Pediatric-type high-grade gliomas with PDGFRA amplification in adult patients with Li-Fraumeni syndrome: clinical and molecular characterization of three cases.

Li-Fraumeni syndrome (LFS) is an autosomal dominant tumor predisposition syndrome caused by heterozygous germline mutations or deletions in the TP53 tumor suppressor gene. Central nervous system tumors, such as choroid plexus tumors, medulloblastomas, and diffuse gliomas, are frequently found in patients with LFS. Although molecular profiles of diffuse gliomas that develop in pediatric patients with LFS have been elucidated, those in adults are limited. Recently, diffuse gliomas have been divided into pediatric- and adult-type gliomas, based on their distinct molecular profiles. In the present study, we investigated the molecular profiles of high-grade gliomas in three adults with LFS. These tumors revealed characteristic histopathological findings of high-grade glioma or glioblastoma and harbored wild-type IDH1/2 according to whole exome sequencing (WES). However, these tumors did not exhibit the key molecular alterations of glioblastoma, IDH-wildtype such as TERT promoter mutation, EGFR amplification, or chromosome 7 gain and 10 loss. Although WES revealed no other characteristic gene mutations or copy number alterations in high-grade gliomas, such as those in histone H3 genes, PDGFRA amplification was found in all three cases together with uniparental disomy of chromosome 17p, where the TP53 gene is located. DNA methylation analyses revealed that all tumors exhibited DNA methylation profiles similar to those of pediatric-type high-grade glioma H3-wildtype and IDH-wildtype (pHGG H3-/IDH-wt), RTK1 subtype. These data suggest that high-grade gliomas developed in adult patients with LFS may be involved in pHGG H3-/IDH-wt. PDGFRA and homozygous alterations in TP53 may play pivotal roles in the development of this type of glioma in adult patients with LFS.

Efficacy and safety of bevacizumab, irinotecan, and temozolomide combination for relapsed or refractory pediatric central nervous system embryonal tumor: a single-institution study.

OBJECTIVE: This study aimed to evaluate the efficacy and safety of combination therapy with bevacizumab (Bev), irinotecan (CPT-11), and temozolomide (TMZ) in children with central nervous system (CNS) embryonal tumor relapse. METHODS: The authors retrospectively examined 13 consecutive pediatric patients with relapsed or refractory CNS embryonal tumors who received combination therapy comprising Bev, CPT-11, and TMZ. Specifically, 9 patients had medulloblastoma, 3 had atypical teratoid/rhabdoid tumor (AT/RT), and 1 had CNS embryonal tumor with rhabdoid features. Of the 9 medulloblastoma cases, 2 were categorized in the Sonic hedgehog subgroup and 6 in molecular subgroup 3 for medulloblastoma. RESULTS: The complete and partial objective response rates were 66.6% in patients with medulloblastoma and 75.0% in patients with AT/RT or CNS embryonal tumors with rhabdoid features. Furthermore, the 12- and 24-month progression-free survival rates were 69.2% and 51.9% for all patients with recurrent or refractory CNS embryonal tumors, respectively. In contrast, the 12- and 24-month overall survival rates were 67.1% and 58.7%, respectively, for all patients with relapsed or refractory CNS embryonal tumors. The authors observed grade 3 neutropenia, thrombocytopenia, proteinuria, hypertension, diarrhea, and constipation in 23.1%, 7.7%, 23.1%, 7.7%, 7.7%, and 7.7% of patients, respectively. Furthermore, grade 4 neutropenia was observed in 7.1% of patients. Nonhematological adverse effects, such as nausea and constipation, were mild and controlled with standard antiemetics. CONCLUSIONS: This study demonstrated favorable survival outcomes in patients with relapsed or refractory pediatric CNS embryonal tumors and thus helped to investigate the efficacy of combination therapy comprising Bev, CPT-11, and TMZ. Moreover, combination chemotherapy had high objective response rates, and all adverse events were tolerable. To date, data supporting the efficacy and safety of this regimen in the relapsed or refractory AT/RT population are limited. These findings suggest the potential efficacy and safety of combination chemotherapy in patients with relapsed or refractory pediatric CNS embryonal tumors.

Paradoxical symptomatic cerebral blood flow decreases after combined revascularization surgery for patients with pediatric moyamoya disease: illustrative case.

BACKGROUND: Transient neurological deficits (TNDs) develop after cerebral revascularization in patients with moyamoya disease (MMD). The authors report a rare pediatric MMD case with extensive decreased cerebral blood flow (CBF) and prolonged TNDs after combined revascularization. OBSERVATIONS: A 9-year-old boy presented with transient left upper limb weakness, and MMD was diagnosed. A right-sided combined surgery was performed. Two years after the surgery, frequent but transient facial (right-sided) and upper limb weakness appeared. The left internal carotid artery terminal stenosis had progressed. Therefore, a left combined revascularization was performed. The patient's motor aphasia and right upper limb weakness persisted for approximately 10 days after surgery. Magnetic resonance angiography showed that the direct bypass was patent, but extensive decreases in left CBF were observed using single photon emission tomography. With adequate fluid therapy and blood pressure control, the neurological symptoms eventually disappeared, and CBF improved. LESSONS: The environment of cerebral hemodynamics is heterogeneous after cerebral revascularization for MMD, and the exact mechanism of CBF decreases was not identified. TNDs are significantly associated with the onset of stroke during the early postoperative period. Therefore, appropriate treatment is desired after determining complex cerebral hemodynamics using CBF studies.

The usefulness and safety of dexmedetomidine for postoperative sedation in pediatric patients with moyamoya disease.

OBJECTIVE: After revascularization surgery in pediatric patients with moyamoya disease (MMD), resting and avoiding crying is important. However, this inaction is often difficult because of pain or anxiety. Dexmedetomidine (DEX), which has sedative and analgesic properties, may be useful in reducing those uncomfortable conditions; however, its common side effects include bradycardia and hypotension, which have a risk of decreasing the cerebral blood flow. The aim of this study was to investigate the efficacy and safety of using DEX for pediatric patients with MMD in the acute period after revascularization surgery. METHODS: This retrospective study included pediatric patients with MMD who underwent revascularization surgery. Based on whether DEX was used for light sedation during postoperative days (PODs) 0-1 after extubation, the patients were divided into DEX or control groups. For neurological outcomes, the incidence of symptomatic cerebral infarction and transient neurological events (TNEs) during PODs 0-1 and the entire hospitalization were investigated. In addition, the Richmond Agitation-Sedation Scale (RASS) was used to assess the effect of DEX, and bradycardia and hypotension were evaluated as side effects. RESULTS: A total of 84 surgical procedures were included in this study (27 in the DEX group and 57 in the control group). During PODs 0-1, symptomatic infarction was not observed in either group. The incidence of TNEs was almost the same in both groups: 2 (7.4%) of the 27 procedures in the DEX group and 4 (7.0%) of the 57 procedures in the control group (p > 0.99). Moreover, the incidences of symptomatic infarction and TNEs during the entire hospitalization did not differ significantly (symptomatic infarction, p > 0.99; TNEs, p = 0.20). Regarding the DEX effect, the median RASS scores during PODs 0-1 were -1.0 (drowsy) in the DEX group and +1.0 (restless) in the control group, showing a significant difference (p < 0.01). Regarding side effects, bradycardia was observed only in 3 (11.1%) of the 27 procedures in the DEX group (p = 0.03), and hypotension was not observed in any of the cases. CONCLUSIONS: In pediatric patients with MMD who are extubated after revascularization surgery, DEX produced appropriate light sedation and analgesia. The risk for symptomatic infarction is almost the same in cases in which DEX is used and those in which it is not; however, neurosurgeons should be cautious of bradycardia and TNEs as potential side effects.

Risk Factors for Cerebral Infarction Early After Revascularization in Children Younger than 5 Years with Moyamoya Disease.

OBJECTIVE: Cerebral revascularization is necessary for pediatric patients younger than 5 years with moyamoya disease (MMD). However, they have a high risk of developing cerebral infarction early after surgery. This study aimed to analyze the risk factors for developing cerebral infarction among these patients. METHODS: The charts of 21 consecutive patients with MMD (39 surgeries) younger than 5 years who had undergone revascularization at our hospital were retrospectively analyzed. Because cerebral infarction occurring within 1 month after surgery was the primary end point, other clinical information was evaluated, including each surgical procedure. Multivariate analysis of the risk factors for postoperative cerebral infarction was performed. RESULTS: Cerebral infarction occurred after 7 of 39 surgeries (17.9%). Of the 39 surgeries, 23 (59%) included direct and indirect combined revascularization. The incidence of cerebral infarction did not differ significantly between the combined (21.7%) and indirect (12.5%) groups (P = 0.46). Logistic regression showed no association between the revascularization procedure and the occurrence of cerebral infarction after surgery (P = 0.3). However, younger age at surgery was correlated with a higher risk of developing cerebral infarction in the early postoperative period (P = 0.05). CONCLUSIONS: No differences were found in the risk of developing cerebral infarction early after surgery as a result of surgical procedures. However, younger patients had higher postoperative risk. Further multicenter research should examine this issue for young pediatric patients with moyamoya at high risk of developing cerebral infarction.

Challenging direct bypass surgery for very young children with moyamoya disease: technical notes.

Cerebral revascularization for moyamoya disease (MMD) is an effective treatment for improving cerebral ischaemia and preventing rebleeding. Although direct bypass surgery is commonly performed on older children and adults, it is challenging in very young children due to the high difficulty level of the procedure. The subjects were MMD patients under 3 years of age on whom surgery was performed by a single surgeon (Y.A.). Preoperative clinical findings, information related to direct bypass surgery, bypass patency, and the incidence of postoperative stroke were investigated. Combined revascularization, including direct bypass surgery, was performed on 3 MMD patients (3 sides) under 3 years of age. The average diameter of the grafts used in direct bypass was 0.8 mm. The average recipient diameter was 0.8 ± 0.17 (range 0.6-1) mm. In all cases, the anastomotic procedure was completed using 11-0 monofilament nylon thread, and patency was confirmed. Direct bypass for MMD patients under 3 years old is technically challenging. However, despite the anatomical differences between very young children and elderly individuals, direct bypass surgery could certainly be completed. In addition, a rapid recovery from cerebral blood flow insufficiency could yield a promising neurological outcome.

Ependymoma-like tumor with mesenchymal differentiation harboring C11orf95-NCOA1/2 or -RELA fusion: A hitherto unclassified tumor related to ependymoma.

Recurrent fusion genes involving C11orf95, C11orf95-RELA, have been identified only in supratentorial ependymomas among primary CNS tumors. Here, we report hitherto histopathologically unclassifiable high-grade tumors, under the tentative label of "ependymoma-like tumors with mesenchymal differentiation (ELTMDs)," harboring C11orf95-NCOA1/2 or -RELA fusion. We examined the clinicopathological and molecular features in five cases of ELTMDs. Except for one adult case (50 years old), all cases were in children ranging from 1 to 2.5 years old. All patients presented with a mass lesion in the cerebral hemisphere. Histologically, all cases demonstrated a similar histology with a mixture of components. The major components were embryonal-appearing components forming well-delineated tumor cell nests composed of small uniform cells with high proliferative activity, and spindle-cell mesenchymal components with a low- to high-grade sarcoma-like appearance. The embryonal-appearing components exhibited minimal ependymal differentiation including a characteristic EMA positivity and tubular structures, but histologically did not fit with ependymoma because they lacked perivascular pseudorosettes, a histological hallmark of ependymoma, formed well-delineated nests, and had diffuse and strong staining for CAM5.2. Molecular analysis identified C11orf95-NCOA1, -NCOA2, and -RELA in two, one, and two cases, respectively. t-distributed stochastic neighbor embedding analysis of DNA methylation data from two cases with C11orf95-NCOA1 or -NCOA2 and a reference set of 380 CNS tumors revealed that these two cases were clustered together and were distinct from all subgroups of ependymomas. In conclusion, although ELTMDs exhibited morphological and genetic associations with supratentorial ependymoma with C11orf95-RELA, they cannot be regarded as ependymoma. Further analyses of more cases are needed to clarify their differences and similarities.

Postoperative stroke and neurological outcomes in the early phase after revascularization surgeries for moyamoya disease: an age-stratified comparative analysis.

Stroke and neurological outcomes in the early phase following revascularization for moyamoya disease (MMD) may depend on the patient's age. In this study, an age-stratified comparative analysis was performed to clarify this issue. We reviewed 105 MMD patients who underwent 179 revascularization surgeries. The demographic characteristics were collected in four age groups (≤ 5 and 6-17 years for pediatric patients and 18-49 and ≥ 50 years for adults). Additionally, we assessed the incidence of subsequent stroke and deterioration of modified Rankin Scale (mRS) score. Then, we evaluated predictors of postoperative stroke and mRS deterioration using logistic regression. The mean patient age was 26.2 ± 18.5 years. No significant difference in the incidence of postoperative stroke was observed between age groups; however, the incidence tended to be increased among patients aged ≤ 5 years (17.9%) and patients aged ≥ 50 years (16.7%). Deterioration of mRS scores was significantly associated with ages ≤ 5 years (17.9%) and ≥ 50 years (11.1%). Logistic regression showed that posterior cerebral artery involvement (odds ratio [OR], 4.6) and postoperative transient neurological events (TNEs) (OR, 5.93) were risk factors for postoperative stroke. Age ≤ 5 years (OR, 9.73), postoperative TNEs (OR, 7.38), and postoperative stroke (OR, 49) were identified as predictors of unfavorable neurological outcomes. The novel feature of this comparative analysis by age group is that membership in the early-childhood MMD patient group (under 5 years old) was an independent risk factor for unfavorable short-term neurological outcomes and was mainly associated with the incidence of postoperative severe cerebral infarction.

Postoperative Cerebral Infarction Risk Factors and Postoperative Management of Pediatric Patients with Moyamoya Disease.

OBJECTIVE: Although revascularization surgery for patients with moyamoya disease can effectively prevent ischemic events and thus improve the long-term clinical outcome, the incidence of postoperative ischemic complications affects patients' quality of life. This study aimed to clarify the risk factors associated with postoperative ischemic complications and to discuss the appropriate perioperative management. METHODS: Fifty-eight revascularization operations were performed in 37 children with moyamoya disease. Patients with moyamoya syndrome were excluded from this study. Magnetic resonance imaging was performed within 7 days after surgery. Postoperative cerebral infarction was defined as a diffusion-weighted imaging high-intensity lesion with or without symptoms. We usually use fentanyl and dexmedetomidine as postoperative analgesic and sedative drugs for patients with moyamoya disease. We used barbiturate coma therapy for pediatric patients with moyamoya disease who have all postoperative cerebral infarction risk factors. RESULTS: Postoperative ischemic complications were observed in 10.3% of the children with moyamoya disease (6 of 58). Preoperative cerebral infarctions (P = 0.0005), younger age (P = 0.038), higher Suzuki grade (P = 0.003), and posterior cerebral artery stenosis/occlusion (P = 0.003) were related to postoperative ischemic complications. Postoperative cerebral infarction occurred all pediatric patients using barbiturate coma therapy. CONCLUSIONS: The risk factors associated with postoperative ischemic complications for children with moyamoya disease are preoperative infarction, younger age, higher Suzuki grade, and posterior cerebral artery stenosis/occlusion. Barbiturate coma therapy for pediatric patients with moyamoya disease who have the previous risk factors is insufficient for prevention of postoperative cerebral infarction. More studies are needed to identify the appropriate perioperative management.

Different rate-limiting activities of intracellular pH regulators for HCO3- secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts.

Intracellular pH (pHi) regulation fundamentally participates in maintaining HCO3- release from HCO3--secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na+-H+ exchanger (NHE) to pHi regulation for HCO3- secretion by cAMP and Ca2+ signals. Resting pHi was dispersed between 7.4 and 7.9. Forskolin consistently decreased pHi showing the dominance of pHi-lowering activities, but carbachol gathered pHi around 7.6. CA inhibition suppressed the forskolin-induced decrease in pHi, while it allowed carbachol to consistently increase pHi by revealing that carbachol prominently activated NHE via Ca2+-calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pHi, which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pHi-lowering CA and pHi-raising NHE, respectively, to regulate pHi for HCO3- secretion.

Membrane potential modulation of ionomycin-stimulated Ca(2+) entry via Ca (2+)/H (+) exchange and SOC in rat submandibular acinar cells.

Ionomycin (IM) at 5 microM mediates the Ca(2+)/H(+) exchange, while IM at 1 microM activates the store-operated Ca(2+) entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K(+) concentration ([K(+)](o)). IM (5 microM, the Ca(2+)/H(+) exchange) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)) to an extremely high value at 151 mM [K(+)](o). However, with increasing [K(+)](o), the rates of Ca(2+) entry decreased in a linear relationship. The reversal potential (E (rev)) for the Ca(2+)/H(+) exchange was +93 mV, suggesting that IM (5 microM) exchanges 1 Ca(2+) for 1 H(+). Thus, depolarization decreases the Ca(2+) influx via the Ca(2+)/H(+) exchange because of its electrogenicity (1 Ca(2+) for 1 H(+)). On the other hand, IM (1 microM, the SOCs) abolished an increase in [Ca(2+)](i) at 151 mM [K(+)](o). With increasing [K(+)](o), the rate of Ca(2+) entry immediately decreased linearly. The E (rev) for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca(2+) over Na(+) (P (Ca)/P (Na) = 8.2). Moreover, an increase in extracellular Ca(2+) concentration (20 mM) enhanced the Ca(2+) entry via the SOCs at 151 mM [K(+)](o), suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca(2+) entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca(2+)](i) via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca(2+)](i) via two pathways depending on its concentration, the exchange of 1 Ca(2+) for 1 H(+) at 5 muM and the SOCs at 1 microM.

Involvement of cytoskeletal integrity in the regulation of Cl- and amylase secretion from rat parotid acinar cells.

The cytoskeleton serves as a signal modulator for Ca2+ and cAMP-regulated cell functions including the secretion of ions and granule contents. The interaction between Ca2+ and cAMP signaling systems potentiates amylase secretion and suppresses Cl- secretion in the parotid glands. In this study, we investigated the role of the cytoskeleton in the modulation of Cl- and amylase secretion from rat parotid acinar cells upon activation of each intracellular signaling system and their interaction. Cytochalasin D markedly inhibited the Ca2+-activated outwardly rectifying Cl- current at positive membrane potentials and carbachol (CCh)-induced Cl- currents in the whole-cell configuration at -80 mV, whereas colchicine enhanced Cl- currents. Cytochalasin D, but not colchicine, markedly inhibited CCh-induced Cl- secretion. Synergistic actions of CCh and forskolin on Cl- and amylase secretion were observed even in the presence of cytochalasin D. These results suggest that the synergistic effects of Ca2+ and cAMP signaling systems on amylase and Cl- secretion do not require actin filament integrity but that secretion by the two signals themselves does require actin filament integrity.

Suppression of carbachol-induced oscillatory Cl- secretion by forskolin in rat parotid and submandibular acinar cells.

Sympathetic stimulation induces weak salivation compared with parasympathetic stimulation. To clarify this phenomenon in salivary glands, we investigated cAMP-induced modulation of Ca(2+)-activated Cl(-) secretion from rat parotid and submandibular acinar cells because fluid secretion from salivary glands depends on the Cl(-) secretion. Carbachol (Cch), a Ca(2+)-increasing agent, induced hyperpolarization of the cells with oscillatory depolarization in the current clamp mode of the gramicidin-perforated patch recording. In the voltage clamp mode at -80 mV, Cch induced a bumetanide-sensitive oscillatory inward current, which was larger in rat submandibular acinar cells than in parotid acinar cells. Forskolin and IBMX, cAMP-increasing agents, did not induce any marked current, but they evoked a small nonoscillatory inward current in the presence of Cch and suppressed the Cch-induced oscillatory inward current in all parotid acinar cells and half (56%) of submandibular acinar cells. In the current clamp mode, forskolin + IBMX evoked a small nonoscillatory depolarization in the presence of Cch and reduced the amplitude of Cch-induced oscillatory depolarization in both acinar cells. The oscillatory inward current estimated at the depolarized membrane potential was suppressed by forskolin + IBMX. These results indicate that cAMP suppresses Ca(2+)-activated oscillatory Cl(-) secretion of parotid and submandibular acinar cells at -80 mV and possibly at the membrane potential during Cch stimulation. The suppression may result in the weak salivation induced by sympathetic stimulation.

Regulation of IL-8 by Irsogladine maleate is involved in abolishment of Actinobacillus actinomycetemcomitans-induced reduction of gap-junctional intercellular communication.

Our previous report has shown that Irsogladine maleate (IM) counters and obviates the reduction in gap junction intercellular communication (GJIC) and the increase in IL-8 levels, respectively, induced by outer membrane protein 29 from Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) in cultured human gingival epithelial cells (HGEC). In addition, IM suppresses the increase in the secretion of IL-8 caused by whole live A. actinomycetemcomitans. These findings implicate the modulation of IL-8 levels by IM in abolishment of the reduction of GJIC in HGEC. Tight junctions are also responsible for cell-cell communication. Zonula occludens protein-1 (ZO-1) is a major tight junction protein. To investigate the regulatory mechanism of intercellular communication mediated by IM, in the present study, we focused on the involvement of IL-8 in A. actinomycetemcomitans-induced change in GJIC and ZO-1 expression in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in levels of Connexin (CX) 43, suggesting that it could abolish the A. actinomycetemcomitans-induced reduction in GJIC in HGEC. CXCR-1 is a receptor of IL-8. The simultaneous addition of A. actinomycetemcomitans and anti-CXCR-1 antibody also abrogated the repression of GJIC and CX43 expression by A. actinomycetemcomitans in HGEC, although the anti-CXCR-1 antibody was less effective than IM. IM inhibited the IL-8-induced reduction in CX43 levels and GJIC in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in the expression of ZO-1, although anti-CXCR-1 antibody did not influence the decrease in ZO-1 mRNA levels caused by A. actinomycetemcomitans. Furthermore, IL-8 had little effect on the mRNA levels of ZO-1. These findings suggest that IL-8 mediates the A. actinomycetemcomitans-induced reduction of GJIC and CX43 expression in HGEC. The regulation of IL-8 levels by IM in HGEC is partially involved in abrogation of the reduction of GJIC and CX43 expression by A. actinomycetemcomitans. Furthermore, the regulatory effect of IM on the expression of CX43 and ZO-1 is different.

Genetic tracing shows segregation of taste neuronal circuitries for bitter and sweet.

The recent discovery of mammalian bitter, sweet, and umami taste receptors indicates how the different taste qualities are encoded at the periphery. However, taste representations in the brain remain elusive. We used a genetic approach to visualize the neuronal circuitries of bitter and sweet tastes in mice to gain insight into how taste recognition is accomplished in the brain. By selectively expressing a transsynaptic tracer in either bitter- or sweet and/or umami-responsive taste receptor cells, and by comparing the locations of the tracer-labeled neurons in the brain, our data revealed the potential neuronal bases that underlie discrimination of bitter versus sweet.

Irsogladine maleate influences the response of gap junctional intercellular communication and IL-8 of human gingival epithelial cells following periodontopathogenic bacterial challenge.

Gingival epithelial cells first encounter periodontopathogenic bacteria and their metabolic products to produce inflammatory cytokines. Gap junctional intercellular communication (GJIC) is thought to play a critical role in cellular coordination in tissue homeostasis. Gap junctions are structured by connexins (CXs). GJIC response of gingival epithelial cells to the bacteria may be involved in the initiation of periodontal disease. Irsogladine maleate (IM) is known to enhance GJIC through cAMP. In the present study, we examined an effect of IM on GJIC response and on interleukin-8 (IL-8) levels in human gingival epithelial cells (HGEC) exposed to a periodontopathogenic bacterium, Actinobacillus actinomycetemcomitans, and its outer membrane protein (OMP) 29 in order to test the hypothesis that IM has the ability to modulate GJIC and inflammatory responses of gingival epithelial cells to periodontopathogenic bacteria. IM countered the OMP29-induced reduction of GJIC, CX43 levels and cAMP levels in HGEC. The simultaneous addition of OMP29 and dibutyryl cAMP also abrogated the repression of GJIC by OMP29. Furthermore, IM obviated the increase in IL-8 levels in HGEC stimulated by whole live A. actinomycetemcomitans and by OMP29. These findings suggest that IM counters the OMP29-induced GJIC reduction in HGEC through cAMP. IM may eliminate initial perturbation of gingival epithelial cells by regulating responses of GJIC and IL-8 to periodontopathogenic bacterial exposure.

Molecular dissection of the butyrate action revealed the involvement of mitogen-activated protein kinase in cystic fibrosis transmembrane conductance regulator biogenesis.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which belongs to the superfamily of ATP-binding cassette transporters and uniquely possesses an additional large cytoplasmic domain [regulatory (R) domain]. CFTR inefficiently folds by means of co- and post-translational interactions with the cytosolic chaperones as well as luminal chaperones in the endoplasmic reticulum (ER). Aberrant folding and defective trafficking of the CFTR protein, which functions as an apical membrane Cl(-) channel, is the principal cause of cystic fibrosis. Recent data indicated that butyrate improves CFTR trafficking partly by regulating molecular chaperones; however, the precise mechanism of butyrate action remains elusive. In the present study, we examine the molecular aspect underlying the butyrate action in CFTR biogenesis by evaluating the expression and localization of the green fluorescent protein (GFP)-tagged CFTR transgenes in Cos7 cells. Our data show that butyrate significantly promoted stability of the ER-located form of GFP-wild-type (wt)-CFTR, followed by an increase in the amount of plasma membrane GFP-wt-CFTR. In contrast, the expression of the R domain deletion mutant GFP-DeltaR-CFTR was slightly increased by butyrate. The butyrate action on wt-CFTR expression was partially blocked by PD98059 (2'-amino-3'-methoxyflavone), a specific inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK), which is the upstream activator of extracellular-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK). Furthermore, activation of ERK/MAPK by the coexpression of constitutively active MAPKK/MEK predominantly augmented the expression of wt-CFTR, but not of DeltaR-CFTR, induced by butyrate. These data suggest that butyrate may facilitate the biogenesis and trafficking of wt-CFTR by requiring the presence of the R domain and further involving active ERK/MAPK in its biogenesis.

Gramicidin-perforated patch recording revealed the oscillatory nature of secretory Cl- movements in salivary acinar cells.

Elevations of cytoplasmic free calcium concentrations ([Ca(2+)](i)) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl(-) through Ca(2+)-activated Cl(-) channels, while Cl(-) enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and/or parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl(-) secretion, we analyzed carbachol (CCh)-activated Cl(-) currents in submandibular acinar cells using the "gramicidin-perforated patch recording configuration." Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl(-) currents in the gramicidin-perforated patch recording were carried by Cl(-) efflux via Cl(-) channels, dependent upon Cl(-) entry through Cl(-) transporters expressed in the acinar cells. CCh-evoked oscillatory Cl(-) currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl(-) currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO(3)(-) had significant effects, suggesting that the cotransporter rather than parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers is the primary Cl(-) uptake pathway. Pharmacological manipulation of the activities of the Ca(2+)-activated Cl(-) channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl(-) currents, while adjusting to the rate imposed by the Ca(2+)-activated Cl(-) channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl(-) movements may effectively provide a driving force for fluid secretion in intact acinar cells.

Extracellular proton sensing of the rat gustatory cyclic nucleotide-gated channel.

Elevations of the intracellular levels of cyclic nucleotides appear to cause the cation influx through gustatory cyclic nucleotide-gated (CNGgust) channels expressed in taste cells. Although changes in the oral pH may directly regulate the activity of the CNGgust channel, the mechanism of pH-dependent control of the channel is not understood. In the present study, we combined the whole-cell patch-clamp recording and the site-directed mutagenesis to investigate the effect of extracellular pH on the ion permeation through CNGgust channels expressed in HEK293 cells. Extracellular acidification strongly inhibited ion permeation through open CNGgust channels. Mutation of Glu(289) remarkably attenuated the pH-dependence of the channel, suggesting that Glu(289) in the pore-forming region is a major proton acceptor site. However, the mutant E289A-CNGgust channel possesses the other residual protonation/deprotonation site. The channel activity, tightly regulated by pH(o) and [cNMP](i), suggests the involvement of its pH(o)-dependent ion permeation in taste signal transduction events.