Isolation and characterization of dental epithelial cells derived from amelogenesis imperfecta rat.

OBJECTIVE: Disruption of the third zinc finger domain of specificity protein 6 (SP6) presents an enamel-specific defect in a rat model of amelogenesis imperfecta (AMI rats). To understand the molecular basis of amelogenesis imperfecta caused by the Sp6 mutation, we established and characterized AMI-derived rat dental epithelial (ARE) cells. MATERIALS AND METHODS: ARE cell clones were isolated from the mandibular incisors of AMI rats, and amelogenesis-related gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Localization of wild-type SP6 (SP6WT) and mutant-type SP6 (SP6AMI) was analyzed by immunocytochemistry. SP6 transcriptional activity was monitored by rho-associated protein kinase 1 (Rock1) promoter activity with its specific binding to the promoter region in dental (G5 and ARE) and non-dental (COS-7) epithelial cells. RESULTS: Isolated ARE cells were varied in morphology and gene expression. Both SP6WT and SP6AMI were mainly detected in nuclei. The promoter analysis revealed that SP6WT and SP6AMI enhanced Rock1 promoter activity in G5 cells but that enhancement by SP6AMI was weaker, whereas no enhancement was observed in the ARE and COS-7 cells, even though SP6WT and SP6AMI bound to the promoter in all instances. CONCLUSION: ARE cell clones can provide a useful in vitro model to study the mechanism of SP6-mediated amelogenesis imperfecta.

Complementary synaptic distribution of enzymes responsible for synthesis and inactivation of the endocannabinoid 2-arachidonoylglycerol in the human hippocampus.

Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders. Their medical significance may be best explained by the emerging concept that endocannabinoids are essential modulators of synaptic transmission throughout the central nervous system. However, the precise molecular architecture of the endocannabinoid signaling machinery in the human brain remains elusive. To address this issue, we investigated the synaptic distribution of metabolic enzymes for the most abundant endocannabinoid molecule, 2-arachidonoylglycerol (2-AG), in the postmortem human hippocampus. Immunostaining for diacylglycerol lipase-α (DGL-α), the main synthesizing enzyme of 2-AG, resulted in a laminar pattern corresponding to the termination zones of glutamatergic pathways. The highest density of DGL-α-immunostaining was observed in strata radiatum and oriens of the cornu ammonis and in the inner third of stratum moleculare of the dentate gyrus. At higher magnification, DGL-α-immunopositive puncta were distributed throughout the neuropil outlining the immunonegative main dendrites of pyramidal and granule cells. Electron microscopic analysis revealed that this pattern was due to the accumulation of DGL-α in dendritic spine heads. Similar DGL-α-immunostaining pattern was also found in hippocampi of wild-type, but not of DGL-α knockout mice. Using two independent antibodies developed against monoacylglycerol lipase (MGL), the predominant enzyme inactivating 2-AG, immunostaining also revealed a laminar and punctate staining pattern. However, as observed previously in rodent hippocampus, MGL was enriched in axon terminals instead of postsynaptic structures at the ultrastructural level. Taken together, these findings demonstrate the post- and presynaptic segregation of primary enzymes responsible for synthesis and elimination of 2-AG, respectively, in the human hippocampus. Thus, molecular architecture of the endocannabinoid signaling machinery supports retrograde regulation of synaptic activity, and its similar blueprint in rodents and humans further indicates that 2-AG's physiological role as a negative feed-back signal is an evolutionarily conserved feature of excitatory synapses.

Structure of electrolyte solutions sorbed in carbon nanospaces, studied by the replica RISM theory.

The replica RISM theory is used to investigate the structure of electrolyte solutions confined in carbonized polyvinylidene chloride (PVDC) nanoporous material, compared to bulk electrolyte solution. Comparisons are made between the models of electrolyte solution sorbed in the carbonized PVDC material and a single carbon nanosphere in bulk electrolyte solution. Particular attention is paid to the chemical potential balance between the species of the sorbed electrolyte solution and the bulk solution in contact with the nanoporous material. As a result of the strong hydrophobicity of the carbonized PVDC material in the absence of activating chemical groups, the densities of water and ions sorbed in the material are remarkably low compared to those in the ambient bulk solution. The interaction between water molecules and cations becomes strong in nanospaces. It turns out that, in carbon nanopores, a cation adsorbed at the carbon surface is fully surrounded by the hydration shell of water molecules which separates the cation and the surface. Distinctively, an anion is adsorbed in direct contact with the carbon surface, which squeezes a part of its hydration shell out. The tendency increases toward smaller cations, which are characterized as "positive hydration" ions. In the bulk, cations are not hydrated so strongly and behave similarly to anions. The results suggest that the specific capacitance of an electric double-layer supercapacitor with nanoporous electrodes is intimately related to the solvation structure of electrolyte solution sorbed in nanopores, which is affected by the microscopic structure of the nanoporous electrode.

Visualization of inositol 1,4,5-trisphosphate receptor type III with green fluorescent protein in living cells.

Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel that releases Ca2+ into the cytosol and its subcellular distribution is believed to have significant effects on Ca2+ signalling. We constructed a plasmid vector containing full-length rat type 3 IP3R linked to GFP (GFP-IP3R) for expression in mammalian cells. Western blot analyses revealed that the expressed fusion protein contained both GFP and full-length type 3 IP3R. Fluorescence confocal microscopy showed that the fluorescence of GFP-IP3R3 was distributed to reticular network structures, even after cell permeabilization with saponin. We further visualized intracellular membranes with DiOC6, a vital fluorescent marker for intracellular membranes, and provide evidence that the distribution of GFP-IP3R3 overlaps with the distribution of the endoplasmic reticulum. Our results indicate that GFP-IP3R3 can be used to visualize IP3R in living cells, and pave the way for subsequent mutational and functional studies.

Possible mechanisms regulating ATP- and thimerosal-induced Ca(2+) oscillations in the HSY salivary duct cell line.

The ATP-induced oscillatory changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)) were analysed in HSY cells, a salivary ductal cell line from human parotid, using a fluorescence ratio imaging system. At concentrations higher than 1 microM, ATP caused sinusoidal [Ca(2+)](i) oscillations due to the periodic release and reuptake of Ca(2+) by intracellular Ca(2+) stores. The phorbol ester 4beta-phorbol 12,13-dibutyrate (PDBu) changed the [Ca(2+)](i) oscillations to a single spike. The inhibitory effect of PDBu on the [Ca(2+)](i) signals was reversed by protein kinase C (PKC) inhibitors such as staurosporine and chelerythrine chloride. However, preincubation of the cells with the PKC inhibitors did not affect the pattern of the ATP-induced [Ca(2+)](i) oscillations. The desensitization of the [Ca(2+)](i) response observed during prolonged stimulation with ATP was also not prevented by the PKC inhibitors. Incubation of HSY cells with the sulphydryl reagent thimerosal, which enhances the sensitivity of inositol 1,4,5-trisphosphate (IP(3)) receptors, caused repetitive Ca(2+) release from intracellular Ca(2+) stores resulting in baseline spikes of [Ca(2+)](i). The thimerosal-induced [Ca(2+)](i) oscillations did not change in the presence of PDBu and the phospholipase C inhibitor U73122. Thus, we could not provide evidence that negative feedback by PKC plays a central role in the regulation of ATP-induced [Ca(2+)](i) oscillations. These results suggest that the [Ca(2+)](i) oscillations, at least the baseline spikes, in HSY cells can be generated without stimulating the formation of IP(3).

[The occurrence of Churg-Strauss syndrome in two patients with remitted asthma].

We report two patients with Churg-Strauss syndrome (CSS) which occurred on the remission stage of bronchial asthma. Case 1 of a 64-year-old woman suffered from asthma in June, 1997, and got relief with treatment. In February, 1998, dysesthesia, pain and severe muscle weakness occurred in the extremities and erythematous rashes appeared on the extremities and back. She was transferred to our hospital on March 3. Peripheral blood eosinophilia was observed and a diagnosis of CSS was made. Eosinophilic tissue infiltration and vasculitis was found in the skin biopsy specimen. She was treated with prednisolone (60 mg/day) with moderate improvement. But the dysesthesia in the extremities, bilateral foot drop and the weakness of the left hand grasping power continued. Case 2 of a 62-year-old woman suffered from asthma in 1995, which improved by treatment. In March 1998, dysesthesia and pain in the lower extremities occurred and progressed. Erythematous rashes appeared on the feet and she was admitted to our hospital in May. Peripheral blood eosinophilia and eosinophilic tissue infiltration in the skin biopsy specimen were observed and a diagnosis of CSS was made. The treatment with prednisolone (60 mg/day) improved the pain but the dysesthesia continued. It is important to know the occurrence of CSS on the remission stage of bronchial asthma.

Clinical pathway for impalpable or small lung lesions treated with coil marking and thoracoscopy.

OBJECTIVES: Advances in computed tomography are detecting increasingly impalpable or small pulmonary lesions. We propose a clinical pathway for managing such lesions. METHODS: We conducted a retrospective study in a community teaching hospital to describe the hospital schedules of 18 patients having 19 lesions 10 mm or less and ground glass attenuation. Under computed tomography, a coil (Complex Helical Fibered Platinum Coil-18) was placed at the proximal side of the lesion. Using thoracoscopy and radiographic fluorography, we conducted partial lung resection targeting the coil the next day, adding lobectomy, if required. RESULTS: Final diagnosis included primary and metastatic lung cancer (n = 14), atypical adenomatous hyperplasia (n = 1), and benignancy (n = 4). Patients were admitted 2* days before surgery (*Numbers are medians). On postoperative day 3, chest tubes were removed. Epidural analgesia was continued for 5 days. On postoperative day 7, patients were discharged. Their admission charge was a total of yen 979,610. CONCLUSIONS: The hospital course above may be applied to the clinical pathway for managing impalpable or small lung lesions.

Characterization of the ca2 + response mediated by activation of beta-adrenoceptors in rat submandibular ducts.

The Ca2+ signaling mediated by activation of beta-adrenoceptors was studied in a purified preparation of ducts from rat submandibular glands. At concentrations above 1 nM, isoproterenol (ISO) caused a small but significant increase in cytosolic Ca2+ concentration ([Ca2+]i). The ISO-induced increase in [Ca2+]i was completely inhibited by the beta-adrenoceptor antagonist propranolol but not by the alpha-adrenoceptor antagonist phentolamine. Forskolin was able to mimic the Ca2+ response to ISO. These results suggest that the ISO-induced increase in [Ca2+]i in rat submandibular ducts is mediated by an accumulation of cAMP resulting from activation of beta-adrenoceptors. In the absence of extracellular Ca2+, ISO or forskolin caused a transient increase in [Ca2+]i, indicating Ca2+ mobilization from intracellular Ca2+ stores. Further, stimulation with ISO failed to mobilize Ca2+ after the depletion of intracellular Ca2+ stores by phenylephrine or carbachol, suggesting that the cAMP-mediated increase in [Ca2+]i is due to a Ca2+ release from inositol trisphosphate (IP3)-sensitive Ca2+ stores. As ISO did not stimulate a detectable production of IP3, the cAMP-mediated Ca2+ mobilization may be evoked by a mechanism different from activation of phosphoinositide hydrolysis.

Evidence that type I, II, and III inositol 1,4,5-trisphosphate receptors can occur as integral plasma membrane proteins.

A number of previous reports have suggested that inositol 1,4, 5-trisphosphate receptors (IP(3)Rs) are present in the plasma membranes of cells. We confirm this directly in the present study by demonstrating that a significant proportion of the IP(3)Rs found in A431 cells, Jurkat cells, and rat parotid acini can be biotinylated by the extracellular application of sulfo-N-hydroxysuccinimide-biotin to intact cells. This labeling cannot be accounted for by the reaction of sulfo-N-hydroxysuccinimide-biotin with intracellular IP(3)Rs since calnexin and the SERCA2 ATPase, both integral membrane proteins of the endoplasmic reticulum, are not labeled under the same experimental conditions. Individual IP(3)R subtypes were detected using subtype-specific antibodies. A431 cells expressed only the type-3 IP(3)R, and 23% of this protein was in the biotinylated (plasma membrane) fraction. Jurkat cells and rat parotid cells expressed all three IP(3)R subtypes. Contrary to earlier results suggesting that only the type-3 IP(3)R might localize to the plasma membrane, we found that significant amounts (5-14%) of all three subtypes could be identified in the biotinylated fractions of Jurkat and rat parotid cells. Our results suggest a role for IP(3)Rs in plasma membrane as well as intracellular membrane function.

Hemopneumothorax and hemoperitoneum in a case with large cell carcinoma of the lung.

Hemopneumothorax and hemoperitoneum coincide rarely in nontraumatic cases. Here, a 70-year-old male presented a left axillary lymph node and was diagnosed as having metastatic squamous cell carcinoma. Under the same diagnosis, another lesion developed in the right femur and was resected. One year later, computed tomography detected another tumor in the left adrenal gland. Shortly afterwards, left pneumothorax developed and a chest operation revealed hemopneumothorax due to a ruptured cavitary form of large cell carcinoma. The serum showed a human chorionic gonadotropin-beta level of 1,100 ng/ml. At three-months later, he died of hemoperitoneum. The autopsy demonstrated hepatic metastases and a ruptured adrenal metastasis; microscopy showed marked trophoblastic and squamous cell changes in these organs. This patient was unique in that the rupture of the pulmonary and the adrenal lesions caused clinical manifestation.

5-hydroxytryptamine induces transient Ca2+ influx through Ni2+-insensitive Ca2+ channels in rat vascular smooth muscle cells.

The effects of Ni2+, a non-selective cation channel inhibitor, on 5-hydroxytryptamine (5-HT)- and angiotensin II (Ang II)-induced intracellular Ca2+ dynamics in rat aortic smooth muscle cells were investigated. Ni2+ (1 mM) significantly inhibited the transient increase in intracellular Ca2+ concentration ([Ca2+]i) induced by Ang II (100 nM) in aortic smooth muscle cells, as measured using fura-2. However, Ni2+ did not suppress the transient increase in Ca2+ influx induced by 5-HT (10 microM), while significantly suppressed the sustained increase. Ca2+ influx evoked by high KCl (80 mM), thapsigargin (TG) (1 microM) or depletion of intracellular Ca2+ store was almost completely suppressed by Ni2+. Ni2+ had no effect on 5-HT-induced inositol triphosphate production and Ca2+ release from the intracellular store(s). These results suggest that 5-HT, but not Ang II, induces transient Ca2+ influx through Ni2+-insensitive Ca2+ channels, which are distinguishable from the voltage-dependent or store-operated Ca2+ channels.

Epiphrenic diverticulum composed of airway components attributed to a bronchopulmonary-foregut malformation: report of a case.

Bronchopulmonary-foregut malformation (BPFM), defined originally as pulmonary sequestration with or without communication to the esophagus, has been acknowledged to include congenital foregut diverticula. We present herein the case of a 43-year-old woman with a 9-year history of dysphagia, in whom a barium meal examination demonstrated a 2.5-cm epiphrenic diverticulum and several fistulae. A laparotomy was performed and the lower esophagus without communication to the lung was pulled down and resected, followed by an esophagogastrostomy carried out with fundopexy. Since her operation, the patient has been free of symptoms. Histologically, the diverticulum was observed to be lined by stratified squamous cells, but its shape was formed by mural cartilage, smooth muscle cells, and three ciliated-cell cysts. The dysphagia was considered to have been derived from the kinked esophagus created by the rigid diverticulum, being the possible developmental arrest of a supernumerary lung bud. These findings indicate that this case may involve BPFM in the broad sense. Although several cases of bronchogenic cysts located beneath or across the diaphragm have been reported as a subgroup of BPFM, congenital epiphrenic diverticula has rarely been described.

Isoproterenol potentiates alpha-adrenergic and muscarinic receptor-mediated Ca2+ response in rat parotid cells.

The effects of the cAMP pathway on the Ca2+ response elicited by phospholipase C-coupled receptor stimulations were studied in rat parotid cells. Although 1 microM isoproterenol (Iso) itself had no effect on the cytosolic Ca2+ concentration, the pretreatment with Iso potentiated Ca2+ responses evoked by phenylephrine. The potentiating effect of Iso was attributed to a shifting of the concentration-response curves of phenylephrine to the left and an increase in the maximal response. Half-maximal potentiation occurred at 3 nM Iso. Iso also potentiated the Ca2+ response elicited by carbachol. The potentiating effect of Iso was mimicked by forskolin (10 microM) and dibutyryl adenosine 3',5'-cyclic monophosphate (2 mM) and was blocked by 10 microM H-89. Iso potentiated the phenylephrine-induced Ca2+ response in the absence of extracellular Ca2+, but Iso did not increase the inositol trisphosphate (IP3) production induced by phenylephrine. These results suggest that the potentiation of the Ca2+ response can be attributed to a sensitization of IP3 receptors by cAMP-dependent protein kinase.

Signet ring cell adenoma of the thyroid with mucin predominance.

A 22-year-old male presented with a 1-year history of a right anterior neck mass. He did not have gastrointestinal cancer. Laboratory examination revealed an elevated serum thyroglobulin level of 120 ng/mL. The neck lesion showed poor uptake on 99mTc scan, but enhanced uptake on 201T1 scan. The patient underwent a hemithyroidectomy; the cut surface of the 7 x 3.5 cm lesion was solid and tanned orange. Postoperatively the serum thyroglobulin level decreased to 26 ng/mL. Microscopy of the tumor showed signet ring cells and microfollides, both of which were positive for mucicarmin and alcian Blue. A small percentage of the follicles were positive for thyroglobulin and periodic acid-Schiff. Our literature search detected 18 patients with signet ring cell lesions positive for thyroglobulin, but none had characteristics similar to ours showing predominance of mucin and poor staining for thyroglobulin.

Interaction of Gli2 with CREB protein on DNA elements in the long terminal repeat of human T-cell leukemia virus type 1 is responsible for transcriptional activation by tax protein.

The long terminal repeat (LTR) of human T-cell leukemia virus type 1 (HTLV-1) has two distinct DNA elements, one copy of TRE2S and three copies of a 21-bp sequence that respond to the viral trans-activator protein, Tax. Either multiple copies of the 21-bp sequence or a combination of one copy each of TRE2S and 21-bp sequence is required for efficient trans activation by Tax. In the trans activation of multiple copies of 21-bp sequence, CREB/ATF protein plays an essential role in forming a complex with Tax. To understand the role of TRE2S in trans activation of one copy of 21-bp sequence, we examined protein binding to the DNA elements by DNA affinity precipitation assay including Gli2 protein binding to TRE2S and CREB protein binding to 21-bp sequence. Binding of CREB to a DNA probe containing both elements, TRE2S-21bp probe, was dependent on Gli2 protein under restricted conditions and was enhanced in a dose-dependent fashion by the binding of Gli2 protein to the same probe. Mutation in either element abolished the efficient binding of CREB. A glutathione S-transferase fusion protein of a fragment of Gli2 was able to bind to CREB. Therefore, Gli2-CREB interaction on the DNA probe is proposed to stabilize CREB binding to DNA. Tax can bind to CREB protein on the DNA; therefore, stabilization of DNA binding of CREB results in more recruitment of Tax onto DNA. Conversely, Tax increased the DNA binding of CREB, although it had almost no effect on the binding of Gli2. These results suggest that Gli2 binds to the DNA element and interacts with CREB, resulting in more recruitment of Tax, which in turn stabilizes DNA binding of CREB. Similar cooperation of the protein binding to TRE2S-21bp probe was also observed in nuclear extract of an HTLV-1-infected T-cell line. Consistent with the Gli2-CREB interaction on the DNA elements, Tax-mediated trans activation was dependent on the size of the spacer between TRE2S and 21-bp sequence. The effective sizes of the spacer suggest that TRE2S in the LTR would cooperate with the second and third copies of the 21-bp sequence and contribute to trans activation of the viral gene transcription.

Constituents in watercress: inhibitors of histamine release from RBL-2H3 cells induced by antigen stimulation.

Histamine release inhibitors in watercress (Nasturtium officinale) were isolated using a monitoring system with antigen-stimulated RBL-2H3 cells. Of the 15 compounds isolated, flavonols and megastigmanes significantly inhibited histamine release. Two flavonols, 3-O-sophorosides of rhamnetin and rhamnazin, were new compounds. To investigate the inhibitory mechanism, the effects of rhamnetin, rhamnetin 3-O-sophoroside and an isolated megastigmane glucoside on the increase in the intracellular free calcium concentration were examined at a concentration providing 60% inhibition of histamine release. The results suggest that these compounds did not affect the calcium influx at that concentration. The structure-activity relationships of the megastigmanes on histamine release were also investigated.

Protective effects of green tea on hepatotoxicity, oxidative DNA damage and cell proliferation in the rat liver induced by repeated oral administration of 2-nitropropane.

To evaluate the benefit of green tea in mitigating hazards caused by repeated exposure of 2-nitropropane (2NP), we examined the effects of the tea on toxic indices, oxidative DNA damage and cell proliferation in the liver of 2NP-treated rats. Male Fischer 344 rats were administered, by gastric intubation, a total of six doses of 60 mg/kg 2NP(L), or alternatively two doses of 90 mg/kg and then four doses of 120 mg/kg 2NP(H) during 2 weeks. Green tea infusion was given to the rats as drinking water 1 week before the 2NP treatments and throughout the experiment. Significant elevation of hepatotoxic indices was evident in the 2NP(H)-treated group, such as an increase of serum glutamic-oxaloacetic transaminase (GOT) activity and of hepatic lipid peroxidation, together with a decrease in hepatic glycogen and serum triglyceride, and degenerative changes in the hepatocytes. A dose-related increase was observed in oxidative DNA damage and cell proliferation in the liver. Green tea effectively inhibited all of above changes induced by 2NP treatment, suggesting that tea intake may be effective for preventing the hepatic injuries after chronic exposure to 2NP.

Activation of beta-adrenoceptors does not cause any change in cytosolic Ca2+ distribution in rat parotid acinar cells.

The effects of the beta-adrenoceptor agonist isoproterenol on the distribution of cytosolic Ca2+ concentrations were studied with digital imaging microscopy in fura-2-loaded rat parotid acinar cells. At concentrations < 10 microM, isoproterenol did not cause any measurable change in cytosolic Ca2+ concentration ([Ca2+]i). Monitoring of [Ca2+]i in selected areas of the acinar cells failed to show that stimulation with isoproterenol causes a localized rise in [Ca2+]i at the apical region close to the lumen. As the maximum response of amylase exocytosis is observed at 0.1 or 1 microM isoproterenol [Tanimura, A., Matsumoto, Y., Tojyo, Y., 1990. Evidence that isoproterenol-induced Ca2+-mobilization in rat parotid acinar cells is not mediated by activation of beta-adrenoceptors. Biochim. Biophys. Acta, 1055, pp. 273-277], the data obtained here indicate that the isoproterenol-induced amylase exocytosis is not accompanied by Ca2+ mobilization. The high concentration (100 microM) of isoproterenol caused a small but significant increase in [Ca2+]i, particularly in the apical region. This response was completely attenuated by the alpha-adrenoceptor antagonist phentolamine, but not by the beta-adrenoceptor antagonist propranolol, indicating that the isoproterenol-induced increase in [Ca2+]i resulted from an activation of alpha-adrenoceptors. Further, the effect of cyclic AMP on Ca2+ release from intracellular Ca2+ stores was studied in saponin-permeabilized acinar cells using the lipophilic Ca2+ indicator Calcium Green C18. Cyclic AMP had no effect on the Ca2+ release, while the same acinar cells responded strongly to inositol 1,4,5-trisphosphate. This result does not support the hypothesis that cyclic AMP directly stimulates Ca2+ mobilization in rat parotid acinar cells.

Analysis of spatial and temporal characteristics of Ca2+ release in permeabilized parotid cells.

Ca2+ waves and oscillations have been observed during stimuli that generate inositol 1,4,5-trisphosphate (IP3) in several exocrine cells, including rat parotid acinar cells. Although a model has been proposed to explain the mechanism of the Ca2+ wave, there exists no direct evidence to prove the model's validity. Analysis with a permeabilized cell system provides direct information about the properties of Ca2+ stores and Ca2+ release channels. We summarize here the experimental techniques that may be used to elucidate the spatial and temporal regulation of Ca2+ in a single permeabilized cell. The future applications of these methods and the possible mechanisms of the Ca2+ wave are also discussed.