The interaction of ATP11C-b with ezrin contributes to its polarized localization.

ATP11C, a member of the P4-ATPase family, translocates phosphatidylserine and phosphatidylethanolamine at the plasma membrane. We previously revealed that its C-terminal splice variant ATP11C-b exhibits polarized localization in motile cell lines, such as MDA-MB-231 and Ba/F3. In the present study, we found that the C-terminal cytoplasmic region of ATP11C-b interacts specifically with ezrin. Notably, the LLxY motif in the ATP11C-b C-terminal region is crucial for its interaction with ezrin as well as its polarized localization on the plasma membrane. A constitutively active, C-terminal phosphomimetic mutant of ezrin was colocalized with ATP11C-b in polarized motile cells. ATP11C-b was partially mislocalized in cells depleted of ezrin alone, and exhibited greater mislocalization in cells simultaneously depleted of the family members ezrin, radixin and moesin (ERM), suggesting that ERM proteins, particularly ezrin, contribute to the polarized localization of ATP11C-b. Furthermore, Atp11c knockout resulted in C-terminally phosphorylated ERM protein mislocalization, which was restored by exogenous expression of ATP11C-b but not ATP11C-a. These observations together indicate that the polarized localizations of ATP11C-b and the active form of ezrin to the plasma membrane are interdependently stabilized.

Antitumor activity of cyclin-dependent kinase inhibitor alsterpaullone in Epstein-Barr virus-associated lymphoproliferative disorders.

Epstein-Barr virus (EBV) is a well-established tumor virus that has been implicated in a wide range of immunodeficiency-associated lymphoproliferative disorders (LPDs). Although rituximab, a CD20 mAb, has proven effective against EBV-associated LPDs, prolonged use of this drug could lead to resistance due to the selective expansion of CD20- cells. We have previously shown that cyclin-dependent kinase (CDK) inhibitors are able to specifically suppress the expression of viral late genes, particularly those encoding structural proteins; however, the therapeutic effect of CDK inhibitors against EBV-associated LPDs is not clear. In this study, we examined whether CDK inhibitors confer a therapeutic effect against LPDs in vivo. Treatment with alsterpaullone, an inhibitor of the CDK2 complex, resulted in a survival benefit and suppressed tumor invasion in a mouse model of LPDs. Inhibition of CDK efficiently induced G1 cell cycle arrest and apoptosis in EBV-positive B cells. These results suggest that alsterpaullone suppresses cell cycle progression, resulting in the antitumor effect observed in vivo.

The cytoplasmic C-terminal region of the ATP11C variant determines its localization at the polarized plasma membrane.

ATP11C, a member of the P4-ATPase family, is a major phosphatidylserine (PS)-flippase located at the plasma membrane. ATP11C deficiency causes a defect in B-cell maturation, anemia and hyperbilirubinemia. Although there are several alternatively spliced variants derived from the ATP11C gene, the functional differences between them have not been considered. Here, we compared and characterized three C-terminal spliced forms (we designated as ATP11C-a, ATP11C-b and ATP11C-c), with respect to their expression patterns in cell types and tissues, and their subcellular localizations. We had previously shown that the C-terminus of ATP11C-a is critical for endocytosis upon PKC activation. Here, we found that ATP11C-b and ATP11C-c did not undergo endocytosis upon PKC activation. Importantly, we also found that ATP11C-b localized to a limited region of the plasma membrane in polarized cells, whereas ATP11C-a was distributed on the entire plasma membrane in both polarized and non-polarized cells. Moreover, we successfully identified LLXY residues within the ATP11C-b C-terminus as a critical motif for the polarized localization. These results suggest that the ATP11C-b regulates PS distribution in distinct regions of the plasma membrane in polarized cells.

Phospholipid flippase ATP11C is endocytosed and downregulated following Ca2+-mediated protein kinase C activation.

We and others showed that ATP11A and ATP11C, members of the P4-ATPase family, translocate phosphatidylserine (PS) and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflets at the plasma membrane. PS exposure on the outer leaflet of the plasma membrane in activated platelets, erythrocytes, and apoptotic cells was proposed to require the inhibition of PS-flippases, as well as activation of scramblases. Although ATP11A and ATP11C are cleaved by caspases in apoptotic cells, it remains unclear how PS-flippase activity is regulated in non-apoptotic cells. Here we report that the PS-flippase ATP11C, but not ATP11A, is sequestered from the plasma membrane via clathrin-mediated endocytosis upon Ca2+-mediated PKC activation. Importantly, we show that a characteristic di-leucine motif (SVRPLL) in the C-terminal cytoplasmic region of ATP11C becomes functional upon PKC activation. Moreover endocytosis of ATP11C is induced by Ca2+-signaling via Gq-coupled receptors. Our data provide the first evidence for signal-dependent regulation of mammalian P4-ATPase.

[Solitary hyoid bone metastasis from hepatocellular carcinoma treated with surgery].

The incidence of bone metastasis in patients with hepatocellular carcinoma (HCC) has reportedly been increasing. The hyoid bone is rarely a target of metastatic HCC. We present a unique case of HCC which showed a single distant metastasis to the hyoid bone. An 81-year-old Japanese man was referred to us from the Department of Gastroenterology complaining of a 1-month history of a painful left neck mass when swallowing. He had a serial history of non-surgical treatments for HCC in the previous six years. Ultrasonography-guided fine needle aspiration cytology yielded a diagnosis of poorly differentiated carcinoma. PET-CT demonstrated a mass (SUV 3.8) which had destroyed the left side of the hyoid body. Based on these cytological and radiological findings of the mass in addition to his medical history of having recurrent HCC, we strongly suspected that the mass was a solitary metastatic HCC of the hyoid bone. The mass was completely resected combined with the hyoid bone under general anesthesia. Postoperative pathologic findings were consistent with metastatic HCC. The patient was relieved from pain in swallowing just after surgery. Minimally invasive surgery can be an effective treatment modality for pain relief even in patients with bone metastasis from HCC.

Visualization and assessment of saccular duct and endolymphatic sinus.

CONCLUSION: The saccular duct and endolymphatic sinus run in the bony groove, before reaching the orifice of the vestibular aqueduct. We first clinically visualized this sulciform groove using three-dimensional (3D) cone beam CT images. This strategy can be useful to assess the condition of the saccular duct and endolymphatic sinus concerning the longitudinal flow system of endolymph. OBJECTIVE: To assess the saccular duct and endolymphatic sinus in the endolymphatic system in order to advance clinical studies on inner ear dysfunction. METHODS: The sulciform groove of the saccular duct and endolymphatic sinus of human subjects was analyzed by cone beam CT and compared with that of a cadaver. RESULTS: We could obtain reconstructed 3D CT images of the sulciform groove of the saccular duct and endolymphatic sinus using several CT window levels.

Blockage of reuniting duct in Meniere's disease.

CONCLUSION: The bony saccular orifice to the bony groove of the ductus reuniens (reuniting duct) could not be visualized in the Meniere's ear with significantly greater frequency compared with normal subjects, which suggests that the reuniting duct is affected by radio-opaque substances in CT findings. OBJECTIVE: This study investigated a more specific, objective, and simpler strategy to diagnose Meniere's disease by assessing the reuniting duct. SUBJECTS AND METHODS: We examined the ears of 12 patients with definitely diagnosed unilateral Meniere's disease in stage 3 based on Meniere's disease criteria proposed by the Committee on Hearing and Equilibrium of the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS), and the ears of 12 normal control subjects using three-dimensional (3D) cone beam CT. RESULTS: The bony groove of the reuniting duct between the saccule and cochlea was visualized in all control subjects. However, the bony saccular orifice to the bony groove could not be visualized in the lesional ear of Meniere's patients with significantly greater frequency compared with those of the contralateral non-lesional ears and control ears (p < 0.01). This orifice was not patent in 66.7% (8 of 12 ears) on the lesional side but all contralateral non-lesional ears of the patients and normal control ears were patent.

Assessment of the reuniting duct by three-dimensional CT rendering.

CONCLUSION: The rendering strategy sometimes induces misunderstanding of the image. We demonstrated a more accurate image of the bony groove of the reuniting duct using three-dimensional (3D) cone beam CT image, which was less affected by artifacts created by the rendering effect. OBJECTIVE: To obtain a suitable image of the groove of the reuniting duct for future morphological study. MATERIALS AND METHODS: The grooves of reuniting ducts in 10 healthy human subjects were analyzed by cone beam CT in comparison with a cadaver study. RESULTS: We could obtain more accurate 3D CT images of the bony groove in human subjects by checking the landmarks of 3D CT images.

[A case of Churg-Strauss syndrome following corticosteroid withdrawal].

A 56-year-old man with bilateral hearing impairment who had taken betamethasone combined with dexchlorpheniramine maleate for 7 years to treat chronic sinusitis developed a dry cough after discontinuing this medication and was diagnosed with asthma, and after which he sensed impaired bilateral hearing. Based on the presence of numerous eosinophilic leukocytes in otorrehea, we made a diagnosis of eosinophilic otitis media, and he was prescribed predonisolone to control the asthma, but discontinued it on his own. He then developed fever, maniphalanx stiffness, testicular pain, and facial hyperesthesia, eruptions, and the lower-limb numbness. The detection of a positive serum reaction for MPO-ANCA and evaluated of eosinophilic leukocyte levels yielded a definitive diagnosis of CSS, for which the man was treated with predonisolone and cyclophosphamide. His symptoms were relieved, even though the onset of neutropenia, necessitated the discontinuation of cyclophosphamade administration.

Three-dimensional images of the reuniting duct using cone beam CT.

CONCLUSION: There is a bony groove under the course of the reuniting duct of the inner ear. Cone beam CT could show three-dimensional (3D) reconstruction images of this groove in a cadaver and living human subjects. OBJECTIVE: To obtain simple and universal images of the reuniting duct in humans for clinical use. MATERIALS AND METHODS: We investigated the reuniting duct macroscopically by observing the temporal bone in cadavers and living human subjects using cone beam CT. 3D reconstruction images of the duct were analyzed by the rendering software IVIEW. RESULTS: The reuniting duct showed a bony groove between the saccule and cecum of the hook portion of the cochlea that could be three-dimensionally visualized by cone beam CT. A bony groove image in a living human subject could also be visualized by cone beam CT.

Cartilage ossiculoplasty by lever method.

CONCLUSION: We invented a new ossicular chain reconstruction by lever methods (OCRLM) in tympanoplasty type III with the canal wall down technique and investigated the postoperative hearing results of the 24 patients who had undergone this surgery. Postoperative air-bone gaps (ABGs) followed up for 2.1-3.2 years gave satisfactory improvement audiometrically compared with former reports. OBJECTIVE: To present newly devised methods applied from the viewpoint of mechanical dynamics for ossicular chain reconstruction in tympanoplasty. MATERIALS AND METHODS: A total of 24 cholesteatoma patients who underwent tympanoplasty with mastoidectomy using the canal wall down technique and ossicular chain reconstruction with OCRLM between May 2003 and April 2004 were investigated. The postoperative ABG of a four-frequency (0.5, 1, 2, and 4 kHz) average of OCRLM followed up for 2 years or more was assessed. RESULTS: Patients who underwent OCRLM showed good postoperative hearing results. Average postoperative ABGs of less than 10, 15, 20, 25, and 30 dB were 4.2, 37.5, 70.8, 91.6, and 100%, respectively. A gap of >30 dB was absent and the mean postoperative ABG was 17.2dB. The OCRLM method was satisfactory to improve hearing and effectively reduce the ABG postoperatively over the period of observation.

Disregard of cholesteatoma in congenital aural stenosis.

Congenital aural stenosis (CAS) is one type of congenital aural atresia (CAA) and is assumed to be a relatively mild type. Although CAS may be associated with cholesteatoma in the external ear canal, little attention has been paid to this association. We present two cases of CAS with middle ear infection due to destructive cholesteatoma. Both had been followed for microtia by a plastic surgeon. These patients had already exhibited cholesteatoma on CT examination, although it had been missed before the emergence of ear symptoms, otorrhea, and otalgia. We present our cases with successive changes in CT findings over time and discuss the problems of CAS with cholesteatoma.

Carotid artery pseudoaneurysm as a rare sequela of surgery for laryngeal cancer.

Formation of carotid artery pseudoaneurysm is uncommon after intensive treatment for head and neck cancer. We encountered a case of postoperative formation of pseudoaneurysm at the left carotid bifurcation in a diabetic man. The risk factors for carotid artery pseudoaneurysm in the patient included diabetes mellitus, previous radiotherapy to the neck, neck dissection, and postoperative Staphylococcus aureus infection secondary to accidental pharyngocutaneous fistula. We successfully obliterated this pseudoaneurysm using a Smart stent and detachable coils without neurological deficits. The possibility of vascular injury after treatment for head and neck cancer must be considered, especially in patients with risk factors for it due to previous treatment.

[Effectiveness of deep breath-hold SPECT in torso area: examination concerning improvement of resolution].

Because SPECT images are acquired under normal respiration, the respiratory motion induces artifacts and decreases resolution. In this study we developed a novel method of acquiring SPECT data during deep inhalation breath-hold (BrST) and assessed its efficacy in reducing motion artifacts and improving resolution. Reproducibility studies found that variations in SPECT image homogeneity were reduced using the BrST method to within a clinically non-problematic range. An experiment using a custom-built respiration phantom showed almost complete elimination of motion artifacts and significant improvement in resolution using the BrST method. Clinical assessment confirmed a significant reduction in motion artifacts along with the improvement in resolution. The BrST method enabled visualization of lesions that previously had been impossible to detect by standard acquisition under normal respiration. The BrST method is expected to both significantly reduce motion artifacts and improve resolution.

Probable function of Boettcher cells based on results of morphological study: localization of nitric oxide synthase.

Boettcher cells lie on the basilar membrane beneath Claudius cells. The cells are considered supporting cells for the organ of Corti, and present only in the lower turn of the cochlea, which responds to high-frequency sound. Boettcher cells interdigitate with each other, and project microvilli into the intercellular space. Their structural specialization suggests that Boettcher cells may play a significant role in the function of the cochlea. Nitric oxide synthase (NOS) has previously been detected in substructures of the cochlea. In the cochlea, it is believed that nitric oxide plays an important role in neurotransmission, blood flow regulation, and induction of cytotoxicity under pathological conditions. Findings concerning detection of NOS on Boettcher cells are rare. We demonstrated here the localization of NOS on Boettcher cells of the rat by immunohistochemistry using polyclonal antibody to NOS. On observation with the light microscope using DAB staining, positive immunostaining to NOS was observed in Boettcher cells. In immunoelectron micrographs, NOS was detected abundantly in the cytoplasm of the interdigitations. This suggests that the interdigitations may play significant roles by using NOS. It follows from this that the nitric oxide (NO) on Boettcher cells may influences neighboring Boettcher cells. The ultrastructure of Boettcher cells suggests that they may be active cells, which perform both secretory and absorptive functions.

Nitric oxide induces apoptosis of the hair cells of cochlea.

OBJECTIVE: Nitric oxide (NO) has a toxic effect on neuronal cells related to glutamate receptors. NO released from post-synaptic cells with glutamate receptors can induce nearby cell death. In this experiment, we examined the effect of NO on cochlear hair cells. METHODS: Two concentrations, 0.5 and 0.1 molar concentration (MC) of 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5), which is a NO donor, were placed on the round window of the guinea pig cochlea and its morphological changes were investigated at 12, 28, 72 h after the treatment. RESULTS: By the trypan blue dye extrusion method, the cell death was recognized in the outer hair cells at the percentage 0, 36.0+/-6.6, 4.9+/-1.2% at 12, 28, 72 h after treatment respectively in the group 0.5 MC NOC-5, but no inner hair cell death was recognized. In the group of 0.1 MC NOC-5 and control any cell death was not detected. Transmission electron microscopy revealed this cell death was characteristic of apoptosis. CONCLUSION: The findings that apoptosis was induced in the outer hair cells of the organ of Corti by NO suggests the possibility that the cochlea is affected when extra NO release occurs there, leading to cochlear dysfunction.

Positional and positioning nystagmus in healthy subjects under videonystagmoscopy.

OBJECTIVE: The eye-focused video camera as sensitive to infrared light is very useful for detecting nystagmus with open eyes in the dark. The existence of physiological nystagmus has been reported. We investigated how frequently physiological positional and positioning nystagmus is seen in healthy individuals under infrared video goggles. MATERIALS AND METHODS: Eighty-nine healthy individuals were examined in this study. Positional and positioning nystagmus was examined with infrared video goggles. RESULTS: Positional nystagmus could be detected in 65 of the 89 subjects under infrared video goggles. The majority of nystagmus was horizontal nystagmus, and vertical nystagmus was also seen in four subjects. Nystagmus was most frequently recognized in right or left shoulder down position. Positioning nystagmus was detected in 40 subjects. CONCLUSIONS: Positional and positioning nystagmus frequently exist in healthy subjects. This finding means that the existence of physiological nystagmus must be considered when diagnosing dizzy patients using this equipment.

Application of modified transmastoid approach methods to congenital atresia of the external ear canal with middle ear infection.

OBJECTIVE: To present our surgical technique for congenital atresia of the external ear canal with middle ear infection. METHODS: A modified transmastoid approach to congenital atresia of the external ear canal with middle ear infection was applied. Our method is to visualize the landmarks of the mastoid cavity such as the antrum, sinodural angle and digastric ridge by the canal-open method to avoid facial nerve injury and then prepare a relatively large external ear canal with reconstruction of the posterior wall of the external ear canal. RESULTS AND CONCLUSION: This method is suitable not only for treating congenital atresia of the external ear canal with middle ear infection, but also for avoiding facial nerve injury and postoperative complications such as re-stenosis of the new ear canal and postoperative middle ear infection.

Concurrent chemoradiotherapy with pirarubicin and 5-fluorouracil for resectable oral and maxillary carcinoma.

OBJECTIVE: We present the response rate and adverse effects of our regimen of concurrent chemoradiotherapy with pirarubicin (THP) and 5-fluorouracil (5-FU) for oral and maxillary carcinoma. PATIENTS AND METHODS: Fifteen patients with oral (10 cases) or maxillary (5 cases) squamous cell carcinoma who underwent our concurrent chemoradiotherapy with the combination of intraarterial pirarubicin, intravenous continuous 5-fluorouracil, and radiation between March 2001 and February 2003 in our department were entered in this study. THP (5 mg/day) was infused into the lingual or maxillary artery one hour before radiation on days 1-5 and 8-12, while intravenous 5-FU (150 mg/m2/day) was instilled continuously on days 1-5, 8-12, 15-19, and 22-26 in accordance with the radiation schedule (2 Gy/day). Consequently, total doses of THP, 5-FU, and radiation were 50 mg, 3000 mg/m2 and 40 Gy, respectively. After the treatment series, response rate and adverse effects were evaluated. RESULTS: Response rate achieved 100% (12 cases exhibited a complete response and the remaining 3 a partial response). Notably, all 10 patients with oral carcinoma exhibited complete response. The main adverse effects were leucopenia (6/15) and mucositis (6/15), both of which were acceptable. CONCLUSIONS: This concurrent chemoradiotherapy is very useful for oral and maxillary carcinoma as a preoperative modality with remarkably high response rate and acceptable adverse events.