Upregulation of Polymeric Immunoglobulin Receptor Expression by the Heat-Inactivated Potential Probiotic Bifidobacterium bifidum OLB6378 in a Mouse Intestinal Explant Model.

We determined whether a potential probiotic bacterium, Bifidobacterium bifidum OLB6378 (BB6378), exerts beneficial effects on the mucosal immune system in a mouse intestinal explant model. The addition of heat-inactivated BB6378 to intestinal explants prepared from embryonic day 18 BALB/c mice increased the expression of polymeric immunoglobulin receptor (pIgR) mRNA by two- to fivefold. These effects were observed on ileal and colonic explants but not on jejunal explants, suggesting that the BB6378-induced pIgR upregulation is site-specific within the mouse intestine. The upregulation of pIgR protein expression in colonic explants was also detected after 24 h of culture. The results of DNA microarray analysis of ileal and colonic samples indicated that BB6378 increased the gene expression of interleukin (IL)-1α and IL-1ß, and IL-1α content in colonic explants was significantly increased after 20 h of culture with BB6378. We then examined the involvement of endogenously induced IL-1α in pIgR mRNA upregulation by using IL-1α knockout (KO) mice. Contrary to our expectations, pIgR mRNA expression was equally upregulated by BB6378 in colonic explants from BALB/c and IL-1α KO mice. Conversely, we examined the involvement of Toll-like receptors in pIgR mRNA upregulation by using MyD88 KO mice. The upregulation of pIgR was completely suppressed in the explants derived from MyD88 KO mice. Taken together, we conclude that in a mouse intestinal explant model, the heat-inactivated potential probiotic BB6378 increases intestinal pIgR expression in a site-specific manner and that the upregulation of pIgR could be explained by a direct microbial effect on the epithelium via Toll-like receptors.

Noradrenergic excitation of a subpopulation of GABAergic cells in the basolateral amygdala via both activation of nonselective cationic conductance and suppression of resting K+ conductance: a study using glutamate decarboxylase 67-green fluorescent protein knock-in mice.

GABAergic interneurons play central roles in the regulation of neuronal activity in the basolateral nucleus of the amygdala (BLA). They are also suggested to be the principal targets of the brainstem noradrenergic afferents which are involved in the enhancement of the BLA-related memory. In addition, behavioral stress has been shown to impair noradrenergic facilitation of GABAergic transmission. However, the noradrenaline (NA) effects in the BLA have not been differentiated among medium- to large-sized GABAergic neurons and principal cells, and remain to be elucidated in terms of their underlying mechanisms. Glutamate decarboxylase 67 (GAD67) is a biosynthetic enzyme of GABA and is specifically expressed in GABAergic neurons. To facilitate the study of the NA effects on GABAergic neurons in live preparations, we generated GAD67-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the control of an endogenous GAD67 gene promoter. Here, we show that GFP was specifically expressed in GABAergic neurons in the BLA of this GAD67-GFP knock-in mouse. Under whole-cell patch-clamp recordings in vitro, we identified a certain subpopulation of GABAergic neurons in the BLA chiefly on the basis of the electrophysiological properties. When depolarized by a current injection, these neurons, which are referred to as type A, generated action potentials at relatively low frequency. We found that NA directly excited type-A cells via alpha1-adrenoceptors, whereas its effects on the other types of neurons were negligible. Two ionic mechanisms were involved in this excitability: the activation of nonselective cationic conductance and the suppression of the resting K+ conductance. NA also increased the frequency of spontaneous IPSCs in the principal cells of the BLA. It is suggested that the NA-dependent excitation of type-A cells attenuates the BLA output for a certain period.

Attenuation by dietary taurine of dextran sulfate sodium-induced colitis in mice and of THP-1-induced damage to intestinal Caco-2 cell monolayers.

The effects of dietary taurine on the experimental colitis induced by dextran sulfate sodium (DSS) in mice were evaluated. C57BL/6 female mice were given 3% DSS in drinking water for 5 d to induce acute colitis. Taurine at 2% was added to the drinking water 5 d before and during the DSS-treatment to investigate its preventive effect. Taurine supplementation significantly attenuated the weight decrease, diarrhea severity, colon shortening, and the increase in the colonic tissue myeloperoxidase activity induced by DSS. Taurine also significantly inhibited the increase in the expression of a pro-inflammatory chemokine, macrophage inflammatory protein 2 (MIP-2), but not of interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha mRNA. Furthermore, taurine significantly protected the intestinal Caco-2 cell monolayers from the damage by macrophage-like THP-1 cells in an in vitro coculture system. These results suggest that taurine prevented DSS-induced colitis partly in association with (1) its inhibitory effects on the secretion of MIP-2 from the intestinal epithelial cells and on the infiltration of such inflammatory cells as neutrophils and (2) its cytoprotective functions on the epithelial barrier from the direct toxicity of DSS and from the inflammatory cell-induced injury.

Tryptophan-19 of beta-lactoglobulin, the only residue completely conserved in the lipocalin superfamily, is not essential for binding retinol, but relevant to stabilizing bound retinol and maintaining its structure.

Residue 19 of tryptophan in bovine beta-lactoglobulin (beta-LG) is the only invariant residue throughout the lipocalin superfamily having two characteristic features: binding ability for small hydrophobic molecules and the unique beta-barrel three-dimensional structure. In this study, we investigated whether this strictly conserved Trp-19 of beta-LG would be indispensable for its structure and function such as maintaining the molecular structure and biological activity of beta-LG. Spectroscopic and enzymatic oxidation experiments on retinol bound to W19Y, in which Tyr was substituted for Trp-19, showed that Trp-19 was not critical for this binding, but was important for stably maintaining the environment surrounding retinol and the bound retinol. An using four anti-beta-LG monoclonal antibodies as probes, revealed a structural change in region 20-29, but not in the reverse region of Trp-19. A guanidine hydrochloride-induced unfolding study showed that the conformational stability of W19Y was greatly reduced by 6.9 kcal/mol compared to that of wild-type beta-LG. These facts indicated that Trp-19 is one of the important residues in correctly maintaining the local structure of beta-LG and stably retaining its overall structure, thereby conserving the bound retinol molecule.

Accelerated secretion of mutant beta-lactoglobulin in Saccharomyces cerevisiae resulting from a single amino acid substitution.

Transformed yeasts producing a mutant form of bovine beta-lactoglobulin (beta-LG), W19Y, in which Trp(19) was replaced with Tyr, were shown to secrete 6 times more than those producing wild type beta-LG. Northern blot analysis suggested that the enhanced level of secretion was not the result of upregulated transcription of W19Y. The ratio of the amount of W19Y secreted into the supernatant to the amount of W19Y remaining inside the cells was much larger than that in the case of wild type beta-LG as shown by immunoblot analysis. A pulse/chase experiment revealed that the speed of secretion of W19Y was significantly accelerated, compared to wild type beta-LG. These results indicated that W19Y was more efficiently and rapidly transported in the course of secretion than wild type beta-LG. Our previous study showed that the DeltaG of unfolding of W19Y in water is 6.9 kcal/mol smaller than that of wild type beta-LG. Furthermore, immunoblot analysis of intracellular beta-LG under non-reducing conditions indicated that W19Y as well as wild type beta-LG maintained a specific folded structure inside the yeast cells, whereas other non-secretable mutant beta-LGs with Phe or Ala at position 19 (W19F and W19A, respectively) did not. These data suggest that low molecular stability and the maintenance of a specific folded structure inside the yeast cells are prerequisites for efficient and rapid secretion. W19Y was more efficiently secreted than wild type beta-LG also in transformed ern1 mutant yeast cells expressing only a basal level of BiP which is considered to function in quality control in the endoplasmic reticulum (ER) by playing an important role in determining the secretion efficiency of secretory proteins. Thus, the reason for the enhanced secretion of W19Y is considered to be that the improved folding ability of W19Y can allow the half-life of the W19Y-BiP complex to become shorter than that of the wild type beta-LG-BiP complex, leading to faster translocation of W19Y into transport vesicles, or that W19Y can fold in a BiP-independent manner in the ER of the yeast cells. Our findings demonstrate that the amount of protein secreted can be improved by alteration of a single amino acid residue crucial for its structure.

Monoclonal antibodies as probes for monitoring the denaturation process of bovine beta-lactoglobulin.

Five monoclonal antibodies (MAbs) of different idiotypes were produced against bovine beta-lactoglobulin (beta-LG). Among them, MAbs 61B4 and 62A6 reacted preferentially to native beta-LG, while MAbs 21B3 and 31A4 reacted more strongly to the reduced carboxymethylated (denatured) beta-LG than to the native material. These two types of MAb were used to analyze the denaturation process of a beta-LG molecule during heating. The binding affinity of MAbs 21B3 and 31A4 with beta-LG was increased by increasing the heating temperature, the transition temperature being 67-68 degrees C, while that of MAbs 61B4 and 62A6 was reduced by increasing the temperature, this transition temperature being about 80 degrees C. Epitopes recognized by MAbs 31A4 and 61B4 were shown to be included in the segments, Lys8-Trp19 (mostly in the random-coil region) and Thr125-Lys135 (helical region), respectively. The heat-induced conformational change of the beta-LG molecule is, therefore, likely to start in random-coil region as Lys8-Trp19, and to be followed by a structural change in a helical region as Thr125-Lys135. This study demonstrates that MAb is a useful probe to monitor local conformational changes of a protein molecule during denaturation.

Selective induction of CD8+ T cell functions by single substituted analogs of an antigenic peptide: distinct signals for IL-10 production.

The CD8+ T cell clone 5F1 produces interleukin 10 (IL-10) and interferon gamma(IFN-gamma) in response to stimulation with a peptide corresponding to region 142-149 of bovine alpha(s1)-casein (p142-149). Ninety analog peptides derived from p142-149 with single amino acid substitutions of putative T cell receptor contact residues were prepared to examine whether production of IL-10 and IFN-gamma by 5F1 can be altered by stimulation with these peptides. We found that some peptides triggered only IL-10 production whereas others induced production of IFN-gamma alone or both of these cytokines. Peptides inducing IFN-gamma production triggered both cytotoxicity and a proliferative response, whereas peptides inducing production of IL-10 but not IFN-gamma triggered neither of these responses. Our results clearly demonstrate that the signaling pathway required for IL-10 production in CD8+ T cells differs from that required for IFN-gamma production. The distinct cellular signals for IL-10 production appear to be independent of those for cytotoxicity and the proliferative response of CD8+ T cells.

Primary response of naive CD4(+) T cells to amino acid-substituted analogs of an antigenic peptide can show distinct activation patterns: Th1- and Th2-type cytokine secretion, and helper activity for antibody production without apparent cytokine secretion.

Naive CD4(+) T cells differentiate into two types of helper T cells showing an interferon-gamma-predominant (Th1) or an interleukin-4-predominant (Th2) cytokine secretion profile after repeated antigenic stimulation. Their differentiation can be influenced by slight differences in the interaction between the T cell receptor (TCR) and its ligand at the time of primary activation. However, the primary response of freshly isolated naive CD4(+) T cells to altered TCR ligands is still unclear. Here, we investigated the primary response of splenic naive CD4(+) T cells derived from transgenic mice expressing TCR specific for residues 323-339 of ovalbumin (OVA323-339) bound to I-A(d) molecules. Naive CD4(+) T cells secreted either Th1- or Th2-type cytokines immediately after stimulation with OVA323-339 or its single amino acid-substituted analogs. Helper activity for antibody secretion by co-cultured resting B cells was also found in the primary response, accompanied by either low-level Th2-type cytokine secretion or no apparent cytokine secretion. Our results clearly indicate that dichotomy of the Th1/Th2 cytokine secretion profile can be elicited upon primary activation of naive CD4(+) T cells. We also demonstrate that the helper activity of naive CD4(+) T cells for antibody production does not correspond to the amounts of the relevant cytokines secreted.

Enhancement of preheparin serum lipoprotein lipase mass by bezafibrate administration.

To clarify the clinical implication of preheparin serum lipoprotein lipase mass (preheparin LpL mass), we studied the relationships between preheparin LpL mass and serum lipids, including midband lipoproteins, which migrate between very low density lipoproteins and low density lipoproteins on polyacrylamide gel disc electrophoresis, in hyperlipidemias. And we also studied the changes of preheparin LpL mass in hypertriglyceridemic patients during bezafibrate administration, which is known to enhance LpL activity in postheparin plasma. Preheparin LpL mass correlated positively with high-density lipoprotein-cholesterol (HDL-C) (r=0.418, P<0.01) and negatively with triglyceride (TG) (r=-0.256, P<0.01), but did not correlate with total cholesterol (TC) in 64 hyperlipidemic (type IIa, IIb and IV) patients. The midband lipoproteins were observed in 80% of hypertriglyceridemic patients (32/40). Preheparin LpL mass in midband lipoprotein-positive subjects was lower significantly than that in midband-negative subjects. When bezafibrate (400 mg/day) was administrated to 40 hypertriglyceridemic patients for 4 months, TG level significantly decreased (-49+/-7%, P<0.01), TC levels decreased (-11+/-4%, not significant), and HDL-C levels increased (+27+/-4%, P<0.01). The midband lipoproteins disappeared in 95% of patients. Preheparin LpL mass significantly increased (+25+/-6%, P<0. 0005). In nine patients who stopped bezafibrate, TG levels significantly increased (+49+/-7%, P<0.01) and HDL-C levels decreased (-27+/-4%, P<0.01). Preheparin LPL mass significantly decreased (-25+/-6%, P<0.0005). These results suggested that bezafibrate administration enhanced preheparin LpL mass. And it might be implicated that enhanced LpL production by bezafibrate could reflect an increase of preheparin LpL mass.

Autocrine B-cell stimulation by interleukin-2 during a cognate interaction with T-cells.

Interleukin-2 (IL-2) secretion as well as expression of IL-2 receptor has been demonstrated for B-cells in response to several activating stimuli. However, the exact role of B-cell-derived IL-2 in the T-cell-dependent antibody response remains to be determined. Here, we have examined the autocrine regulatory roles of IL-2 secreted from B-cells. Splenic resting B-cells were stimulated with a fixed pre-activated Th1 clone, G1.19, in the presence of a single amino acid-substituted peptide (pD129A; Ala-129 substituted for Asp-129), an analog of the original ligand (p119-133, derived from bovine beta-lactoglobulin) recognized by G1.19 cells. pD129A allowed a cognate interaction between B-cells and fixed pre-activated G1.19 T-cells, but pD129A had no agonistic activity against G1.19 T-cells. Thus, the level of expression of B-cell-activating molecules on T-cells remained unchanged after stimulation with pD129A. Regardless of the lack of ability to induce IL-2 secretion in the case of T-cells, pD129A significantly enhanced antibody secretion from B-cells, and this was partially blocked by anti-IL-2 antibody. Furthermore, IL-2 secretion from B-cells was modestly upregulated in response to added pD129A. Taken together, these data suggest that helper signals from interacting cognate T-cells induce IL-2 secretion by B-cells, which can enhance antibody secretion in an autocrine manner.

MHC class II/T-cell receptor interactions potentiate secretion of IgG but not IgM in response to T-dependent antigens.

We have examined whether the interaction of peptide-loaded MHC molecules on the surface of B-cells with antigen-specific T-cell receptors (TCRs) enhances Ig secretion in the presence of other antigen-independent interactions in vitro. B-cells specific for region 25-40 of beta-lactoglobulin (beta-LG) were stimulated in a T-cell dependent manner using plasma membranes (PM) derived from two different T-helper (Th) clones, culture supernatants of activated Th2 cells and beta-LG as a specific antigen. PM were obtained from either the beta-LG-specific T-cell clone H1.1 which can mediate specific TCR/MHC class II interactions as well as antigen-independent ones or from the D10 clone which bears a TCR of an irrelevant specificity and thus, can only mediate antigen-independent interactions. IgG, but not IgM, secretion was specifically enhanced by H1.1 PM, but not D10 PM in the presence of beta-LG. Furthermore, a blockade of TCR/MHC class II interactions using either anti-T-cell receptor, beta or anti-CD4 monoclonal antibodies inhibited this enhanced IgG secretion in response to beta-LG. The results show that while antigen-independent interactions between T- and B-cells can enhance secretion of IgM antibodies, specific interactions between TCRs and peptide:MHC complexes stimulate B-cells to enhance secretion of IgG but not IgM antibodies. This mechanism may contribute to antibody secretion only from B-cells activated through cognate interaction in vivo.

Capacity of circulating neutrophils to produce reactive oxygen species after exhaustive exercise.

To investigate the cause of disagreement within the large body of literature concerning the effect of exercise on the capacity of circulating neutrophils to produce reactive oxygen species (ROS), 10 male endurance-trained athletes underwent maximal exercise. The generation of superoxide radical (O2-.) by neutrophils was first detected on a cell-by-cell basis by using histochemical nitro blue tetrazolium tests performed directly on fresh unseparated blood, which showed that responsive neutrophils under several stimulatory conditions relatively decreased after exercise. Similarly, O2-. detected with bis-N-methylacridinium nitrate (lucigenin)-dependent chemiluminescence (CL) of a fixed number of purified neutrophils on stimulation with opsonized zymosan was decreased slightly after exercise. In contrast, the 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol)-dependent CL response of the neutrophils indicative of the myeloperoxidase (MPO)-mediated formation of highly reactive oxidants was significantly enhanced after exercise. It therefore suggests that the pathway of neutrophil ROS metabolism might be forwarded from the precursor O2-. production to the stages of more reactive oxidant formation due to the facilitation of MPO degranulation. In addition, these phenomena were closely associated with the exercise-induced mobilization of neutrophils from the marginated pool into the circulation, which was mediated by the overshooting of catecholamines during exercise. These findings indicate that the use of different techniques for detecting ROS or the different stages of neutrophil ROS metabolism could explain some of the disparate findings of the previous studies.

Endurance exercise causes interaction among stress hormones, cytokines, neutrophil dynamics, and muscle damage.

We analyzed adaptation mechanisms regulating systemic inflammatory response of the stressed body by using an experimental challenge of repeated exercise bouts and accompanying muscle inflammation. Eight untrained men bicycled at 90 W for 90 min, 3 days in a row. Exercise induced peripheral neutrophilia with a leftward shift of neutrophil nucleus and neutrophil priming for oxidative activity determined by luminol-dependent chemiluminescence. Plasma growth hormone and interleukin-6 rose significantly after exercise and were closely correlated with the neutrophil responses. Serum creatine kinase and myoglobin levels as muscle damage markers rose after exercise in "delayed onset" and were closely correlated with the preceding neutrophil responses. These exercise-induced responses were strongest on day 1, but the magnitude gradually decreased with progressive daily exercise. In contrast, the magnitude of catecholamine responses to exercise sessions gradually rose, possibly suppressing neutrophil oxidative responses. These results indicate that stress-induced systemic release of bioactive substances may determine neutrophil mobilization and functional status, which then may affect local tissue damage of susceptible organs.

Effect of temocapril hydrochloride on serum lipid levels in patients with hypertensive type 2 diabetes mellitus.

The effect of angiotensin converting enzyme inhibitor, temocapril hydrochloride on the serum lipoproteins, and especially on the size of low density lipoproteins (LDL) of hypertensive diabetic patients, were studied. Temocapril hydrochloride (5 mg/day) was administered to 32 hypertensive type 2 diabetes patients for 16 weeks. During treatment, systolic and diastolic blood pressures decreased significantly from 162/95 mmHg to 138/76 mmHg at 16 weeks (p<0.001), and serum levels of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) showed significant reduction, but those of HbA1c, triglycerides (TG) and high density lipoprotein cholesterol (HDL-C) showed no significant changes. LDL particle size evaluated by polyacrylamide gel disc electrophoresis was normalized from small size. It is concluded that temocapril hydrochloride favorably affects the serum lipoprotein metabolism of hypertensive type 2 dependent diabetes mellitus patients.

The direct cloning of the immunoglobulin VH genes from primary cultured B cells specific for a short peptide.

A new and simple method was devised to obtain immunoglobulin VH genes directly from primary cultured B cells specific for a short peptide. Peptide-specific B cells were separated from splenocytes of peptide-immunized BALB/c mice with antigen-coated magnetic beads, and were cloned by a limitedly diluted culture in the presence of lipopolysaccharide, recombinant interleukin (rIL) -2, rIL-4 and rIL-5, and 3T3 fibroblasts as filler cells for 7 days. Seventeen clones were obtained from 3 x 10(3) fractionated cells by screening the positive wells containing anti-peptide antibody-secreting cells by an enzyme-linked immunosorbent assay (ELISA). The VH cDNAs of these clones were amplified by a set of primers; a primer complementary to the mu-chain constant region gene and the other with high complementarity to most of the VH genes. This is the first report of success in obtaining unknown VH genes directly from primary B cell clones, after their antigen-specificity has been confirmed by ELISA. This new method will provide a powerful tool for designing specific recombinant antibodies.

The effect of insulin sensitizer, troglitazone, on lipoprotein lipase mass in preheparin serum.

Lipoprotein lipase mass exists in preheparin serum, even though the activity is scarcely found. The implication of this is unclear. We studied the effect of an insulin sensitizer, troglitazone, on this preheparin serum lipoprotein lipase mass (preheparin LPL mass) in non-insulin-dependent diabetes mellitus (NIDDM) patients as well as on serum lipid levels and low density lipoproteins (LDL) particle size. Thirty-one NIDDM patients with poor control were administered troglitazone 400 mg/day. Hemoglobin A1c had significantly decreased (13%, P < 0.001) 2 months later. Preheparin LPL mass had gradually increased and a 69% increase (P<0.01) was observed 4 months later. Triglyceride significantly decreased (23%, P < 0.01) and high density lipoprotein-cholesterol increased (10%, P < 0.01), whereas total cholesterol and LDL levels did not change 4 months later. The size of LDL increased significantly (P < 0.01). These results were consistent with the idea that preheparin LPL mass might be relating to the insulin sensitivity enhanced by troglitazone, as well as LDL particle size.

Branched-chain amino acids metabolic support in surgical patients: a randomized, controlled trial in patients with subtotal or total gastrectomy in 16 Japanese institutions.

A prospective, randomized, controlled trial of nutritional effects of branched-chain-enriched amino acid (BCAA) solution was undertaken in 173 surgical patients with gastric cancer. Eighty-six and 87 patients underwent subtotal and total gastrectomy, respectively. The effects were evaluated in total parenteral nutrition (TPN) in an isocaloric/isonitrogenous setting where the major difference between the group was the amount of BCAA received. Each 80 patients in the control and the BCAA groups completed the trial. The group receiving BCAA-enriched amino acid solution demonstrated a statistically significant improvement on days 2 and 3 in nitrogen balance in patients with total gastrectomy. Three-methyl-histidine excretion gradually decreased after day 1, and the values on day 7 were significantly lower than those on day 1 in the BCAA group in both those receiving subtotal and total gastrectomy. There were no significant differences of serum albumin and rapid turnover proteins between the control and BCAA groups in both those receiving subtotal and total gastrectomy. Plasma BCAA level and BCAA to aromatic amino acid (AAA) ratio were significantly higher, and AAA level was significantly lower in the BCAA group than in the control group. There were no serious complications encountered during the observation period in both groups. These results indicated that a BCAA-enriched amino acid solution can improve metabolism and maintains good nitrogen retention without increasing side effects as compared with a conventional amino acid solution for nutritional support of patients who have received subtotal or total gastrectomy.

Two types of Ca channels in smooth muscle cells isolated from guinea-pig taenia coli.

1) Two types of voltage dependent Ca channels with different conductances and inactivation kinetics were identified from cell-attached patch clamp recordings. One type, with a larger conductance of 25pS, had a threshold of activation near -40mV and the mean current inactivated slowly. A second type of Ca channel, with a smaller conductance of 12pS channel, but the averaged mean current inactivated rapidly. 2) Cadmium ions inhibited the large conductance Ca channel currents, while the small conductance Ca channel currents was not blocked. 3) Large conductance Ca channel current selectively inhibited by dihydropyridine derivative, Nifedipine. Small conductance Ca channel current is abolished in dose dependent manner by pyrethroid insecticide, tetranethrine.

Supernumerary teeth with eumorphism in the lower incisor region: a report of five cases and a review of the literature.

Five cases of supernumerary teeth with eumorphism in the lower incisor region of the permanent dentition are reported. The patients were two males and three females. One (a 31-year-old woman) of them had bilateral supernumerary teeth. The review of the English language literature yielded only one such bilateral supernumerary teeth in the lower incisor region of the permanent dentition was reported.

An unusual staining of the tooth roots: a case report with histological and micro-analytical studies.

The discoloration of tooth roots is rare. We report here a 22-year-old Japanese woman with blackish-brown staining of the roots of the upper and lower third molars. Staining was found in the dentin and cementum. Electron probe X-ray microanalysis showed no significant difference in the composing elements between the stained tooth root and control tooth. Fluorescent bands coincided with staining in the dentin of the root and cementum along the incremental lines under confocal laser-scanning microscope.