Evaluation of 4β-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice

Cytochrome P450 3A (CYP3A) enzymes metabolize approximately half of all drugs on the market. Since the endogenous compounds 4β-hydroxycholesterol (4β-HC) and 25-hydroxycholesterol (25-HC) are generated from cholesterol via CYP3A enzymes, we examined whether the plasma levels of 4β-HC and 25-HC reflect hepatic CYP3A4 activity by using a CYP3A-humanized mouse model, in which the function of endogenous Cyp3a was genetically replaced by human CYP3A. CYP3A-humanized mice have great advantages for evaluation of the relationship between hepatic CYP3A protein levels and plasma and hepatic levels of 4β-HC and 25-HC. Levels of CYP3A4 protein in the liver microsomes of CYP3A-humanized mice were increased by treatment with pregnenolone-16α-carbonitrile, a CYP3A inducer. Hepatic and plasma levels of 4β-HC and 25-HC normalized by cholesterol were significantly correlated with hepatic CYP3A4 protein levels. In addition, in vitro studies using human liver microsomes showed that the formation of 4β-HC was strongly inhibited by a CYP3A inhibitor, while the inhibitory effect of the CYP3A inhibition on the formation of 25-HC was weak. These results suggested that CYP3A mainly contributed to the formation of 4β-HC in human liver microsomes, whereas other factors may be involved in the formation of 25-HC. In conclusion, the in vivo studies using CYP3A-humanized mice suggest that plasma 4β-HC and 25-HC levels reflect hepatic CYP3A4 activity. Furthermore, taking the results of in vitro studies using human liver microsomes into consideration, 4β-HC is a more reliable biomarker of hepatic CYP3A activity.     

A rare case of pulmonary typical carcinoid with prominent acinic cell differentiation,resembling acinic cell carcinoma

We herein describe a rare case of low‐grade endobronchial tumor that exhibited two distinct features of typical carcinoid and acinic cell carcinoma (ACC) by immunohistochemical and ultrastructure study. ACC was suspected on transbronchial biopsy. The resected specimen showed that the tumor surface comprised an acinic cell component (40% of the tumor), and the central area comprised typical carcinoid (60% of the tumor). The acinic cell component was positive for chromogranin A, synaptophysin and alpha‐1‐antichymotrypsin. Additionally, this component showed focal apical membranous staining for DOG1 and weak positivity for BCL10 and SOX10. Conversely, the carcinoid component was negative for all proteins except for chromogranin A and synaptophysin. Electron microscopy indicated zymogen‐type granules (600–800 nm in diameter) in the acinic cell component, whereas neuroendocrine‐type granules (200–300 nm in diameter) were observed in the carcinoid component. Nuclear NR4A3 immunostaining, which is highly specific for ACC of the salivary gland, was negative in this case. We conclude that the pulmonary carcinoid tumor with true zymogen‐type granules could be seen but showed superficial similarities to ACC based on negative nuclear staining for NR4A3. Pulmonary carcinoids encompass a wide morphological spectrum and may exhibit prominent acinic cell differentiation.     

DENTAL MATURITY AS AN INDICATOR OF CHRONOLOGICAL AGE: RADIOGRAPHIC ASSESSMENT OF DENTAL AGE IN A BRAZILIAN POPULATION

Objective:

The purpose of this investigation was to evaluate the applicability of the methods proposed by Nolla and by Nicodemo and colleagues for assessing dental age and its correlation to chronological age.

Methods:

Panoramic radiographs of 360 patients from the city of Fortaleza (CE, Brazil) aged 7-15 years were used to assess the associations between dental and chronological age. Data were submitted to statistical analysis using the BioEstat 2.0 (2000) software. Student-Neuman-Keuls test was performed and Pearson''s correlation coefficients were calculated at 5% significance level.

Results:

When the Nolla method was applied, the mean difference between true and estimated age for males and females was underestimated. The use of the method proposed by Nicodemo and colleagues also resulted in underestimation, although it was more evident in male subjects. The correlation coefficients between chronological age and estimated dental age were high, with mean values ranging between 0.87 and 0.91 for males and between 0.84 and 0.93 for females.

Conclusion:

Although both methods proved to be reliable in estimating age, the use of correction factors is recommended.     

Subcloning of the 200-kDa Porphyromonas gingivalis antigen gene and inhibition of hemagglutination by an antibody against the recombinant protein

Porphyromonas gingivalis is a major etiologic agent of periodontitis and exhibits hemagglutinating and adherence activities. We previously succeeded in molecular cloning the 200-kDa cell-surface antigenic protein (200-k AP), designated pMD101, that is recognized in sera from periodontitis patients, and identified the 200-k AP as a hemagglutinin A (HagA) derivative. HagA is one of the hemagglutinins known to be a useful vaccine against periodontitis. HagA has four large, contiguous, direct repeats and the repeat unit is believed to contain the hemagglutinin domain. Because production of 200-k AP was low in the Escherichia coli host, it was difficult to obtain large amounts of recombinant protein. In this study, we attempt to subclone the gene encoding the useful antigen from pMD101 in an effort to obtain large quantities. A subclone, designated pMD160, encoding a fusion protein of 80-kDa HagA and maltose-binding protein was successfully constructed, and the novel clone produced relatively large amounts of recombinant protein. DNA nucleotide sequences of the pMD160 insert demonstrated that the 80-kDa protein contained a short hemagglutinin motif and a direct repeat unit region. The recombinant protein was purified to homogeneity and rabbit antiserum was raised. The antibody was capable of inhibiting the hemagglutinating activity of P. gingivalis. These findings suggest that novel 80-kDa HagA derivative proteins can be produced efficiently from E. coli hosts and these may be useful in developing immunotherapy against periodontitis infected by P. gingivalis.     

Comparison of depth of dentin etching and resin infiltration with single-step adhesive systems

OBJECTIVES: Adhesion of resin composites to dentin is currently believed to result from impregnation of adhesive resin into superficially demineralized dentin. The purpose of this study was to use micro-Raman spectroscopy and scanning electron microscopy (SEM) to investigate the extent of resin penetration into etched dentin with single-step adhesive systems. METHODS: Adhesive systems used were One-Up Bond F (Tokuyama Dental) and Reactmer Bond (Shofu, Inc.). A self-etching primer system Mac Bond II (Tokuyama Dental) was employed as a control. Resin composites were bonded to bovine dentin with the adhesive systems, and specimens were sectioned parallel to dentinal tubules. Raman spectra were successively recorded along a line perpendicular to the dentin-adhesive interface in steps of 0.2 microm and the spectra were obtained. SEM observations of the resin-dentin interface were also conducted. RESULTS: The dentin-resin interface of single-step adhesive systems showed a gradual transition in the relative amount of adhesive from the resin side to dentin side. The widths of resin penetration into demineralized dentin detected by Raman microscopy were greater than those obtained by the morphological analysis using SEM. CONCLUSIONS: From the results of this study, a gradual variation in the composition of the dentin-resin interface was detected, and the degree of resin impregnation observed with SEM observation was less than that detected with the Raman microscopy.     

Evaluation of 2 thione primers and 3 resin adhesives for silver-palladium-copper-gold alloy bonding.

OBJECTIVE: The purpose of this study was to evaluate 2 thione primers and 3 resin adhesives for enhancement of bonding strength to a silver-palladium-copper-gold alloy. METHOD AND MATERIALS: Two different sized disk specimens (10- and 8-mm diameter by 2.5-mm thick) were prepared from a silver-palladium-copper-gold alloy (Castwell M.C. 12, GC). The specimens were airborne-particle abraded with 50-microm-grain alumina, conditioned either with a thiouracil primer (Metaltite, Tokuyama Dental) or with a triazine dithione primer (V-Primer, Sun Medical), and then bonded with 1 of 3 acrylic resins: a benzoyl peroxide-amine redox-initiated resin adhesive (Multi-Bond, Tokuyama Dental) or a tri-nbutylborane-initiated resin adhesive (Super-Bond C&B and Super-Bond Quick, Sun Medical). For each adhesive, unprimed specimens were prepared as experimental controls. Shear bond strength was determined after thermocycling (100,000 cycles). RESULTS: Use of primers significantly (P < .05) enhanced the bond strength of specimens in all adhesives. Irrespective of the type of primer, the strength of Multi-Bond adhesive was significantly (P < .05) lower than that of Super-Bond C&B and Super-Bond Quick adhesives. The strength of the 2 tri-n-butylborane-initiated adhesives did not differ significantly (P > .05). The mean strength of the Super-Bond C&B adhesive was 40.4 MPa with Metaltite and 37.8 MPa with V-Primer; that of Super-Bond Quick adhesive was 40.9 MPa with Metaltite and 36.5 MPa with V-Primer. CONCLUSION: Use of thione primers effectively enhanced the strength of the bond to the silver-palladium-copper-gold alloy. Furthermore, the combinations of primers and tri-n-butylborane-initiated adhesives were found to be more efficient for bonding.