesive were significantly lower than those receiving 2-4 coatings. Single coats produced more nanoleakage than multiple coats. However, no correlation was found between the bond strengths and nanoleakage between the different adhesives (total-etch adhesive with different conditions or self-etch adhesives).

Cystatin S: a cysteine proteinase inhibitor of human saliva.

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.

Convergence of input from tongue and palate to the parabrachial nucleus neurons of rats.

Receptive fields (RFs) in the oral cavity were examined for 24 taste and 20 mechanoreceptive neurons in the parabrachial nucleus (PB) of anesthetized rats. All of the units studied had an RF on the ipsilateral half of the oral cavity. About half of the units in each category had at least two separate RFs. Most of the 'taste neurons', i.e. units receiving taste inputs, were activated by mechanical stimulation and some of them had a complex RF, e.g. separated into two parts, one on the tongue for taste and one on the palate for mechanical stimulation. The present findings show that the afferent inputs of two modalities from the tongue and palate converge on the PB neurons.

Fluid movement across the resin-dentin interface during and after bonding.

This study evaluated the extent of water penetration through resin-dentin interfaces before and after being sealed with adhesives. Four adhesive resin systems (2 total-etch adhesives and 2 self-etching primer adhesives) were used in this study. Dentin disks were placed in a split-chamber device, and in situ fluid movement across dentin was measured, with and without physiological pressure, during bonding procedures or 24 hrs after bonding. The fluid movement across dentin occurs via dentin tubules after acid-etching. Large outward or inward fluid shifts across dentin were observed during air-drying and light-curing for resin application. The amount of fluid movement across resin-bonded dentin when total-etch adhesives were used was significantly greater than that with self-etching adhesives. The milder acid-etching effects of self-etching primers may retain hybridized smear plugs within the tubules that reduce outward fluid flow, resulting in superior dentin sealing.

Collagen degradation by host-derived enzymes during aging.

Incompletely infiltrated collagen fibrils in acid-etched dentin are susceptible to degradation. We hypothesize that degradation can occur in the absence of bacteria. Partially demineralized collagen matrices (DCMs) prepared from human dentin were stored in artificial saliva. Control specimens were stored in artificial saliva containing proteolytic enzyme inhibitors, or pure mineral oil. We retrieved them at 24 hrs, 90 and 250 days to examine the extent of degradation of DCM. In the 24-hour experimental and 90- and 250-day control specimens, we observed 5- to 6-microm-thick layers of DCM containing banded collagen fibrils. DCMs were almost completely destroyed in the 250-day experimental specimens, but not when incubated with enzyme inhibitors or mineral oil. Functional enzyme analysis of dentin powder revealed low levels of collagenolytic activity that was inhibited by protease inhibitors or 0.2% chlorhexidine. We hypothesize that collagen degradation occurred over time, via host-derived matrix metalloproteinases that are released slowly over time.

Cooling effect on excimer laser photorefractive keratectomy.

PURPOSE: To evaluate the effect of cooling on pain, corneal haze, and refractive outcome after excimer laser photorefractive keratectomy (PRK). SETTING: Tokyo Medical and Dental University Hospital, Tokyo, Japan. METHODS: The corneal surface was cooled before, during, and after laser ablation using a method called cooling PRK. Thirty-eighty highly myopic eyes of 38 patients whose spherical errors ranged from -8.00 to -18.75 diopters (D) were randomized into 2 groups: 16 eyes with conventional PRK and 22 eyes with cooling PRK. Postoperative pain was measured using the Visual Analogue Scale (VAS). Refraction, visual acuity, and complications were followed for up to 2 years. All data were analyzed and compared between groups to evaluate the cooling effect on PRK. RESULTS: One day postoperatively, patients in the cooling PRK group had significantly less pain (P < .01). At 3 months, the haze score in the cooling PRK group was significantly less than in the conventional PRK group (P < .01). The residual refractive error was not significantly different between the 2 groups until 2 years, when it was greater in the conventional PRK (mean -5.09 D +/- 2.11 [SD]) than the cooling PRK group (-4.64 +/- 2.27 D). Ten eyes (62.5%) in the conventional PRK group and 15 eyes (68.2%) in the cooling PRK group were within +/- 1.00 D of the intended refraction. There were no serious complications in the cooling PRK group. Two eyes in the conventional PRK group had severe corneal haze and lost 2 Snellen lines of best corrected visual acuity. CONCLUSION: Corneal cooling on PRK effectively reduced postoperative pain, corneal haze, and myopic regression.

Recurrent giant longitudinal duodenal ulcer with massive hemorrhage in a Helicobacter pylori-negative patient.

A 67-year-old man, in whom a linear ulcer running from the duodenal bulb to the descending part had been noted 3 years previously, was admitted to our hospital because of abdominal pain and melena. Duodenoscopy revealed a bleeding giant longitudinal ulcer, which was more extensive than before. Tests for Helicobacter pylori (Hp) were negative. The ulcer was cured by endoscopic hemostasis and repeated blood transfusions. Attention must be paid to Hp-negative post-bulbar duodenal ulcers because of the frequent complications including hemorrhage.

Neural activities in the fronto-opercular cortex of macaque monkeys during tasting and mastication.

We recorded responses from 298 units in the fronto-opercular cortex (Fop) of three Japanese macaque monkeys. The units were activated while animals were drinking or eating. Three types of units could be defined: units with transient responses to the delivery of all the fluid into the mouth (ON-type units; n = 123); units activated in association with certain oral movements, e.g., licking (MO-type units; n = 90); and units responsive specifically to a certain fluid (taste units; n = 85). In both ON-type and MO-type units were included mechanoreceptive units with receptive fields in the oral cavity, mainly on the tongue and teeth. All but two taste units responded exclusively to gustatory stimuli. ON-type units were found mainly in the precentral opercular area (PrCO), and MO-type units were recorded predominantly in PrCO and area 6. Taste units were concentrated at the gustatory area and at a part of area 1-2. These results indicate the lack of convergence of somatic and taste input on single neurons. In awake monkeys, Fop units of a given function tended to be clustered at certain loci, thereby forming separate functional regions.