Abstract: Objective To elucidate the direct role of human T cells in the induction of osteoclastogenesis in rheumatoid arthritis (RA), by studying human monocytes and the pathogenetic roles of receptor activator of nuclear factor κB ligand (RANKL), RANK, and osteoprotegerin (OPG). Methods Synovial tissue obtained at total knee replacement was stained immunohistologically using anti-RANKL, CD3, and CD4 antibodies. Synovial fluid was obtained from patients with RA, osteoarthritis (OA), gout, or trauma. Concentrations of the soluble form of RANKL (sRANKL) and OPG in the synovial fluid were measured by enzyme-linked immunosorbent assay. Activated T cells from peripheral blood mononuclear cells (PBMC) of healthy volunteers were cultured with human monocytes from PBMC. Results Immunostaining of the synovial tissue of RA patients demonstrated that RANKL-positive cells were detected in a subset of fibroblast-like synoviocytes and infiltrating mononuclear cells. Double immunostaining revealed that RANKL-positive cells were detected in a subset of CD3+ cells and CD4+ cells. An increased concentration of sRANKL and a decreased concentration of OPG were detected in synovial fluid from RA patients. The ratio of the concentration of sRANKL to that of OPG was significantly higher in synovial fluid of RA patients than in synovial fluid of patients with OA or gout. The activated T cells expressing RANKL induced osteoclastogenesis from autologous peripheral monocytes. The role of RANKL in this osteoclastogenetic process was confirmed by dose-dependent inhibition by OPG. Conclusion The present study is the first to demonstrate osteoclastogenesis using human-derived T cells and monocytes. In addition, the present findings suggest that excess production of RANKL by activated T cells increases the level of sRANKL in synovial fluid and may contribute to osteoclastic bone resorption in RA patients.

Abstract: Compared to MICs (more than 800 μg/ml) of (−)-epigallocatechin gallate (EGCg) against Escherchia coli, MICs of EGCg against methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) were 100 μg/ml or less. Furthermore, less than 25 μg EGCg per ml obviously reversed the high level resistance of MRSA to all types of tested β-lactams, including benzylpenicillin, oxacillin, methicillin, ampicillin, and cephalexin. EGCg also induced a supersusceptibility to β-lactams in MSSA which does not express mecA, encoding penicillin-binding protein 2′ (PBP2′). The fractional inhibitory concentration (FIC) indices of the tested β-lactams against 25 isolates of MRSA were from 0.126 to 0.625 in combination with 6.25, 12.5 or 25 μg of EGCg per ml. However, no synergism was observed between EGCg and ampicillin against E. coli. EGCg largely reduced the tolerance of MRSA and MSSA to high ionic strength and low osmotic pressure in their external atmosphere, indicating damage of the cell wall. Unlike dextran and lipopolysaccharide, peptidoglycan from S. aureus blocked both the antibacterial activity of EGCg and the synergism between EGCg and oxacillin, suggesting a direct binding of EGCg with peptidoglycan on the cell wall. EGCg showed a synergistic effect with dl-cycloserine (an inhibitor of cell wall synthesis unrelated to PBP2′) but additive or indifferent effect with inhibitors of protein and nuclear acid synthesis. EGCg did not suppress either PBP2′ mRNA expression or PBP2′ production, as confirmed by reverse transcription-PCR and a semiquantitative PBP2′ latex agglutination assay, indicating an irrelevance between the synergy and PBP2′ production. In summary, both EGCg and β-lactams directly or indirectly attack the same site, peptidoglycan on the cell wall. EGCg synergizes the activity of β-lactams against MRSA owing to interference with the integrity of the cell wall through direct binding to peptidoglycan.

Abstract: Craniometaphyseal dysplasia (CMD) is a rare skeletal disorder characterized by progressive thickening and increased mineral density of craniofacial bones and abnormally developed metaphyses in long bones. Linkage studies mapped the locus for the autosomal dominant form of CMD to an ∼5-cM interval on chromosome 5p, which is defined by recombinations between loci D5S810 and D5S1954. Mutational analysis of positional candidate genes was performed, and we describe herein three different mutations, in five different families and in isolated cases, in ANK, a multipass transmembrane protein involved in the transport of intracellular pyrophosphate into extracellular matrix. The mutations are two in-frame deletions and one in-frame insertion caused by a splicing defect. All mutations cluster within seven amino acids in one of the six possible cytosolic domains of ANK. These results suggest that the mutated protein has a dominant negative effect on the function of ANK, since reduced levels of pyrophosphate in bone matrix are known to increase mineralization.

Abstract: Background: It is important to clarify the participation of periodontal ligament (PDL) cells in the regeneration of alveolar bone to establish a reliable approach for obtaining periodontal regeneration. The aim of this study was to determine whether PDL cells play an important role in alveolar bone repair during the course of periodontal regeneration. Methods: In an in vitro study, the expression of the osteoblast phenotype, such as alkaline phosphatase activity and parathyroid hormone-dependent 3′,5′-cyclic adenosine monophosphate accumulation, was investigated in dog PDL cells (DPLC) and dog bone cells isolated from mandibles (DBC). In a related study, the roots of mandibular third premolars extracted from aged dogs were divided into a PDL(+) group, in which the PDL was preserved, and a PDL(-) group, in which the PDL was removed. These roots were respectively transplanted into surgically created bone cavities with buccal and interproximal bone defects in an edentulous area, prepared in advance by extrac...

Abstract: Background Nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. Previously, we found that the CYP2A6 gene was deleted homozygously in one subject who was deficient in cotinine formation from nicotine. Objective Our objective was to clarify the relationship between interindividual differences in nicotinemetabolism and CYP2A6 genetic polymorphism. Methods Nicotine was administered to 92 healthy Japanese subjects in the form of 1 piece of nicotine gum to investigate the potency of nicotine metabolism. The cotinine-nicotine ratio of the plasma concentration 2 hoursafter chewing was calculated as an index of nicotine metabolism. The genotypes of CYP2A6 gene, CYP2A6*1A, CYP2A6*1B, CYP2A6*2, CYP2A6*3, CYP2A6*4, and CYP2A6*5, were determined with polymerase chain reaction–restriction fragment length polymorphism. Results A large interindividual difference in nicotine metabolism was observed. Allele frequencies of CYP2A6*1A, CYP2A6*1B, and CYP2A6*4 were 42.4%, 37.5%, and 20.1%, respectively. The CYP2A6*2, CYP2A6*3, and CYP2A6*5 alleles were not found. Three subjects genotyped as CYP2A6*4/CYP2A6*4 were completely deficient in cotinine formation. The heterozygotes of the CYP2A6*4 allele tend to show lower capacities for cotinine formation. The subjects with CYP2A6*1A/CYP2A6*1B appeared to have higher capacities of cotinine formation than subjects with CYP2A6*1A/CYP2A6*1A, although the difference was not significant. The probit plot of the cotinine-nicotine ratio was not linear; this possibly indicated the existence of a novel mutation in the CYP2A6 gene genotyped as CYP2A6*1B/CYP2A6*4. Conclusions The relationship between interindividual differences in nicotine metabolism and CYP2A6 genetic polymorphism in humans was proved. Clinical Pharmacology & Therapeutics (2001) 69, 72–78; doi: 10.1067/mcp.2001.112688

Abstract: Publisher Summary This chapter introduces the recent advances in the diversity, enzymatic properties, and functions of the sPLA 2 family emphasizing particularly on the roles of each sPLA 2 in eicosanoid generation in the context of functional coupling between other eicosanoid-biosynthetic enzymes in different phases of cell activation. There are four major families of PLA 2 : secretory PLA 2 s (sPLA 2 s), cytosolic PLA 2 s (cPLA 2 s), Ca2 + -independent PLA 2 s (iPLA 2 s), and PAF acetylhydrolases (PAF-AH). As the overproduction of these lipid mediators causes various diseases and tissue disorders, it is important to understand the mechanisms that regulate the functions of PLA 2 . Recent advances in molecular and cellular biology have led to the identification of a number of mammalian PLA 2 enzymes that are subdivided into several groups based on their structures, enzymatic characteristics, subcellular distributions, and cellular functions. With the cloning of 10 sPLA 2 s, it is obvious that a diversity of sPLA 2 s exists in mammals. These sPLA 2 s exhibit different tissue distributions and inducibility by stimuli that also differ according to animal species. Moreover, sPLA 2 s can function not only as enzymes but also as ligands. Furthermore, it is important to understand the biological functions of the different members of the growing family of sPLA 2 s.

Abstract: In 1997, Lo et al. (1) first described the presence of fetal DNA in maternal plasma and serum. Cell-free DNA offered a new source of fetal genetic material for noninvasive prenatal diagnosis. They developed a real-time, quantitative PCR assay to measure the concentration of fetal DNA and analyzed SRY, a single-copy Y-chromosome-specific sequence, to quantify the number of genome-equivalents per milliliter of blood when a woman carries a male fetus (2). The results revealed that the concentrations of fetal DNA in maternal plasma DNA during the first and third trimesters were 3.4% and 6.2%, respectively. They estimated a mean of 25.4 copies of fetal DNA circulates per milliliter of maternal plasma samples during early pregnancy. They also demonstrated that after delivery, cell-free fetal DNA is cleared very rapidly from the maternal circulation, with a half-life in the order of minutes (3). Thus, the relatively high concentration of fetal cell-free DNA suggests that extensive and time-consuming fetal DNA enrichment procedures would not be necessary when using fetal DNA from maternal plasma for diagnostic purposes. Although the potential of fetal cell-free DNA analysis for the prenatal diagnosis of fetal gender (2) and rhesus D status (4)(5) has been presented, the accuracy of prenatal diagnosis has not been described. If fetal gender could be determined, the number of invasive procedures required to determine X-linked genetic disorders would be reduced. The present study evaluated the diagnostic accuracy of fetal gender determination using cell-free DNA from maternal plasma obtained at early gestation. Pregnant women (n = 302), carrying a single fetus between 7 and 16 weeks of gestation, …

Abstract: Background Underlying mechanisms behind opioid-induced respiratory depression are not fully understood. The authors investigated changes in burst rate, intraburst firing frequency, membrane properties, as well as presynaptic and postsynaptic events of respiratory neurons in the isolated brainstem after administration of opioid receptor agonists. Methods Newborn rat brainstem-spinal cord preparations were used and superfused with mu-, kappa-, and delta-opioid receptor agonists. Whole cell recordings were performed from three major classes of respiratory neurons (inspiratory, preinspiratory, and expiratory). Results Mu- and kappa-opioid receptor agonists reduced the spontaneous burst activity of inspiratory neurons and the C4 nerve activity. Forty-two percent of the inspiratory neurons were hyperpolarized and decreased in membrane resistance during opioid-induced respiratory depression. Furthermore, under synaptic block by tetrodotoxin perfusion, similar changes of inspiratory neuronal membrane properties occurred after application of mu- and kappa-opioid receptor agonists. In contrast, resting membrane potential and membrane resistance of preinspiratory and majority of expiratory neurons were unchanged by opioid receptor agonists, even during tetrodotoxin perfusion. Simultaneous recordings of inspiratory and preinspiratory neuronal activities confirmed the selective inhibition of inspiratory neurons caused by mu- and kappa-opioid receptor agonists. Application of opioids reduced the slope of rising of excitatory postsynaptic potentials evoked by contralateral medulla stimulation, resulting in a prolongation of the latency of successive first action potential responses. Conclusions Mu- and kappa-opioid receptor agonists caused reduction of final motor outputs by mainly inhibiting medullary inspiratory neuron network. This inhibition of inspiratory neurons seems to be a result of both a presynaptic and postsynaptic inhibition. The central respiratory rhythm as reflected by the preinspiratory neuron burst rate was essentially unaltered by the agonists.

Abstract: Objective: It is well known that both gastric and intestinal phenotypic cell markers are expressed in gastric carcinomas, irrespective of their histologic type. However, the clinico

Abstract: Compared with ampicillin, oxacillin, cefmetazole and imipenem, the combination of ampicillin and sulbactam at a constant ratio of 2:1 showed the greatest effect against 28 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), but MICs of ampicillin/sulbactam were still above the resistance breakpoint. When ampicillin/sulbactam was further combined with epigallocatechin gallate (EGCg, a main constituent of tea catechins), the MIC90 of ampicillin/ sulbactam was reduced to 4 mg/L, the susceptibility breakpoint. The fractional inhibitory concentration indices were between 0.19 and 0.56 in combination with 6.25 and 25 mg/L EGCg, respectively, indicating that ampicillin/sulbactam and EGCg combination may be effective against MRSA infections.

Abstract: Nausea and vomiting of moderate intensity are especially common complaints in early gestation. Hyperemesis gravidarum (HG), which is the most severe form of this disorder, occurs in 0.5–2% of pregnancies and is associated with weight loss, ketonemia, ketonuria, electrolyte imbalance, dehydration, and possible hepatic and renal damage. Recently, the presence of cell-free fetal DNA in maternal plasma or serum has been demonstrated (1). It has been reported that the concentration of fetal DNA in maternal plasma is increased in pregnancies involving preterm delivery (2), preeclampsia (3)(4), and trisomy 21 (5)(6). In the present study, we evaluated the concentration of fetal DNA in maternal plasma in HG patients. Pregnant women diagnosed with HG and admitted to Showa University Hospital were recruited. In this study, HG was defined according to the following criteria: ( a ) persistent nausea and vomiting; ( b ) weight loss (>2 kg); and ( c ) ketonuria (>2+, urine dipstick). Maternal blood samples were obtained at the time of admission from 35 patients with HG carrying a single fetus between 7 and 16 weeks of gestation. Sixteen of 35 blood samples collected from the HG patients carrying a male fetus were …

Abstract: Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKC zeta in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKC zeta to associate specifically with the GLUT4 compartments and that PKC zeta together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKC zeta and GLUT4 recycled independently of one another. To further establish the importance of PKC zeta in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKC zeta (DNPKC zeta) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKC zeta was associated with a marked increase in the activity of this isoform. The overexpressed, active PKC zeta coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKC zeta caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKC zeta induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKC zeta disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKC zeta regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.

Abstract: Hic-5 is a paxillin homologue that is localized to focal adhesion complexes. Hic-5 and paxillin share structural homology and interacting factors such as focal adhesion kinase (FAK), Pyk2/CAKb/RAFTK, and PTP-PEST. Here, we showed that Hic-5 inhibits integrin-mediated cell spreading on fibronectin in a competitive manner with paxillin in NIH 3T3 cells. The overexpression of Hic-5 sequestered FAK from paxillin, reduced tyrosine phosphorylation of paxillin and FAK, and prevented paxillin-Crk complex formation. In addition, Hic-5-mediated inhibition of spreading was not observed in mouse embryo fibroblasts (MEFs) derived from FAK 2/2 mice. The activity of c-Src following fibronectin stimulation was decreased by about 30% in Hic-5-expressing cells, and the effect of Hic-5 was restored by the overexpression of FAK and the constitutively active forms of Rho-family GTPases, Rac1 V12 and Cdc42 V12, but not RhoA V14. These observations suggested that Hic-5 inhibits cell spreading through competition with paxillin for FAK and subsequent prevention of downstream signal transduction. Moreover, expression of antisense Hic-5 increased spreading in primary MEFs. These results suggested that the counterbalance of paxillin and Hic-5 expression may be a novel mechanism regulating integrin-mediated signal transduction.

Abstract: The transradial approach (TRA) has been used for diagnostic and interventional cardiology. It has not previously been determined how many times the same radial artery can be cannulated without complications. A total of 812 patients (502 men and 310 women) underwent angiography or angioplasty via the TRA between 1997 and 1999 at our institution with a total of 1,438 procedures. Sheaths were 5 (55%) or 6 Fr (45%). Dropout rates of 3.5% and 7.9% were found at the second TRA attempt in the men and the women, respectively. Of the 62 TRA failures, 56 (90%) were due to narrowing or occlusion of the radial artery after the previous TRA procedure. A third TRA procedure was possible in 90% of the men and 80% of the women. A fifth TRA procedure was possible in 70% of the men and 50% of the women. The dropout rates for TRA increased as successive punctures were performed. This was primarily due to vessel narrowing and occlusion occurring as a function of multiple punctures.

Abstract: Objective: This investigation was performed to evaluate the acid resistance of lased enamel and dentin by Er,Cr:YSGG laser to artificial caries-like lesions by spectrophotometry, and the ultrastructure of lased areas was investigated by scanning electron microscopy (SEM) in vitro. Background Data: In recent years, many studies have been performed to evaluate the effects of Er,Cr:YSGG laser on dental hard tissues. However, there have been only a few studies to determine if this laser is suitable for caries preventive treatments. Methods: An Er,Cr:YSGG laser was used to irradiate the enamel or dentin samples from 30 extracted human molars at 6 W (67.9 J/cm2) or 5 W (56.6 J/cm2) pulse energy, respectively, with or without water mist. Samples were subjected to 2 μl of 0.1 M lactic acid solution (pH 4.8) for 24 h at 36°C. The parts per million (ppm) of calcium ion (Ca2+) dissolved in each solution was determined by atomic absorption spectrophotometery, and the morphological changes were investigated by SEM. Re...

Abstract: Vitamin D has been shown to exert manifold immunomodulatory effects. Type 1 diabetes mellitus (T1DM) is regarded to be immune-mediated and vitamin D prevents the development of diabetes in the NOD mouse. We studied the association between T1DM and the initiation codon polymorphism in exon 2 of the vitamin D receptor gene in a Japanese population. We also investigated associations between the vitamin D receptor polymorphism and GAD65-antibody (Ab) positivity. We carried out polymerase chain reaction-restriction fragment length polymorphism analysis in 110 Japanese T1DM patients and 250 control subjects. GAD65 antibodies were assessed in 78 patients with T1DM. We found a significantly higher prevalence of the F allele / the FF genotype in the patients compared to the controls (P = 0.0069 and P = 0.014, respectively). Genotype and allele frequencies differed significantly between GAD65-Ab-positive patients and controls (P = 0.017 and P = 0.012, respectively), but neither between GAD65-Ab-negative patients and controls (P = 0.68 and P = 0.66, respectively) nor between GAD65-Ab-positive and -negative patients (P = 0.19 and P = 0.16, respectively). Our findings suggest that the vitamin D receptor initiation codon polymorphism influences genetic susceptibility to T1DM among the Japanese. This polymorphism is also associated with GAD65-Ab-positive T1DM, although the absence of a significant difference between GAD65-Ab-negative patients and controls might be simply due to the small sample size of patients tested for GAD65 antibodies.

Abstract: The purpose of this study was to determine the accuracy of Gleason scores in prostate needle biopsy diagnosis and to investigate factors affecting the accuracy of the tumor grade. A single pathologist reviewed 116 sets of prostate cancer biopsies and radical prostatectomy specimens. The following factors were examined to determine their effect on the accuracy of the biopsy Gleason scores: (i) relative tumor differentiation; (ii) pathological stage; (iii) amount of tissue in the biopsy specimen; (iv) amount of cancer tissue in the biopsy specimen; (v) tumor heterogeneity; (vi) clinical findings (prostate specific antigen value and digital rectal examination); and (vii) interobserver variability. In 53 cases the Gleason score of biopsy specimens was identical to the score of prostatectomy specimens (45.7%). Fifty-four cases (46.6%) of biopsy specimens were undergraded. The most common discrepancy was diagnosis of well-differentiated carcinoma in the biopsy but diagnosis of moderately differentiated tumor in the corresponding prostatectomy specimen. This discrepancy occurred when the amount of tumor in the biopsy was 3 mm or less. Biopsy and prostatectomy results showed less agreement when the original biopsy tumor grade rendered by nine different pathologists was used, suggesting that interobserver variability can adversely affect the accuracy of tumor grade. Clarifying the histologic criteria for distinguishing each grade, especially between Gleason grades 2 and 3, is important for accurate grading.

Abstract: Background Mouse imprinted gene Peg3 encodes a large C2H2 type zinc finger protein with unique characteristics. Peg3 knockout mice were found to show an impairment in maternal behaviour of the adult female. Mouse Peg3 is located on the proximal region of chromosome 7 which is syntenic to the long arm of human chromosome 19. It has been reported that a loss of heterozygosity (LOH) of chromosome 19q occurs frequently in several glioma types. Results We isolated human PEG3 cDNA. Both human and mouse PEG3 were strongly expressed in the adult brain and the Peg3 protein was localized in the nuclei of both neurones and glial cells. A significant decrease in PEG3 expression was more commonly observed in glioma cell lines as compared with that in primary cultures of astrocytes. Transfection of PEG3 cDNA in a glioma cell line resulted in a loss of tumorigenicity in nude mice. Conclusions The human PEG3 gene is a paternally expressed imprinted gene. Introduction of PEG3 cDNA into the glioma cells suggests that human PEG3 protein functions as a tumour suppressor. Human PEG3 is located on 19q13.4 and is one of the candidates for tumour suppressor genes that are predicted to be sited in gliomas.

Abstract: Objective: The purpose of this investigation was to compare the surface roughness of enamel and dentin following the Er,Cr:YSGG laser irradiation and acid etching. Background Data: Laser-roughened enamel or dentin surfaces have been expected to enhance restorative materials bond strength. Materials and Methods: Er,Cr:YSGG laser irradiation was performed in one half of each polished enamel or dentin sample at 3 W (33.9 J/cm2, with air 70% and water 20%,) pulse energy for 6 sec. Then the other half was treated with 37% phosphoric acid for 30 sec. Surface roughness and morphological studies were performed. Results: It was found that surface roughness was significantly increased with the laser system. Scanning electron microscopy analysis showed that irradiated surface produces a rough surface that was completely lacking of a smear layer; there was also no cracking of enamel or dentin. Conclusion: Er,Cr:YSGG laser irradiation could provide an effective and alternative method to the acid etch technique.

Abstract: Although it is well known that the hippocampal CA1 subfield is highly vulnerable to ischemic injury, cellular mechanisms leading to this neuronal degeneration are not fully understood. Using organotypic cultures of rat hippocampal slices, we determined whether phospholipase A2 (PLA2) is activated in response to ischemic conditions (OGD; oxygen and glucose deprivation). The PLA2 activity in the pyramidal cell layer increased immediately following a 35-min exposure to OGD, which was likely to be mediated by selective activation of cytosolic Ca 2+ -dependent PLA2 subtype (cPLA2). This enhancement lasted for at least 24 h. Interestingly, no apparent increase was detected in the dentate gyrus. Twenty-four hours after the OGD exposure, neuronal death was detected mainly in the CA1 region of hippocampal slices. To examine whether the PLA2 activation is causally or protectively involved in the ischemic injury, we investigated the effect of pharmacological blockade of PLA2 on the OGD-induced neuronal death. The PLA2 inhibitor bromophenacyl bromide efficiently prevented the cell death in a concentration-dependent manner. Similar results were obtained for the selective cPLA2 inhibitor AACOCF3. However, the Ca 2+ -independent PLA2 inhibitor bromoenol lactone and the secretory PLA2 inhibitor LY311727 were virtually ineffective. These results suggest that cPLA2 plays a causative role in the neuronal death following OGD exposure. Thus, the present study may provide novel therapeutic targets for the development of neuroprotective agents.

Abstract: In this study, we investigated a change in the excretory content of 2,7,8-trimethyl-2(2′-carboxyethyl)-6-hydroxychroman (γ-CEHC), a γ-tocopherol (γ-Toc) metabolite, in rat urine and bile by using a new high-performance liquid chromatography-elelectrochemical detection (HPLC-ECD) method. In this determination, CEHC [α- and γ-CEHC, where α-CEHC-2,5,7,8-tetramethyl-2(2′-carboxyethyl)-6-hydroxychioman] in the biological specimens were treated with 3 N methanolic HCl to hydrolyze conjugates and to promote esterification. The methylated samples were extracted by n-hexane/water (1∶2). The analyses of the methyl esters of α-CEHC and γ-CEHC were performed by an HPLC-ECD using an ODS-3 column at 35°C. The mobile phase was acetonitrile/water (45∶55, vol/vol) containing 50 mM sodium perchlorate. After rat urine and bile samples, respectively, were methylated as described above, methylated biliary metabolites were identified by liquid chromatography mass spectrometry as methyl esters of γ-CEHC. Furthermore, we examined the differences in the excretion of γ-CEHC between rat urine and bile after an oral administration of γ-Toc or α- +γ-Toc by the above HPLC method. In the γ-Toc group, each vitamin E-deficient rat was given 0.5 mL of a stripped corn oil preparation containing 10 mg of γ-Toc. In the α- +γ-Toc group, the rat was given 10 mg of α-Toc and 10 mg of γ-Toc. The content of γ-CEHC in rat urine from the α- +γ-Toc group was increased more in comparison to the γ-Toc group at 18–36 h after oral administration. Moreover, the content of γ-CEHC in rat bile in the α- +γ-Toc group was increased more in comparison to the γ-Toc group at 6–18 h after oral administration. Therefore, we have suggested that γ-CEHC was shifted mainly to urinary excretion after γ-CEHC had been excreted into the bile. Furthermore, we assume that α-Toc may affect the metabolism of γ-Toc to γ-CEHC in the body.

Abstract: Objective: The purpose of this study was to investigate the effect of Nd:YAG laser irradiation on the acid demineralization of enamel and dentin by spectrophotometry. A mechanism of acquired acid resistance is also proposed. Summary Background Data: The ability of Nd:YAG laser irradiation to the enhanced resistance to artificial caries formation is still controversial. Methods: A pulsed Nd:YAG laser at 1.064-µm wavelength was used to irradiate the human enamel and dentin samples from 20 extracted human molars at the parameters of 1, 2, and 3 W and 20 pps for a total of 9 sec after painting with black ink. Samples were then subjected to 2 µl of 0.1 M lactic acid solution (pH 4.8) for 24 h at 36°C. The parts per million (ppm) of calcium ion (Ca2+) dissolved in each solution was determined by atomic absorption spectrophotometry and the morphological changes were also investigated by scanning electron microscopy (SEM). Results: The lowest mean Ca2+ ppm was recorded in the samples irradiated at 3 W, in those b...